Preliminary assessment of bacterial antibiotic resistance and candidatus liberibacter asiaticus titer in three Florida commercial citrus groves

Authors: DE GRACIA COQUEREL, MARIE - University Of Florida; WEGERIF, JIELI - University Of Florida; MCAULEY, ANDREW - University Of Florida; READ, QUENTIN - US Department Of Agriculture (USDA); CHOWDHURY, NABIL - University Of Florida; JEONG, KWANG CHEOL - University Of Florida; MORRIS, J. GLENN - University Of Florida; MARTINS, SAMUEL - University Of Florida; GOSS, ERICA - University Of Florida; Ascunce, Marina

Submitted to: Crop Protection
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/15/2023
Publication Date: 7/17/2023
Citation: De Gracia Coquerel, M., Wegerif, J., Mcauley, A., Read, Q.D., Chowdhury, N., Jeong, K., Morris, J., Martins, S.J., Goss, E., Ascunce, M.S. 2023. Preliminary assessment of bacterial antibiotic resistance and candidatus liberibacter asiaticus titer in three Florida commercial citrus groves. Crop Protection. 172. https://doi.org/10.1016/j.cropro.2023.106350.
DOI: https://doi.org/10.1016/j.cropro.2023.106350

Interpretive Summary: Antimicrobials are used in agriculture to treat microbial diseases of plants. However, nowadays, there is a growing concern about the effect of their use on the emergence and spread of antimicrobial resistance. In addition, there is an increase knowledge of the beneficial role of a healthy microbiome among organisms including plants. Thus, to assess the impact of antimicrobial, studies need to evaluate their effects on both the pathogen to be treated as well as the whole microbial community. In this study, we focused in analyzing the use of antibiotics to treat Huanglongbing (HLB) or citrus greening disease. In the last 18 years, the citrus industry has been devastated by this disease caused by Candidatus Liberibacter asiaticus (CLas), an unculturable bacterium transmitted by the insect citrus psyllid upon feeding on citrus leaves. One of the methods to control this disease is the spray of two antibiotics: streptomycin (STR) and oxytetracycline (OTC). In this work, we compared the amount of the pathogen and total bacteria colonies among groves that have been sprayed with the antibiotics and another that was no sprayed. We found that there was no difference in the amount of pathogen among any of the groves independently of the antibiotic management. Our results agree with previous studies that have shown that antibiotic spray might not be effective to control HLB due to the inability of the used antibiotics to enter the tree vascular system. More studies are needed to develop alternative ways to control bacterial diseases in trees.

Technical Abstract: Over the last 16 years, the U.S. citrus industry has been devastated by citrus greening or Huanglongbing (HLB) disease. The causal agent, Candidatus Liberibacter asiaticus (CLas), is a bacterium transmitted by the invasive Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae), and by grafting. As a last resort to control citrus greening, growers were approved to spray antibiotics: streptomycin (STR) and oxytetracycline (OTC) in commercial citrus groves in Florida. This study analyzed the impact of antibiotic sprays on citrus trees by quantifying CLas titer and bacterial colony count from leaf, root+rhizosphere, and soil samples collected in three citrus groves. Two used antibiotic spray treatment (two conventional groves) and one used no antibiotic treatment (one organic grove). Samples were plated onto TSA medium containing either, STR (1 mg/mL), OTC (1 mg/mL) or no antibiotic (control) and colony-forming units (CFU) per gram of sample were estimated. For leaf samples in addition to the plating, DNA was extracted from leaves and used to quantify CLas titer. Among leaf samples, there were no significant differences in CFU or CLas titer based on grove. For soil samples, we found a larger number of resistant CFU in the organic grove. Roots+rhizosphere samples from all groves (organic and conventional) showed high prevalence of both streptomycin and oxytetracycline-resistant bacteria indicating that plant selective processes during root colonization might be favoring antibiotic resistant acquisition. Despite antibiotic sprays, there were no significant differences in CLas titer among the three groves suggesting that antibiotic spray might not be effective to control CLas, in agreement with other studies . This research further highlights the need for continued monitoring of antibiotic efficacy to control plant pathogens in commercial settings.