Developing second generation antimicrobial treatments for the control of citrus greening disease Annual report, January 1, 2017 – December 30, 2017.

Developing second generation antimicrobial treatments for the control of citrus greening disease Annual report, January 1, 2017 - December 30, 2017.

Report Date: 04/15/2018
Project: 16-009   Year: 2017
Category: Horticultural & Management
Author: Eric Triplett
Sponsor: Citrus Research and Development Foundation

Major accomplishments: 1. The frequency of streptomycin resistance in Liberibacter crescens was determined in the lab. One in 500 million cells are spontaneously resistant to streptomycin. 2. The mutations responsible for spontaneous streptomycin resistance in Liberibacter crescens were identified. They are all present in one of two codons in the rpsL gene, as is typically the case for Gram-negative bacteria. 3. A high-throughput method has been developed to rapidly assess the extent of streptomycin in the field. This was done by simply adapting our 16S rRNA sequencing method to another gene, the rpsL gene. We are now working diligently to develop the database needed to analyze the data. This approach will be used to assess the level of resistance in the CLas and non-target bacteria in the field. 4. No spontaneous mutants of L. cresens resistant to oxytetracycline were recovered despite several attempts with billions of cells. We now know that the frequency of spontaneous resistance to oxytetracycline is less than one cell in 10 billion. This is because no single mutation can provide resistance to oxytetracycline and that L. crescens and CLas lack any gene that could confer oxytetracycline resistance. Such genes are often transferred from one bacterium to another through a process called lateral gene transfer. However, the restricted habitats of CLas, the Asian citrus psyllid and citrus phloem, do not provide little opportunity to acquire tetracycline resistance genes from other bacteria. 5. The spontaneous streptomycin-resistant mutants of L. crescens are not resistant to oxytetracyline. 6. In addition to the discovery of the loci involved in spontaneous streptomycin resistance in L. crescens, whole genome sequencing of the streptomycin resistant mutants also showed that a streptomycin resistant mutant has increased methylation of an efflux protein often implicated in antibiotic resistance. Also, the methylation pattern of L. crescens changes with streptomycin treatment itself. We don’t yet know the implications of these results for the control of HLB. 7. Progress was made toward the culturing of the citrus greening pathogen, Ca. Liberibacter asiaticus (CLas). Using the closest cultured relative of CLas, L. crescens, we discovered Liberibacter prefers citrate as major carbon and energy source. The optimal level of citrate required to culture L. crescens was very similar to the amount of citrate found in citrus phloem and the haemolymph of the Asian citrus psyllid. This discovery suggests a simple, inexpensive, and unregulated means to control HLB by foliar phosphate fertilization. This is described in the discussion section of our recent paper (Cruz-Munoz M, Petrone JR, Cohn AR, Munoz-Beristain A, Killiny N, Drew JC, Triplett EW, Development of chemically defined media reveals citrate as preferred carbon source for Liberibacter growth. Frontiers in Microbiology, 2018; 9:668). In summary, foliar phosphate fertilization is expected to dramatically reduce citrate levels in phloem, thereby starving the pathogen. Plants load citrate in phloem in response to P deficiency. We are pursuing this idea now and seeking external support to test it in the field.

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