Optimization of a Loop-Mediated Isothermal Amplification Assay for On-Site Detection of Fusarium fujikuroi in Rice Seed

Fusarium fujikuroi, causing bakanae disease, is one of the most important seedborne pathogens of rice, the detection of which is paramount for seed certification and for preventing field infections. Molecular tests—qPCR and loop-mediated isothermal amplification (LAMP)—are replacing the blotter test in seed health procedures, due to higher sensitivity, specificity, fast turnaround results delivery, on-site application and the possibility of quantifying endophytic seed infections. A LAMP test, which had been previously developed with primers designed to target the elongation factor 1-α sequence of F. fujikuroi, was validated according to the international validation standard (EPPO, PM7/98) on thirty-four rice seed lines of different levels of susceptibility to the disease, thus comparing it to the blotter test and with two different DNA extraction procedures. The use of crude extracted DNA provided more sensitive results than the DNA extracted with the commercial kit Omega E.Z.N.A® Plant DNA kit. The results showed that the endophytic infection of F. fujikuroi is essential for the development of the disease in the field and that the minimum amount of the pathogen necessary for the development of the disease corresponds to 4.17 × 104 cells/µL. This study confirms the applicability of the LAMP technique on-site on rice seeds with fast and quantitative detection of the pathogen

Guidelines for improving statistical analyses of validation datasets for plant pest diagnostic tests

peer reviewedAppropriate statistical analysis of the validation data for diagnostic tests facilitates the evaluation of the performance criteria and increases the confidence in the conclusions drawn from these data. A comprehensive approach to analysing and reporting data from validation studies and inter-laboratory comparisons such as test performance studies is described. The proposed methods, including statistical analyses, presentation and interpretation of the data, are illustrated using a real dataset generated during a test performance study conducted in the framework of the European project, VALITEST. This analytical approach uses, wherever possible and whenever applicable, statistical analyses recommended by international standards illustrating their application to plant health diagnostic tests. The present work is addressed to plant health diagnosticians and researchers interested and/or involved in the validation of plant diagnostic tests, and also aims to convey the necessary information to those without a statistical background. Detailed statistical explanations are provided in the Appendices

What did we achieve with VALITEST an EU project on validation in plant pest diagnostics?

peer reviewedEnsuring the reliability of diagnostic activities is an essential cornerstone of Plant Health strategies to reduce the risk of entry and spread of plant pests in a region and ultimately their impacts. Diagnostic tests should be validated to ensure that they are fit for purpose. Validation is usually done by diagnostic laboratories although companies commercializing diagnostic kits also produce validation data for their products. Due to the high number of pest , matrix and method combinations and given the significant resources required to validate tests, it is essential that validation data are shared with the entire diagnostic community and produced in a harmonized way to facilitate their use by different stakeholders. Indeed, the selection of tests to be used in specific contexts is not the sole responsibility of diagnostic laboratories and also involve National Plant Protection Organizations. The VALITEST EU project (2018-2021) was established to tackle all these issues. New validation data for tests targeting important pests for the EPPO region were produced. Guidelines to improve and harmonize the validation framework were developed. Sharing of validation data and experience was ensured through the development of new or existing databases, the organization of training courses and the dissemination of the project outputs in scientific publications and Standards. Finally, the involvement of researchers, diagnosticians, policy makers, inspectors, industries etc. and the establishment of the European Plant Diagnostic Industry Association were important actions to strengthen the interactions between Plant Health stakeholders

Global Fusarium networking

Fusarium Head Blight (FHB) caused by several species of Fusarium fungi continues to be a major wheat disease worldwide, causing direct grain yield losses and heath concerns due to mycotoxins. Although chemical intervention can reduce losses, replacing susceptible cultivars with resistant ones is the best recommended control strategy. CIMMYT has been working on FHB for more than 20 years. Progress has been obtained through the shuttle breeding program between CIMMYT and China, and a network of collaborations has been developed over years with advanced research institutes in USA, Japan, Europe, South America and Australia. Although new significant investments in basic and applied FHB research generated worldwide a wealth of knowledge on the pathogen(s) and genetics of resistance in recent years, sources of host resistance are limited and progress toward releasing new resistant varieties in farmers’ fields remains challenging. The concept of a Global Fusarium Initiative (GFI) was presented at the 2 nd International Symposium on FHB in Orlando (2004) and a workshop was held in 2006 in Mexico to foster international collaboration. The need for a GFI facilitated by CIMMYT and informed by technical advisory committees was endorsed. CIMMYT’s assets in Mexico include the identification and selection for resistance in broadly adapted germplasm, phenotyping of mapping populations, and a unique platform for germplasm exchange and distribution. Most critical research needs and opportunities for collaborations consist of three areas: FHB-QTL research, study of the Fusarium pathogen and a joint international scab nursery consortium. Actions and decisions are needed to resource this ambitious project and make it sustainable