INnovative DIagnostiCs for BANana paThogens Surveillance

Boosted by the globalization of economy, emerging and invasive plant pests and pathogens are a major threat to the sustainability of agricultural industries and food security. Timely surveillance of pathogens associated with their accurate identification and sensitive detection from host are key for prevention or informed management of plant disease outbreaks. Banana is world's most important fruit in terms of production, with over 114 million tonnes produced globally each year. International banana trade remains a major economic pillar for many countries, providing income and sustenance to millions of people. Nevertheless, diseases of banana are becoming increasingly important globally, resulting in reduced yields and escalated disease management costs. The most important vascular diseases include Fusarium wilt disease, caused by Fusarium oxysporum f. sp. cubense, particularly the TR4 strains (FocTR4). Banana Xanthomonas wilt (BXW) caused by Xanthomonas vasicola pv. musacearum (Xvm), Moko disease (MOKO) and banana blood disease (BDB) caused by Rasltonia solanacearum and Ralstonia syzygii subsp. celebesensis, respectively. Efficient surveillance and disease management require the availability of Point-of-Care (POC) diagnostics that can be operated on-site. The objectives of the project were to develop some POC diagnostic tools like LAMP (loop-mediated isothermal amplification) for these diseases, to validate them through inter-laboratory tests and missions in the field and to collaborate with a private company to produce some ready-to-use kits. All these scientific objectives have been successfully achieved, along with those related to training and the two-way transfer of knowledge between the researcher and the host.

The main results achieved are:
-The development of LAMP assays for the four banana pathogens. Specific DNA targets were selected for each bacterial pathogen using in silico comparative genomic analysis of target and non-target genomes. Candidate regions were further screened based on their size, function, and specificity using BLAST on NCBI databases. Several sets of LAMP primers were designed from the best candidate regions and screened on a few strains to select the most efficient ones. Two DNA regions were targeted for the MOKO LAMP because of the high diversity of strains. One hundred percent specificity was demonstrated for each LAMP assay evaluated on a wide collection of target and non-target DNAs. Additionally, high sensitivity was observed for each LAMP assay when tested on both serially diluted DNA and healthy banana tissues spiked with calibrated suspensions of target strains.
-The fine tuning of RPA (recombinase Polymerase amplification)-CRISPR Cas12a assay for the specific diagnostic of TR4 strains of Fusarium oxysporum f. sp. cubense. This highly specific and sensitive molecular tool was developed as a Proof of Concept and will be further validated as a real POC tool.
-The selection of a NaOH-based simplified extraction method for implementing the POC assays in the field. This very simple and cheap technique was shown to be adapted to the LAMP detection of all the pathogens from field samples.
-The validation of LAMP assays . The four LAMP protocols have been validated on inoculated plants as well as field samples. MOKO disease was diagnosed by LAMP in Jamaica and from DNA samples supplied by Ecuador. Missions in Mozambique and Indonesia have been carried out for FocTR4 LAMP, in Uganda for BXW, Indonesia for BDB. Samples collected in banana plantations were directly tested by LAMP in the field or in non-laboratory environment. Reference PCR/qPCR detection were carried out by the local partners in their laboratory to compare the results. Additionally, an inter-laboratory test involving eight international laboratories, aiming to transfer the protocols and assess their reproducibility have been performed . Prototypes LAMP kits manufactured by the industrial partner Qualiplante, randomized samples as well as protocols and guides were sent to the laboratories. Very high reproducibility, repeatability, diagnostic sensitivity and specificity values were obtained.
-Some actions of dissemination and transfer. Some results have been disseminated to various audiences: (i) scientific community, through two accepted publications and two others in progress, the participation to several conferences , (ii) the general public, via press and scientific events like European researcher’s night, and (iii) courses to master's students. Information and training sessions on the LAMP technique and the plant diseases have been carried out for local partners and plant health stakeholders in the visited countries. A closing meeting was organized in La Réunion, bringing together different partners involved in plant surveillance. Also, the transfer of the four protocols has been made to the industrial partner.

We have developed Lamp tests for four economically significant diseases affecting bananas. Instead of focusing on a single pathogen, our approach considers the characteristics of the pathogens from the outset of research, leading to the development of a common extraction protocol applicable before each specific Lamp test. Our efforts extend beyond the development of LAMP tests, as we have implemented the entire process from sample collection to the final diagnostic result, with validation in the field and through interlaboratory tests. Protocols have been transferred to Qualiplante company for ready-to-use kits manufacturing and MOKO and FocTR4 kits are already commercially available. We have also developed a multiplex LAMP assay for MOKO and FocTR4, as these diseases can be found in the same countries and plots. This molecular tool will be validated in the future.
Furthermore, we are pioneering in the development of an innovative diagnostic tool, RPA-Crispr Cas12a, for FocTR4 strains detection. This technique is relatively uncommon in plant pathology, marking a significant "proof of concept" that demonstrates the feasibility of developing and utilizing such tools in plant health.
This work on LAMP development will help raise awareness of this technique and will demonstrate its usefulness for a quick and reliable diagnosis. Future steps will consist of integrating these protocols into European and international diagnostic standard methods.
The research outputs will primarily contribute to improving pathogen control by providing efficient and valuable tools to improve pathogen surveillance within territories and at borders as well as promoting the use of disease-free material for planting. Moreover, through training and information dissemination, this research will strengthen the capacity and skills of national plant protection services and organizations involved in surveillance.
Accurate pathogen detection can result in significant economic benefits, usually achieved through reduced pesticide misuse and increased crop production and quality due to properly timed interventions. International trade will also benefit, as these diagnostic tools will support safe, pathogen-free trade and ensure compliance with the sanitary and phytosanitary requirements of importing countries. Therefore, our research contributes to the global transition toward sustainable food systems promoted by Europe, enhancing crop protection, agricultural productivity, food security, and even human and environmental health by potentially reducing pesticide use.