Joining Forces to Save Citrus: French-South African research team explores new resources for better citrus greening detection

Developing bilateral cooperation to produce genomic resources to improve diagnosis of citrus greening

CONTEXT

Greening or Huanglongbing (HLB) disease is the world’s most serious phytosanitary constraint for citrus crops, killing tens of millions of trees over the last decade and causing billions of dollars in economic losses.

This disease is caused by three non-cultivable bacteria of the alpha-proteobacterium ‘Candidatus Liberibacter’, i.e., ‘Ca. Liberibacter asiaticus’ (CLas), ‘Ca. Liberibacter americanus’ (CLam), and ‘Ca. Liberibacter africanus’ (CLaf). This degenerative disease has no effective treatment and therefore severely affects citrus varieties as well as some related genera, causing death within a few years. The disease is transmitted by insects of the psyllid group, Diaphorina citri (Kuwayama)and Trioza erytreae (Del Guercio), and is present in Southeast Asia and on the American and African continents, but is still absent from Europe, although D. citri and T. erytreae have become established in Cyprus and the Iberian Peninsula respectively in recent years. In France’s overseas territories, it is particularly present in the West Indies and on Reunion Island, where it causes severe damage.

Accurate, sensitive detection tests for ‘Ca. Liberibacter’ species associated with HLB are the cornerstone of HLB surveillance and management strategies, and require ongoing development and validation. However, the non-culturable nature of these bacteria represents a major hindrance to the laboratory diagnostic process and hence to fundamental studies on understanding epidemics and developing means of control. The latest advances in molecular technology open up new possibilities for the development of control measures to complement existing efforts. Current reference diagnostic methods suffer from a lack of specificity. New, more specific detection methods are needed, but the definition of optimal targets will require the new genomic resources, particularly for CLaf.

To date, five CLaf subspecies have been identified in various rutaceae species. CLaf has been affecting the two northern provinces of South Africa for almost 100 years, but has since been reported in several countries in central, eastern and southern Africa, as well as on several islands in the south-western Indian Ocean. However, the detailed molecular characterization of the milder African form of HLB caused by CLaf is far inferior to that of the more severe Asian form caused by CLas, even though both species represent a major threat to citrus-growing countries in Europe and the wider Mediterranean basin. Indeed, while over forty genomes are available for the bacterium responsible for the more severe Asian form of HLB (CLas), only two genomes (one of which is incomplete) are available for CLaf, and they concern only one of the subspecies described in the literature. Greater availability of CLaf genomes, and more specifically those of its subspecies, will fill these gaps and provide the new knowledge needed for preventive screening and epidemic detection, to enable early control interventions.

OBJECTIVES

The aim of this project is to help produce the new genomic resources needed to improve the diagnostic tools for the ‘Ca. Liberibacter’ species and subspecies associated with HLB. This improved availability of CLaf genomes will open up new possibilities for innovative control strategies. The availability of these genomic resources is also a prerequisite for improving academic knowledge of the mechanisms involved in plant-bacteria-insect interactions, and of the mechanisms involved in horizontal gene transfer and ‘Ca. Liberibacter’ genome plasticity, enabling us to better characterize the adaptive potential of this pathogens.

TEAMS

This project was initiated thanks to funding from the Partenariat Hubert Curien (PHC) PROTEA bilateral program between France and South Africa. The French partner, CIRAD (UMR PVBMT), has been working for over three years to develop and evaluate bacterial sequence enrichment protocols with the aim of producing high-throughput genomic resources of the species responsible for HLB. The South African partner, University of Stellenbosch (Citrus Biotechnology Laboratory), involves experts in graft-transmissible diseases of perennial fruit crops actively researching the HLB pathosystem in South Africa. This program coordinates interdisciplinary research to cover the different facets of this disease and provides a focal point for planning and coordinating research to meet the needs of industry in the face of the challenges posed by HLB. The complementary nature of the South African and French teams will enable exchanges of biological material, high-throughput sequencing data, bioinformatics expertise and technical skills and knowledge to produce new ‘Ca. Liberibacter’ genomic resources, particularly for the five CLaf subspecies identified in various rutaceae species.