Periodic Reporting for period 2 - PRE-HLB (PREVENTING HLB EPIDEMICS FOR ENSURING CITRUS SURVIVAL IN EUROPE)
Reporting period: 2020-12-01 to 2022-05-31
The EU citriculture is threatened by the emerging disease Huanglongbing (HLB, also known as Greening), considered the most devastating citrus disease due to its rapid dispersal, severity and fast progression of symptoms, huge losses in fruit production and quality, cost and difficulty of preventing new infections, lack of resistant citrus varieties and feasible treatments for infected trees, and absence of durable control mechanisms. HLB generates multimillion economic losses to the citrus industry.
HLB-research in the EU is mandatory because the vector Trioza erytreae is already spreading in Spain and Portugal. The overarching objective of Pre-HLB is to develop and implement a holistic contingency plan to protect the EU citrus sector from HLB disease drivers and to co-create new solutions to manage the disease through a multidisciplinary approach and in collaboration with experienced partners from America and Asia. The Pre-HLB work plan includes three specific objectives to monitor progress:
a) Pre-HLB preventive actions and development of mid-term tools will limit HLB potential introduction, reducing the potential economic impact in EU by 40%
b) The consortium will contribute to improve current HLB surveillance, contingency and control strategies.
c) Excellent European research centres and associates will join forces to fill research gaps in vector biology, host/vector/pathogen interactions, Aurantioideae genetics and genomics, as well as to develop new biotechnological tools, such as new effectors, metabolites, bio-stimulants and biopesticides.
HLB-research in the EU is mandatory because the vector Trioza erytreae is already spreading in Spain and Portugal. The overarching objective of Pre-HLB is to develop and implement a holistic contingency plan to protect the EU citrus sector from HLB disease drivers and to co-create new solutions to manage the disease through a multidisciplinary approach and in collaboration with experienced partners from America and Asia. The Pre-HLB work plan includes three specific objectives to monitor progress:
a) Pre-HLB preventive actions and development of mid-term tools will limit HLB potential introduction, reducing the potential economic impact in EU by 40%
b) The consortium will contribute to improve current HLB surveillance, contingency and control strategies.
c) Excellent European research centres and associates will join forces to fill research gaps in vector biology, host/vector/pathogen interactions, Aurantioideae genetics and genomics, as well as to develop new biotechnological tools, such as new effectors, metabolites, bio-stimulants and biopesticides.
Consortium has implemented short-term measures with the creation of the project website and the incorporation of a wide variety of content for all stakeholders interested in HLB. Publication of practice abstracts is disseminating this through the EIP-AGRI.
We have developed a new sampling methodology to assist and optimize monitoring and containment of the spread of T. erytreae. Studies on its life cycle and reproductive biology as well as host range are underway. T. erytreae flight behaviour and spread along the Iberian Peninsula are also being investigated.
Brazilian partners work on Candidatus Liberibacter asiaticus (CLas) and Diaphorina citri host ranges revealed sources of full resistance to CLas in Citrus relatives, including some Australian species sexually compatible with Citrus. Moreover, some of these genotypes showed resistance to D. citri. Some sample extracts were securely transferred to Europe to initiate the identification of secreted peptides by D. citri. Cirad-Reunion was able to rear D. citri and to set up a system for CLas transmission by this insect using excised citrus leaves.
Pre-HLB is quantifying the risks of entry, establishment and spread of T. erytreae/D. citri vectors and HLB-associated bacteria in the EU citrus‐growing areas. Areas most favorable for Candidatus Liberibacters (CLs)/psyllids illegal entry into the EU are being tracked based on host density and environmental suitability maps, linked with potential routes of infected plant material. Climatic models are being used to assess the risks of establishment.
Citrus Health Management Areas (CHMAs) are being set up over European citrus main areas, by collating periodic information about regional psyllid population and presence of new shoots in citrus trees. This will allow growers identifying places of psyllid occurrence and critical moments of spread. CHMAs are being established in Spain, Portugal and Sicily (Italy) from where data are available, although with different level of detail. Another challenge is the development of a kit for diagnosis of CLs with the aim of preventing an unintended spread of the disease.
Treatments disrupting host finding ability of T. erytreae (kaolin, reflective mulches) are being successful controlling psyllid landing and feeding activity. New antimicrobial and biopesticides developed within the project, and chimeric anti-HLB defensin proteins may provide novel biological products for effective control or either bacteria or psyllid vectors. Lab experiments performed in Brazil with fusion proteins produced in England are showing promising results in killing D. citri nymphs and adults. High throughout sequencing technologies of T. erytreae insects from different geographic areas has already allowed identification of an endosymbiont bacterium and viruses belonging to the Densovirus, Anphevirus and Mivirus genera that may allow development of new psyllid control strategies.
Pre-HLB is also focused on breeding Citrus for resistance to CLas. De novo sequencing of Australian citrus species identified as CLas-resistant is underway as well as transcriptomic analysis of them and of some Murraya species showing different responses to CLas. Almost 300 accessions of Citrinae are being subjected to genotyping by sequencing (GBS) to identify diagnostic SNPs (dSNPs) related to CLas resistance. Those Citrinae species resulting to be resistant to CLas are being tested directly as possible rootstocks or interstocks and/or used to produce somatic hybrids with citrus for the same purpose. New biotechnological tools to obtain HLB-resistant non-transgenic edited citrus are being set up.
We have developed a new sampling methodology to assist and optimize monitoring and containment of the spread of T. erytreae. Studies on its life cycle and reproductive biology as well as host range are underway. T. erytreae flight behaviour and spread along the Iberian Peninsula are also being investigated.
