The development of a multiplex PCR assay for the detection of Fusarium oxysporum f. sp. cubense lineage VI strains in East and Central Africa

P Ndayihanzamaso,A Viljoen,G Mahuku,F Beed,R Swennen,P Karangwa,G Blomme,D Mostert

doi:10.1007/s10658-020-02092-9

Abstract

Banana is a staple food and cash crop grown in East and Central Africa (ECA). The main banana varieties grown in ECA are the East African highland bananas (EAHB), although dessert/beer bananas such as Sukari Ndizi, Kayinja (Pisang Awak) and Gros Michel are also produced due to their high value at local markets. The Fusarium wilt fungus Fusarium oxysporum f. sp. cubense (Foc) causes disease of susceptible dessert/beer bananas, which significantly reduces yields. Banana Fusarium wilt is managed by excluding the pathogen from disease-free areas and by planting disease-resistant varieties in infested fields. Six phylogenetically closely-related vegetative compatibility groups (VCGs) of Foc, VCGs 0124, 0125, 0128, 01212, 01220 and 01222 are present in ECA, which all group together in Foc Lineage VI. Rapid and accurate detection of Foc Lineage VI strains is thus important to prevent its spread to disease-free areas. In this study, molecular markers specific to Foc Lineage VI were therefore developed. Primer sets were then combined in a multiplex PCR assay, and validated on a worldwide population of 623 known Foc isolates, other formae speciales and non-pathogenic Fusarium oxysporum isolates. The Foc Lineage VI multiplex PCR was used to identify Foc isolates collected in banana fields at five locations in Uganda and Tanzania. Foc Lineage VI DNA was detected at a concentration as low as 0.1 ng/μl, both in the absence and presence of banana DNA, and can therefore be used as an accurate diagnostic tool for Foc Lineage VI strains.

Highlights

Banana (Musa spp.) is produced as a staple food and cash crop in Uganda, Kenya, Tanzania, Burundi, Rwanda and the Democratic Republic of the Congo (DRC)
When the specificity of both primer sets was tested on seven Foc strains it exclusively amplified CAV 2260, an isolate that representing vegetative compatibility groups (VCGs) 0124 in Foc Lineage VI, but not the isolates that represent the other six Foc lineages (Fig. 3)
The primers could detect fungal DNA as low as 0.1 ng/μl in the absence and presence of 50 ng of banana DNA, and were sensitive enough to detect the isolates of the Foc Lineage VI in diseased plants (Fig. 4)

Summary

Introduction

Banana (Musa spp.) is produced as a staple food and cash crop in Uganda, Kenya, Tanzania, Burundi, Rwanda and the Democratic Republic of the Congo (DRC). This area is collectively known as East and Central Africa (ECA), and produced 16.8 million of the 153.2 million tons of bananas produced worldwide in 2017 (FAOSTAT 2017). In Uganda, Burundi and Rwanda, annual consumption levels per capita are approximately 131, 233 and 297 kg, respectively (FAOSTAT 2013). This is substantially more than the average annual global consumption of 20 kg per person (FAOSTAT 2013). The low yields in ECA are primarily caused by poor soil fertility, water shortage, pests and diseases, poor management and socio-economic constraints (Karamura et al 1998; Wairegi et al 2010; Van Asten et al 2011)

Objectives

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Results

Conclusion