Tools and methods for the early detection, detoxification, and prevention of Huanglongbing (HLB) infection of Citrus and their integration into commercial production practices

Pathology; Genetics and Genomics; Plant Sciences

The project aims to develop plant remediation therapies for Citrus huanglongbing (HLB) disease using novel bio-engineered enzymes to suppress Candidatus Liberibacter asiaticus infection.

Citrushuanglongbing (HLB), previously called citrus greening disease, is one of themost destructive diseases of citrus worldwide. Originally thought to be caused by a virus, it is now known to be causedby unculturable phloem-limited bacteria, CandidatusLiberibacter asiaticus (CLas).  Thereare three forms of greening that have been described.  The African form produces symptoms only undercool conditions and is transmitted by the African citrus psyllid Triozaerytreae, while the Asian form prefers warmer conditions and is transmitted bythe Asian citrus psyllid Diaphorina citri. Recently a third American form transmitted by the Asian citrus psyllidwas discovered in Brazil.  This Americanform of the disease apparently originated in China.  In North America, the psyllid vector,Diaphorina citri, of HLB is found in Florida, Louisiana, Georgia, SouthCarolina, Texas and Hawaii, and recently arrived in Southern California fromMexico. There is at present no known resistance in citrus cultivars for thedisease and as a result control measures are limited to moving production underscreenhouses, costly pesticide treatments for the vector with a needed 3-6treatments per year, and costly eradication programs to remove infected trees toprevent spread.

Candidatus Liberibacter asiaticus (CLas) caninfect most citrus cultivars, species and hybrids and even some citrusrelatives.  Leaves of newly infectedtrees develop a blotchy mottle appearance. On chronically infected trees, the leaves are small and exhibitasymmetrical blotchy mottling. Fruit from HLB-infected trees are small,lopsided, poorly colored, and contain aborted seeds. The juice from affectedfruit is low in soluble solids, high in acids and abnormally bitter.  The fruit retains its green color at thenavel end when mature, which is the reason for the common name "citrusgreening disease."  This fruit is ofno value because of poor size and quality. 

Ourobjectives are as follows. We will develop plant remediation therapies based onthe deployment of novel bioengineered enzymes that can prevent or suppressCandidatus Liberibacter asiaticus (CLas) infection. Since surfacelipopolysaccharides (LPS) are key to bacterial cell wall integrity and act as adefensive barrier, their removal weakens the bacterial cell wall, reducinggrowth and causing cell death. We propose to engineer an enzyme capable ofkilling Clas using strategies previously used in the laboratory to kill thecyanobacterial pathogen M. aeruginosa. We will then develop topically appliedsprays/ coatings/ injections of stable bioengineered enzyme formulations suitablefor use to decontaminate Citrus scions, rootstocks and micropropagated materialto reduces occurrence and spread of disease. Stable enzyme formulations will bedeveloped using methods effective previously for long-term (days to weeks)release of active enzyme against other pathogens and liposomal nanoformulationsshown previously to permeate plant tissue when sprayed directly onto plantsurfaces.  Second approach will be to generatenovel Citrus cultivars with tolerance / resistance to all CLas pathotypes by genetictransformation of engineered enzymes into susceptible Citrus genotypes usingAgrobacterium-based transformation methods and high level constitutive and phloem-specificpromoters. We believe that in vivo expressed enzyme could potentially preventCLas infection or reduce the severity of HLB disease progression in maturetrees (i.e., curative). 


Desired outcomes

Theanticipated project deliverables are as follows: (i) Bioengineered microbes andenzymatic disinfectants and protocols for whole plant, tissue and fruitdecontamination as therapeutics for existing infected Citrus) (ii) Novel Citrusgermplasm and genotypes with broadly acting engineered resistance/tolerance toHLB disease (iii) Understanding the molecular mechanism behind disease kineticsand HLB resistance.