2019-06_16-009C – Developing a second generation antimicrobial treatments for the control of citrus greening disease

Developing a second generation antimicrobial treatments for the control of citrus greening disease

Report Date: 01/23/2020
Project: 16-009C   Year: 2019
Category: CLas Bacteria
Author: Eric Triplett
Sponsor: Citrus Research and Development Foundation, Inc.

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. Gene mutation identified for streptomycin resistance – the rpsL gene.
3. Developed rapid method to assess frequency of streptomycin in the field for CLas and non-target bacteria.
4. No spontaneous resistance observed for oxytetracycline suggesting that CLas resistance to oxytetracyline will take a very long time to occur.
5. Streptomycin resistance strains of L. crescens are not resistant to oxytetracycline.
6. Resistance to currently available antimicrobials should not arise quickly in the field.
7. Developed a defined culture medium for L. crescens that alow for more rapid and accurate antimicrobial testing.
8. Discovered that HLB symptoms may be the result of ammonia production by the pathogen.
9. Discovery of preferred carbon sources of L. crescens led to discovery of citrate as the preferred carbon source.
10 Optimal citrate concentrations for the growth of L. crescens are very close to the levels of citrate in phloem and the psyllid hemolymph.
11. Citrate use by Liberibacter leads to discovery that foliar phosphate fertilization may reduce CLas infection.
12. A new antimicrobial assay for L. crescens was developed for rapid antimicrobial discovery. New assay was shared with representatives from Bayer.
13. Sulbactim, erythromycin, and thiamphenicol considered primary candidates for CLas inhibition based on their probable phloem mobility and their inhibition of L. crescens at low concentrations.
14. Glyphosate was found to inhibit L. crescens at levels below that required to kill plants.
15. Citrus sensitivity to glyphosate was tested in the field. Although citrus is less sensitive to glyphosate than most plants, it is still sensitive enough to prevent the use of glyphosate for HLB control.
16. With Prof. Mou, an altered citrus ESPS synthase gene was made and transformed into Arabidopsis and citrus. The altered gene conferred glyphosate resistance in both plants.
17. The number of glyphosate resistant citrus plants are not being propogated to get enough plant to test the effect of glyphosate on HLB infection.
18. A cisgenic line of glyphosate resistance is now being generated by the CREC citrus transformation lab.
19. We are confident that glyphosate applications to glyphosate resistance citrus, will prevent HLB.
Cruz-Munoz, M., Petrone, J. R., Cohn, A. R., Munoz-Beristain, A., Killiny, N., Drew, J. C., & Triplett, E. W. 2018. Development of chemically defined media reveals citrate as preferred carbon source for Liberibacter growth. Frontiers in Microbiology, 9, 668.
Killiny, N. 2017. Metabolite signature of the phloem sap of fourteen citrus varieties with different degrees of tolerance to Candidatus Liberibacter asiaticus. Physiol. Mol. Plant Pathol. 97, 20-29.
Cruz-Munoz, M., Munoz-Beristain, A., Petrone, J.R., Robinson, M.A., & Triplett, E.W. 2019. Growth parameters of Liberibacter crescens suggest ammonium and phosphate as essential molecules in the Liberibacter-plant host interface. BMC Microbiology 19:222.

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