Development of a hydrolysis probe-based real-time assay for the detection of tropical strains of Fusarium oxysporum f. sp. cubense race 4.
Fusarium oxysporum f. sp. cubense (Foc) is one of the most important threats to global banana production. Strategies to control the pathogen are lacking, with plant resistance offering the only long-term solution, if sources of resistance are available. Prevention of introduction of Foc into disease-free areas thus remains a key strategy to continue sustainable banana production. In recent years, strains of Foc affecting Cavendish bananas have destroyed plantations in a number of countries in Asia and in the Middle East, and one African country. One vegetative compatibility group (VCG), 01213/16, is considered the major threat to bananas in tropical and subtropical climatic conditions. However, other genetically related VCGs, such as 0121, may potentially jeopardize banana cultures if they were introduced into disease-free areas. To prevent the introduction of these VCGs into disease-free Cavendish banana-growing countries, a real-time PCR test was developed to accurately detect both VCGs. A previously described putative virulence gene was used to develop a specific combination of hydrolysis probe/primers for the detection of tropical Foc race 4 strains. The real-time PCR parameters were optimized by following a statistical approach relying on orthogonal arrays and the Taguchi method in an attempt to enhance sensitivity and ensure high specificity of the assay. This study also assessed critical performance criteria, such as repeatability, reproducibility, robustness, and specificity, with a large including set of 136 F. oxysporum isolates, including 73 Foc pathogenic strains representing 24 VCGs. The validation data demonstrated that the new assay could be used for regulatory testing applications on banana plant material and can contribute to preventing the introduction and spread of Foc strains affecting Cavendish bananas in the tropics.
ANSES, Laboratoire de la Santé des Végétaux-LSV, Unité de mycologie. Domaine de Pixérécourt, Malzéville, France.
Jaime
http://orcid.org/0000-0002-7552-0655
J
Aguayo
Department of Plant Pathology, Faculty of AgriSciences. Stellenbosch University. Matieland, South Africa.
Diane
D
Mostert
ANSES, Laboratoire de la Santé des Végétaux-LSV, Unité de mycologie. Domaine de Pixérécourt, Malzéville, France.
Céline
C
Fourrier-Jeandel
ANSES, Laboratoire de la Santé des Végétaux-LSV, Unité de mycologie. Domaine de Pixérécourt, Malzéville, France.
Isabelle
I
Cerf-Wendling
ANSES, Laboratoire de la Santé des Végétaux-LSV, Unité des ravageurs et agents pathogènes tropicaux. Saint-Pierre, Ile de la Réunion, France.
Bruno
B
Hostachy
Department of Plant Pathology, Faculty of AgriSciences. Stellenbosch University. Matieland, South Africa.
Altus
A
Viljoen
ANSES, Laboratoire de la Santé des Végétaux-LSV, Unité de mycologie. Domaine de Pixérécourt, Malzéville, France.
Renaud
R
Ioos
D000704:Analysis of Variance; D015342:DNA Probes; D004271:DNA, Fungal; D021901:DNA, Intergenic; D005670:Fusarium / Q000145:classification* / Q000235:genetics*; D028521:Musa / Q000382:microbiology; D010935:Plant Diseases / Q000382:microbiology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D015342:DNA Probes; D004271:DNA, Fungal; D021901:DNA, Intergenic