Detection of Oxytetracycline in Citrus Phloem and Xylem Saps Using Europium-Based Method.

Faraj Hijaz,Nabil Killiny,Yasser Nehela,Ozgur Batuman

doi:10.3390/antibiotics10091036

Faraj Hijaz, Nabil Killiny + Show 2 more

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https://doi.org/10.3390/antibiotics10091036

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Abstract

Oxytetracycline (OTC) has been used for the control of several plant diseases and was recently approved for the control of Huanglongbing, the citrus greening disease. Huanglongbing is caused by the phloem limited ‘Candidatus Liberibacter asiaticus’. Determination of OTC in the xylem and phloem of citrus plants is of great interest as they are the main routes of translocation in citrus. In addition, the determination of the level of OTC in the phloem sap is necessary for the control of the ‘Ca. L. asiaticus’ pathogen, which resides in the phloem. Herein, we demonstrated that the level of OTC in the citrus phloem and xylem saps obtained using the centrifugation method can be successfully measured using the europium (Eu) method directly or with cleanup by solid-phase extraction (SPE). Recovery of OTC from spiked sap samples purified by solid-phase extraction (SPE) was higher than 90%, while recovery from saps without SPE cleanup were nearly 100%. The ‘Ca. L. asiaticus’-infected leaf and phloem sap samples showed higher inhibition of the fluorescence intensity of the OTC standard compared to non-infected control leaf and phloem samples. In agreement with this finding, the levels of phenols and flavonoids in ‘Ca. L. asiaticus’-infected leaves were higher than those controls and were shown to interfere with the Eu method. Therefore, the SPE cleanup step only improved OTC recovery from leaf samples containing the interfering compounds. The Eu method was then used to determine OTC levels in the phloem and xylem sap of OTC-treated plants, and the results were similar whether measured directly or after SPE. Visualization under ultraviolet light (400 nm) showed the presence of OTC in citrus xylem and phloem saps with and without the use of SPE.

Highlights

Huanglongbing, known as citrus greening disease, is caused by a fastidious, phloem-limited, plant pathogenic bacterium ‘Candidatus Liberibacter’ spp
Our results showed that the OTC in the phloem sap and xylem sap of citrus plants can be directly visualized under ultraviolet light (400 nm) by complexing it with Eu(III) and citrate (Figure 6C)
Xylem movement is the most efficient way for the translocation of insecticides as it results in a consistent distribution throughout the canopy, whereas the phloem movement is best for herbicides as it results in the accumulation of herbicides in the new shoots [15]

Summary

Introduction

Huanglongbing, known as citrus greening disease, is caused by a fastidious, phloem-limited, plant pathogenic bacterium ‘Candidatus Liberibacter’ spp. Three Liberibacter species are associated with HLB; ‘Ca. L. africanus’ in Africa, ‘Ca. L. americanus’ in Brazil, and ‘Ca. L. asiaticus’ in the Americas [1]. The ‘Ca. L. asiaticus’ is considered the most dangerous species as it is the most dominant, causing massive destruction to the citrus industry in different countries [1]. The ‘Ca. L. africanus’ is transmitted by the African psyllid citrus, Trioza erytreae Del Guercio (Hemiptera: Triozidae), whereas ‘Ca. L. americanus’ and ‘Ca. L. asiaticus’ are transmitted by the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae). The ‘Ca. L. asiaticus’-infected trees show a wide range of symptoms including blotchy and mottled leaves [1], lopsided, small-sized, asymmetric, bitter, and sour fruits. The roots of ‘Ca. L. asiaticus’-infected trees are poorly developed with very few fibrous roots, and infected trees look stunted and smaller than healthy trees. Infected trees may develop twig and limb dieback, which often result in the tree’s death

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