Brazil is the biggest producer of sweet oranges and the main exporter of concentrated orange juice in the world. Among the diseases that affect citriculture, Asiatic citrus canker, caused by the bacterial pathogen Xanthomonas citri, represents one of the most significant threats. The current Brazilian legislation regulating the control of citrus canker no longer requires the eradication of affected trees in states where the incidence of the disease is high. Instead, control involves disease control measures, including periodic preventative spraying of copper compounds. The long‐term use of copper for plant disease control has raised concerns about environmental accumulation and toxicity, as well as the selective pressure it exerts leading to the emergence of copper‐resistant X. citri strains. Here, we evaluated hexyl gallate (G6) as an alternative to copper compounds for citrus plant protection. G6 was able to protect citrus nursery trees against X. citri infection. Thirty days after inoculation, the trees treated with G6 developed 0.5 lesions/cm2 leaf area compared with the 2.84 lesions/cm2 observed in the untreated control trees. Also, G6 did not interfere with germination and root development of tomato, lettuce, and arugula, which is consistent with our previous data showing that G6 is safe for tissue culture cell lines. Membrane permeability tests showed that the primary target of G6 is the bacterial outer membrane. Finally, we could not isolate spontaneous X. citri mutants resistant to G6 nor induce resistance to G6 after long‐term exposures to increasing concentrations of the compound, which suggests that G6 may have multiple cellular targets. This study demonstrated that G6 is a promising candidate for the development and use in citrus canker management.
Aims: The aim of the study is to evaluate hexanoic acid (HA) as an alternative to manage citrus canker. Methods and Results: The minimal growth inhibitory concentration of HA against Xanthomonas citri subsp. citri was determined at 2Á15 mmol l −1 using a respiratory activity assay. Growth curves at different pH values showed that growth inhibition was not due to media acidification induced by HA. The germination rate and root elongation of Lactuca sativa seeds exposed to different concentrations of HA (varying from 0Á86 to 5Á16 mmol l −1 ) were assessed to screen for phytotoxicity. The acid exhibited low phytotoxicity for L. sativa at 1Á29 and 2Á58 mmol l −1 . To evaluate the ability of HA to protect citrus against X. citri infection, leaves of Citrus sinensis were sprayed with the acid and subsequently challenged with X. citri. HA at 3Á44 mmol l −1 was able to protect citrus against infection, showing a reduction of three orders of magnitude in the number of citrus canker lesions per cm 2 when compared to the untreated negative control. Conclusion: HA is a potential alternative to copper for citrus canker management. Significance and Impact of the Study: HA inhibits X. citri growth, exhibits low phytotoxicity and is an alternative to copper for the protection of citrus plants against bacterial infection.
Xanthomonas citri subsp. citri (X. citri) is an important phytopathogen and causes Asiatic Citrus Canker (ACC). To control ACC, copper sprays are commonly used. As copper is an environmentally damaging heavy metal, new antimicrobials are needed to combat citrus canker. Here, we explored the antimicrobial activity of chalcones, specifically the methoxychalcone BC1 and the hydroxychalcone T9A, against X. citri and the model organism Bacillus subtilis. BC1 and T9A prevented growth of X. citri and B. subtilis in concentrations varying from 20 µg/mL to 40 µg/mL. BC1 and T9A decreased incorporation of radiolabeled precursors of DNA, RNA, protein, and peptidoglycan in X. citri and B. subtilis. Both compounds mildly affected respiratory activity in X. citri, but T9A strongly decreased respiratory activity in B. subtilis. In line with that finding, intracellular ATP decreased strongly in B. subtilis upon T9A treatment, whereas BC1 increased intracellular ATP. In X. citri, both compounds resulted in a decrease in intracellular ATP. Cell division seems not to be affected in X. citri, and, although in B. subtilis the formation of FtsZ-rings is affected, a FtsZ GTPase activity assay suggests that this is an indirect effect. The chalcones studied here represent a sustainable alternative to copper for the control of ACC, and further studies are ongoing to elucidate their precise modes of action.
