Annals of Applied Biology ISSN 0003-4746 RESEARCH ARTICLE Lessons learned from the virus indexing of Musa germplasm: insights from a multiyear collaboration C. De Clerck 1,† , K. Crew 2,† , I. Van den houwe 3 , L. McMichael 2 , C. Berhal 1 , L. Lassois 1 , M. Haissam Jijakli 1 , N. Roux 4 , J. Thomas 5 & S. Massart 1 1 Laboratory of Integrated and Urban Phytopathology, Gembloux Agro-Bio Tech, University of Liège, Gembloux, Belgium 2 Queensland Department of Agriculture and Fisheries, Brisbane, Australia 3 Bioversity Transit Centre, c/o Laboratory of Tropical Crop Improvement, Leuven, Belgium 4 Bioversity International, Commodity Systems and Genetic Resources Programme, Montpellier Cedex, France 5 The University of Queensland, Queensland Alliance for Agriculture and Food Innovation, Ecosciences Precinct, Brisbane, Australia Keywords Banana viruses; diagnostic test; virus indexing; phytosanitary certification; molecular diagnostic; electron microscopy; virus miniprep. Correspondence S. Massart, Laboratory of Integrated and Urban Phytopathology, Gembloux Agro-Bio Tech, University of Liège, Passage des déportés, 2, 5030 Gembloux, Belgium. E-mail: sebastien.massart@ulg.ac.be; and J. Thomas, The University of Queensland, Queensland Alliance for Agriculture and Food Innovation. Ecosciences Precinct, GPO Box 267, Brisbane, Queensland 4001, Australia. E-mail: j.thomas2@uq.edu.au † Equal first authors. Received: 17 November 2016; revised version accepted: 10 January 2017. doi:10.1111/aab.12353 Abstract The Bioversity International Transit Center (ITC) for banana hosts more than 1500 accessions largely covering the genetic diversity of the genus Musa. Its objective is to conserve this genetic diversity and to supply plant materials to users worldwide. All the Musa accessions must be tested for virus presence and, if infected, virus elimination must be attempted, to enable the supply of virus-free plant material. An international collaborative effort launched under the auspices of Bioversity International (2007–2013) finally led to the implementation of a two-step process to test the accessions. The first step, called pre-indexing, involved only molecular tests and was designed as a pre-screen of new germplasm lines or existing accessions to reduce the need for post-entry virus therapy and repeated virus indexing. The second step, called full indexing, was performed on either older existing accessions or newer accessions which tested negative during pre-indexing, and involved molecular tests, transmission electron microscopy (TEM) and symptom observation. In total, 270 germplasm lines (434 samples) were pre-indexed; while full indexing was carried out on 243 accessions (68 of which had been pre-indexed). A significant proportion of the samples tested during pre-indexing was infected with at least one virus (68%), showing the utility of this early pre-screening step. Banana streak OL virus and Banana mild mosaic virus were the most commonly detected viruses during both pre- and full indexing. For 22 accessions, viral particles were observed by TEM in full indexing while the molecular tests were negative, underlining the importance of combining various detection techniques. After full indexing, viruses were not detected in 166 accessions, which were then released for international distribution from the ITC. This publication exemplifies how the practical application of diagnostic protocols can raise fundamental questions related to their appropriate use in routine practice and the need for their continuous monitoring and improvement after their first publication. Introduction Banana is a perennial herbaceous plant and is important for food security, feeding millions of small holders, and as a cash crop in many developing countries. Musa acuminata Colla subspecies (A genome) and M. balbisiana Colla (B genome) are the ancestors of the parthenocarpic Musa diploid and polyploid cultivars grown around the world. In 2012, they were grown on an area of 10.4 million ha worldwide, of which 48% was for dessert banana and 52% for cooking bananas, including plantain (FAO, 2014). They are among the 10 most important staple food crops worldwide with a global production of 139.5 million Ann Appl Biol (2017) 1 © 2017 Association of Applied Biologists