Brazilian partners work on Candidatus Liberibacter asiaticus (CLas) and Diaphorina citri host ranges revealed sources of full resistance to CLas in Citrus relatives, including some Australian species sexually compatible with Citrus. Moreover, some of these genotypes showed resistance to D. citri. Some sample extracts were securely transferred to Europe to initiate the identification of secreted peptides by D. citri. Cirad-Reunion was able to rear D. citri and to set up a system for CLas transmission by this insect using excised citrus leaves.
Pre-HLB is quantifying the risks of entry, establishment and spread of T. erytreae/D. citri vectors and HLB-associated bacteria in the EU citrus‐growing areas. Areas most favorable for Candidatus Liberibacters (CLs)/psyllids illegal entry into the EU are being tracked based on host density and environmental suitability maps, linked with potential routes of infected plant material. Climatic models are being used to assess the risks of establishment.
Citrus Health Management Areas (CHMAs) are being set up over European citrus main areas, by collating periodic information about regional psyllid population and presence of new shoots in citrus trees. This will allow growers identifying places of psyllid occurrence and critical moments of spread. CHMAs are being established in Spain, Portugal and Sicily (Italy) from where data are available, although with different level of detail. Another challenge is the development of a kit for diagnosis of CLs with the aim of preventing an unintended spread of the disease.
Treatments disrupting host finding ability of T. erytreae (kaolin, reflective mulches) are being successful controlling psyllid landing and feeding activity. New antimicrobial and biopesticides developed within the project, and chimeric anti-HLB defensin proteins may provide novel biological products for effective control or either bacteria or psyllid vectors. Lab experiments performed in Brazil with fusion proteins produced in England are showing promising results in killing D. citri nymphs and adults. High throughout sequencing technologies of T. erytreae insects from different geographic areas has already allowed identification of an endosymbiont bacterium and viruses belonging to the Densovirus, Anphevirus and Mivirus genera that may allow development of new psyllid control strategies.
Pre-HLB is also focused on breeding Citrus for resistance to CLas. De novo sequencing of Australian citrus species identified as CLas-resistant is underway as well as transcriptomic analysis of them and of some Murraya species showing different responses to CLas. Almost 300 accessions of Citrinae are being subjected to genotyping by sequencing (GBS) to identify diagnostic SNPs (dSNPs) related to CLas resistance. Those Citrinae species resulting to be resistant to CLas are being tested directly as possible rootstocks or interstocks and/or used to produce somatic hybrids with citrus for the same purpose. New biotechnological tools to obtain HLB-resistant non-transgenic edited citrus are being set up.
This is an updated summary list of Pre-HLB main expected results:
- Web platform about HLB with training materials for all the stakeholders
-Guidelines for IPM and sustainable farming
-Psyllid population and dynamics over Europe
-New sampling methods and bioecology of T. erytreae
-Identification of citrus relatives resistant to HLB-associated bacteria (CLs) and/or to D. citri
-Identification of effectors and elicitors of CLs and the insect vectors
-Identification of metabolites critically involved in citrus-psyllid- CLs interactions
-Relative transmission efficiency and Acquisition Access Period of CLs by D. citri and T. erytreae
-Algorithms for epidemiological modelling of HLB spread
-Simulation results to predict the invasion of vectors and HLB into and across EU
-New management tools and control strategies against HLB
-Knowledge about natural enemies against T. erytreae
-Peptides formulations effectiveness against CLas
-Use of novel biopesticide(s) to control the psyllid vectors
-New diagnosis Kit for CLs and improved sampling and detection protocol
-De novo sequence of Microcitrus and Eremocitrus assembled in pseudomolecule
-Identification of candidate genes for resistance against CLas
-Haplotypic and genotypic diversity of CLas resistance candidate genes.
-SNPs for further pangenomic selection of resistant genotypes
-Identification of the most promising citrus relative interstocks/rootstocks.
-Tetraploid somatic hybrids with potential use as resistance rootstocks
-Procedures for citrus non-transgenic CRISPR-edition.
-Identification of rationally designed and stable anti-CLas proteins.
-Catalogue of viruses infecting T. erytreae populations from different geographic origin.
-Characterization of endosymbionts and prophages/phages associated with T. erytreae.
- Web platform about HLB with training materials for all the stakeholders
-Guidelines for IPM and sustainable farming
-Psyllid population and dynamics over Europe
-New sampling methods and bioecology of T. erytreae
-Identification of citrus relatives resistant to HLB-associated bacteria (CLs) and/or to D. citri
-Identification of effectors and elicitors of CLs and the insect vectors
-Identification of metabolites critically involved in citrus-psyllid- CLs interactions
-Relative transmission efficiency and Acquisition Access Period of CLs by D. citri and T. erytreae
-Algorithms for epidemiological modelling of HLB spread
-Simulation results to predict the invasion of vectors and HLB into and across EU
-New management tools and control strategies against HLB
-Knowledge about natural enemies against T. erytreae
-Peptides formulations effectiveness against CLas
-Use of novel biopesticide(s) to control the psyllid vectors
-New diagnosis Kit for CLs and improved sampling and detection protocol
-De novo sequence of Microcitrus and Eremocitrus assembled in pseudomolecule
-Identification of candidate genes for resistance against CLas
-Haplotypic and genotypic diversity of CLas resistance candidate genes.
-SNPs for further pangenomic selection of resistant genotypes
-Identification of the most promising citrus relative interstocks/rootstocks.
-Tetraploid somatic hybrids with potential use as resistance rootstocks
-Procedures for citrus non-transgenic CRISPR-edition.
-Identification of rationally designed and stable anti-CLas proteins.
-Catalogue of viruses infecting T. erytreae populations from different geographic origin.
-Characterization of endosymbionts and prophages/phages associated with T. erytreae.