BACKGROUND The largest and most profitable market for citrus is the production of fresh fruit. Xanthomonas citri subsp. citri is a Gram‐negative plant pathogen and the etiological agent of citrus canker, one of the major threats to citrus production worldwide. In the early stages of infection, X. citri can attach to plant surfaces by means of biofilms. Biofilm is considered an essential virulence factor, which helps tissue colonization in plants. Thus, sanitization of citrus fruit is mandatory in packinghouses before any logistic operation as packing and shipment to the market. The aim of this study was to evaluate electrolysed water (EW) as a sanitizer for the disinfection of citrus fruit in packinghouses. RESULTS Using a protocol to monitor cell respiration we show that EW, obtained after 8 and 9 min of electrolysis, sufficed to kill X. citri when applied at a concentration of 500 μL mL−1. Furthermore, microscopy analysis, combined with time‐response growth curves, confirmed that EW affects the bacterial cytoplasmatic membrane and it leads to cell death in the first few minutes of contact. Pathogenicity tests using limes to simulate packinghouse treatment showed that EW, produced with 9 min of electrolysis, was a very effective sanitizer capable of eliminating X. citri from contaminated fruit. CONCLUSION It was possible to conclude that EW is significantly effective as sodium hypochlorite (NaClO) at 200 ppm. Therefore, EW could be an alternative for citrus sanitization in packinghouses. © 2020 Society of Chemical Industry
Citrus canker is a quarentenary disease caused by Xanthomonas citri subsp. citri (X. citri). Thus, sanitization of fresh fruit is a necessary measure before any commercial activity. Therefore, we evaluated the clove essential oil (CEO), as an alternative sanitizer for the disinfection of citrus fruit in packinghouses. Tests in vitro and in vivo were carried out to determine the cell inhibitory concentration and to verify the efficacy of the oil for the disinfection of citrus fruits. In in vitro tests, CEO was able to inhibit X. citri when used at 0.75% (v/v). In experiments that simulate the sanitization process used in packinghouses, 5% CEO was as effective as the recommended sanitization product based on sodium hypochlorite. GC‐MS results showed a high presence of eugenol derivatives as the major compounds of CEO. All results proved that CEO is a potential sanitizer that could be used as an alternative to sodium hypochlorite. Novelty impact statement Experimental evidence shows that the clove essential oil (CEO) has the same sanitization efficacy as sodium hypochlorite, which makes of CEO an alternative sanitizer for the decontamination of citrus fruits to be exported to the European Union. CEO is a safer and more sustainable sanitizer for the postharvest disinfection of citrus fruit.
Plant Essential Oils and their constituents are well-known for their properties as antimicrobial agents and are labeled as Generally Recognized as Safe (GRAS), prompting studies around their usage in the control of food-borne microorganisms and phytopathogens. In this study, we evaluated Oregano Essential Oil (OEO), Thymol (THY) and Carvacrol (CAR) for the control of Xanthomonas citri subsp. citri (X. citri). In vitro antibacterial assays revealed that CAR and THY inhibit X. citri growth at the concentrations of 100 µg.mL-1 and 114 µg.mL-1, lower than OEO (136 µg.mL-1). Bactericidal effects were observed at 400 µg.mL-1 for OEO and 200 ug.mL-1 for CAR and THY. Investigating potential cellular targets for the compounds showed that after 30 minutes of exposure up to 84% of the cells had their membranes disrupted, implicating the membrane as the primary target. Phytotoxicity evaluations using Lactuca sativa and Solanum lycopersicum seeds showed an acute toxic effect in all treatments above 200 µg.mL-1, except for OEO and THY in S. lycopersicum at lower concentrations. Regarding their protective effect on citrus leaves, CAR showed no effect when compared to the untreated control (0.39 and 0.50 lesions per cm2, respectively). OEO and THY were able to reduce significantly citrus canker symptoms (0.18 and 0.11 lesions per cm2, respectively). In addition, no toxic effects were observed on citrus leaves in all treatments. THY inhibits X. citri growth and the development of citrus canker lesions. These results show that THY as a viable alternative to be used in citrus canker management.
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