Article Cite This: J. Agric. Food Chem. 2019, 67, 6970−6977
pubs.acs.org/JAFC
Antimicrobial Zn-Based “TSOL” for Citrus Greening Management: Insights from Spectroscopy and Molecular Simulation Shih-Hsien Liu,†,∥ Takat B. Rawal,‡,∥ Mikhael Soliman,§ Briana Lee,§ Tyler Maxwell,§ Parthiban Rajasekaran,§ Hajeewaka C. Mendis,§ Nicole Labbe,́ † Swadeshmukul Santra,§,⊥ Laurene Tetard,§,# and Loukas Petridis*,‡,∥ †
Center for Renewable Carbon, The University of Tennessee, Knoxville, Tennessee 37996, United States Department of Biochemistry & Cellular and Molecular Biology, The University of Tennessee, Knoxville, Tennessee 37996, United States § NanoScience Technology Center, University of Central Florida, Orlando, Florida 32826, United States ∥ UT/ORNL Center for Molecular Biophysics, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States ⊥ Department of Chemistry, Department of Materials Science & Engineering and Burnett School of Biomedical Sciences, University of Central Florida, Orlando, Florida 32826, United States # Department of Physics, University of Central Florida, Orlando, Florida 32826, United States
Downloaded via BUFFALO STATE on July 27, 2019 at 03:32:46 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
‡
S Supporting Information *
ABSTRACT: Huanglongbing (HLB), also known as citrus greening, is a bacterial disease that poses a devastating threat to the citrus industry worldwide. To manage this disease efficiently, we developed and characterized a ternary aqueous solution (TSOL) that contains zinc nitrate, urea, and hydrogen peroxide. We report that TSOL exhibits better antimicrobial activity than commercial bactericides for growers. X-ray fluorescence analysis demonstrates that zinc is delivered to citrus leaves, where the bacteria reside. FTIR and Raman spectroscopy, molecular dynamics simulations, and density functional theory calculations elucidate the solution structure of TSOL and reveal a water-mediated interaction between Zn2+ and H2O2, which may facilitate the generation of highly reactive hydroxyl radicals contributing to superior antimicrobial activity of TSOL. Our results not only suggest TSOL as a potent antimicrobial agent to suppress bacterial growth in HLB-infected trees, but also provide a structure− property relationship that explains the superior performance of TSOL. KEYWORDS: Huanglongbing, Candidatus Liberibacter asiaticus, antimicrobial agent, spectroscopy, molecular simulation
■
INTRODUCTION Huanglongbing (HLB) is a devastating disease of citrus that represents a major threat to the global citrus industry. HLB causes undersized, lopsided, and greenish fruits that often drop prematurely.1 The most widespread pathogens of HLB are the Gram-negative bacteria Candidatus Liberibacter asiaticus (CLas) that propagate within the barely accessible phloem (i.e., inside the vascular system) of citrus plants, making HLB a “systemic” disease that is difficult to cure.2−6 Common treatments for managing citrus diseases such as canker use film-forming and surface-restricted Cu-based compounds7,8 or Cu/ZnO mixtures, and are thus nonsystemic.9 Also, the frequent use of Cu-based agrochemicals in fields has accelerated the development of bacterial resistance to Cu.10,11 It is thus imperative to develop a Cu alternative for disease management. One potential measure being evaluated to control HLB is to provide Zn to infected citrus trees.1 The rationale behind this strategy is that HLB-affected citrus leaves have significantly reduced Zn2+ concentration compared to healthy ones,12 Zn deficiencies have similar symptoms to HLB,1,12 and Zn2+ ions are known to inhibit bacterial growth.13,14 Although some studies have shown that applying Zn-based compounds does not lead to significant reduction on CLas in vivo,15,16 other © 2019 American Chemical Society
studies have shown that Zn-based formulations exhibit comparable or superior antimicrobial activity and significantly lower phytotoxicity than Cu-based treatments.17 Recent studies suggest that ZnO nanoparticle formulations release Zn2+ ions in plants13 and have better antimicrobial activity against Escherichia coli (E. coli) than Cu-based chemicals.18,19 However, regulations of nanoparticles may considerably delay the deployment of such nanobased treatment into a commercial product available to growers in the short term. We consider here a comprehensive HLB management solution that employs a ternary solution (TSOL) consisting of zinc nitrate, urea and hydrogen peroxide (H2O2) in an aqueous solution.20 TSOL has been envisioned as an antimicrobial and nutritional product, in which Zn2+ and H2O2 would prevent CLas proliferation while urea would act as a nitrogen fertilizer in foliar spray treatments. However, fundamental properties of TSOL, such as the antimicrobial efficacy and the potency of delivery of the treatment to the phloem, have not been reported yet. The mechanism of action Received: Revised: Accepted: Published: 6970
April 18, 2019 May 16, 2019 May 31, 2019 May 31, 2019 DOI: 10.1021/acs.jafc.9b02466 J. Agric. Food Chem. 2019, 67, 6970−6977
Article
Journal of Agricultural and Food Chemistry
0.75 M of HCl for 30 s and a final rinse in DI water were performed. The plant was then dried in an oven at 60 °C for 24 h. After drying, leaves, stems, and roots of the plant were manually separated into different groups. Each group was ground into fine powder using a standard spice and nut grinder for an equal duration to homogenize the size of the powder. XRF measurements were conducted using a Malvern Panalytical Epsilon 1 XRF Spectrometer. The Zn content in ppm (μg/mL) was obtained by building a calibration model in Epsilon 3 software as described by Norrish and Hutton,23 where cellulose was used as the matrix material. Statistical significance analysis on the Zn content increase in leaves between untreated control and 24 h-treated seedlings was conducted using multiple analysis of variance (ANOVA) and post hoc Tukey test, where a pvalue 1600 n/a
Zinkicide zinc oxide cuprous oxide Nordox 30/30 Kocide 3000 SecA inhibitor SA7
31 n/a n/a n/a n/a 256
X. alfalfae (ATCC 49120) 6.25 25 6.25 >1600 13−26 (Zn)/16-31 (Cu) 62.5 62−125 62−125 125−250 250−500 n/a
n/a: not available.
considered in this study, we suggest that the synergistic effect of H2O2 and Zn2+ enhances antimicrobial activity of TSOL. As discussed in the computational results below, the synergy between H2O2 and Zn2+ arises from their colocalization: H2O2 is found in the second solvation shell of Zn2+. Further, we compare TSOL with other Zn-based, Cu-based and aromatic antimicrobial compounds. In inhibiting E. coli growth, TSOL does better than aromatic compounds,48 but shows less antimicrobial efficacy than the Zn-based formulation Zinkicide.18,19 In inhibiting X. alfalfae growth, TSOL is superior to all Zn and Cu-based formulations (i.e., ZnOnCuSi,9 Zinkicide,18,19 zinc oxide and cuprous oxide,9 and commercial bactericides Nordox 30/30 and Kocide 300017). The detailed antimicrobial mechanism of TSOL and Zinkicide warrants further investigation, which is ongoing. Zn Delivery in Citrus Leaves Following TSOL Treatment. An elemental analysis of the citrus leaves, stems, and roots was conducted to determine the Zn content in the plant after TSOL treatment (Figure 2). Upon spraying of TSOL, Zn content significantly increases in leaves (p-value
