Periodic Reporting for period 3 - PRE-HLB (PREVENTING HLB EPIDEMICS FOR ENSURING CITRUS SURVIVAL IN EUROPE)
Okres sprawozdawczy: 2022-06-01 do 2023-11-30
The EU citriculture is threatened by 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 citriculture worldwide.
HLB-research in the EU is mandatory because it is considered a priority pest, the vector Trioza erytreae is already spreading in Spain and Portugal and the other vector Diaphorina citri is spreading in Israel and Cyprus. 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 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 tools which would limit HLB potential introduction.
b) Pre-HLB 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.
HLB-research in the EU is mandatory because it is considered a priority pest, the vector Trioza erytreae is already spreading in Spain and Portugal and the other vector Diaphorina citri is spreading in Israel and Cyprus. 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 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 tools which would limit HLB potential introduction.
b) Pre-HLB 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.
The consortium has implemented short-term measures with the implementation of the project website, a blog, and a mobile phone app with a wide variety of content for all stakeholders. Practice abstracts are disseminating this also through EIP-AGRI. A new contingency plan including important outcomes from Pre-HLB have been proposed to the EU.
We have developed a new sampling methodology to optimise monitoring of the spread of T. erytreae. Studies on its life cycle, reproductive biology and host range as well as flight behaviour, spatial pattern distribution and spread along the Iberian Peninsula have been set-up. Spread of D. citri has been followed in Israel and management methods have been proposed to local authorities.
Regarding the medium-term actions, a stochastic, landscape scale mathematical model has been developed to simulate HLB spread in the EU, and using the developed epidemiological model, Citrus Health Management Areas (CHMAs) were proposed for Spain so far. An efficient, user-friendly, field applicable detection protocol with new confident sets of primers has been demonstrated to detect 100% of Candidatus Liberibacters (CLs) in naturally-infected samples. Detailed molecular characterisation of T. erytreae endosymbionts has allowed to trace back in Stellenbosch (South Africa) the origin of T. erytreae populations present in the EU. Another important achievement has been the finding that T. erytreae acquires, multiplies, and transmits Candidatus Liberibacter asiaticus (CLas), the most aggressive bacterial species, equally as well as D. citri.
Treatments disrupting host finding ability of T. erytreae (kaolin, UV-absorbing nets) as well as biological control with natural enemies and organic-derived insecticides are being successful in controlling this psyllid. Other pest management strategies, such as entomopathogenic fungi, antimicrobial peptides or fusion protein-based bio-pesticides have also generated very promising results, opening the way to new Integrated Pest Management (IPM) strategies in the coming future. For example, specific peptides contained in artificial diets or applied by spraying under greenhouse conditions induced high levels of mortality in D. citri landing and feeding activity in Brazil.
With the aim of developing long-term strategies to control HLB, different approaches have been set-up. Studies on psyllid-citrus-bacterial interactions have revealed sources of full-resistance to CLas or to D. citri. Oceanian citrus species identified as CLas-resistant have been subjected to de novo genome sequencing, gene annotation and search for resistance/susceptibility gene candidates. Transcriptomic and metabolomics experiments aimed at analysing the interaction of CLas with resistant vs. susceptible genotypes have been performed to shed light on HLB-resistance/susceptibility mechanisms. Pre-HLB is also focused on breeding citrus for resistance to CLas. Challenge-inoculation with CLas and parallel GBS (Genotyping By Sequencing) and QTL (Quantitative Trait Locus) analysis of the segregating populations is allowing the identification of genes of interest in relation to resistance/susceptibility to CLas. Species 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 set up. Moreover, the discovery and detailed characterisation of T. erytreae viruses and endosymbionts might be potentially useful to develop new strategies to attempt controlling the spread of the disease vector in the future. New antimicrobials as defensin proteins may provide novel biological anti-HLB products.
We have developed a new sampling methodology to optimise monitoring of the spread of T. erytreae. Studies on its life cycle, reproductive biology and host range as well as flight behaviour, spatial pattern distribution and spread along the Iberian Peninsula have been set-up. Spread of D. citri has been followed in Israel and management methods have been proposed to local authorities.
Regarding the medium-term actions, a stochastic, landscape scale mathematical model has been developed to simulate HLB spread in the EU, and using the developed epidemiological model, Citrus Health Management Areas (CHMAs) were proposed for Spain so far. An efficient, user-friendly, field applicable detection protocol with new confident sets of primers has been demonstrated to detect 100% of Candidatus Liberibacters (CLs) in naturally-infected samples. Detailed molecular characterisation of T. erytreae endosymbionts has allowed to trace back in Stellenbosch (South Africa) the origin of T. erytreae populations present in the EU. Another important achievement has been the finding that T. erytreae acquires, multiplies, and transmits Candidatus Liberibacter asiaticus (CLas), the most aggressive bacterial species, equally as well as D. citri.
Treatments disrupting host finding ability of T. erytreae (kaolin, UV-absorbing nets) as well as biological control with natural enemies and organic-derived insecticides are being successful in controlling this psyllid. Other pest management strategies, such as entomopathogenic fungi, antimicrobial peptides or fusion protein-based bio-pesticides have also generated very promising results, opening the way to new Integrated Pest Management (IPM) strategies in the coming future. For example, specific peptides contained in artificial diets or applied by spraying under greenhouse conditions induced high levels of mortality in D. citri landing and feeding activity in Brazil.
With the aim of developing long-term strategies to control HLB, different approaches have been set-up. Studies on psyllid-citrus-bacterial interactions have revealed sources of full-resistance to CLas or to D. citri. Oceanian citrus species identified as CLas-resistant have been subjected to de novo genome sequencing, gene annotation and search for resistance/susceptibility gene candidates. Transcriptomic and metabolomics experiments aimed at analysing the interaction of CLas with resistant vs. susceptible genotypes have been performed to shed light on HLB-resistance/susceptibility mechanisms. Pre-HLB is also focused on breeding citrus for resistance to CLas. Challenge-inoculation with CLas and parallel GBS (Genotyping By Sequencing) and QTL (Quantitative Trait Locus) analysis of the segregating populations is allowing the identification of genes of interest in relation to resistance/susceptibility to CLas. Species 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 set up. Moreover, the discovery and detailed characterisation of T. erytreae viruses and endosymbionts might be potentially useful to develop new strategies to attempt controlling the spread of the disease vector in the future. New antimicrobials as defensin proteins may provide novel biological anti-HLB products.
This is an updated summary list of Pre-HLB main expected results:
- Web platforms about HLB with training materials for all the stakeholders
- Guidelines for new contingency plans in EU
- Psyllid population dynamics over EU
- 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, elicitors and metabolites in citrus-psyllid-CLs interactions
- Relative transmission efficiency and Acquisition Access Period of CLs by D. citri
- Algorithms for epidemiological modelling of HLB spread
- Simulation results to predict the invasion of vectors and HLB into EU
- New management tools (CHMAs) 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
- De novo sequence of Microcitrus and Eremocitrus assembled in pseudomolecule
- Breeding and transcriptomics for identification of candidate genes for resistance to CLas
- 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 platforms about HLB with training materials for all the stakeholders
- Guidelines for new contingency plans in EU
- Psyllid population dynamics over EU
- 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, elicitors and metabolites in citrus-psyllid-CLs interactions
- Relative transmission efficiency and Acquisition Access Period of CLs by D. citri
- Algorithms for epidemiological modelling of HLB spread
- Simulation results to predict the invasion of vectors and HLB into EU
- New management tools (CHMAs) 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
- De novo sequence of Microcitrus and Eremocitrus assembled in pseudomolecule
- Breeding and transcriptomics for identification of candidate genes for resistance to CLas
- 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