## 4 Discussion  
Citrus canker may seem, at first glance, a \"simple\" disease. Unable to survive outside of plants, it is not clear how bacterial population and disease rates rapidly increased after many years of suppression, nor is it clear how these inocula could have survived in the field enduring harsh environmental conditions. Based on the results presented in this study, we suggest that persistence in _X. citri_ might be an essential biological step that has been preventing conventional management from successfully eradicating canker disease. In the present work, we studied the persister phenotype in _X. citri_, one of the most important phytopathogenic bacteria for the citrus industry. We used standardized methodology, largely used for _E. coli_ persistence studies [49; 51], to determining specific triggers that can: (1) induce persistence (chemicals, high temperature), (2) kill persisters (copper, mitomycin C, cisplatin), (3) induce resuscitation/waking of persister cells (proline), and (4) prevent persisters from waking (isoleucine). We found that copper, the main chemical used for plant disease control in the field [56] can both induce and kill persister cells in _X. citri_ depending on the conditions used. The  
Figure 5: Effect of each group of amino acids on the regrowth of _X. citri_ persister cells (**A**) amino acids group #1 (Ala, Arg, Cys, Phe, Ser); group #2 (Gly, His, Thr, Val, Tyr); group #3 (Asn, Ile, Lys, Pro, Trp) and group #4 (Asp, Glu, Gln, Leu, Met). (**B**) Same experiment as shown in A, but with M9 minimal medium containing individual amino acids of the fast-growing group #3 and group #4. Photographs show colonies observed with proline (group #3), glutamine (group #4) and aspartic acid (group #4). (**C**) To observe the inhibitory effect of isoleucine on _X. citri_ persisters, a suspension of _X. citri_ persister cells (same experiment as in A) were plated on M9-agar plates supplemented with only isoleucine; isoleucine + proline or only proline. recommended concentrations of metallic copper to control citrus canker in the field is 0.5-0.7 g/L [57], therefore copper is used in the field at higher concentrations than we used in the present work. However, it is virtually impossible to precisely determine the copper concentration _X. citri_ cells will be exposed to in the field. After being deposited on plant surfaces, copper products may be washed away from the leaves by rain and other meteorological events and may be deposited in water bodies and soils where other toxic effects can be observed [58]. In addition, copper can easily bind to organic compounds, changing its availability to bacterial cells on the leaves [59; 60; 61]. Therefore, residual amounts of copper could lead to dormancy, inducing persistence cycles in _X. citri_ and requiring even higher amounts of chemicals to eradicate these still viable cells from the field. Contrary to the widespread notion that persisters cannot be killed [62], we demonstrate that other chemicals besides copper can also eradicate _X. citri_ persister cells. As already demonstrated for _E. coli_ persisters, we found that molecules with a metabolism-independent mode of action such as cisplatin [48] and mitomycin C [49] can be used to control _X. citri_ persister. However, due to their cost and toxicity, these compounds are not practical for large-scale use to control phytopathogens in the field. The potential physical stresses that can induce persistence in bacteria are frequently overlooked. Since citrus orchards are grown under environments of high temperature, and _X. citri_ cannot grow and complete its cell cycle above 35-37 \({}^{\circ}\)C [46], heat stress is one of the main challenges _X. citri_ cells must overcome. This finding points to an interesting scenario, where daily heat-cold cycles may be regulating cell physiology and influencing dynamic disease processes. Apparently, _X. citri_ cells may enter into persistence during the warmest periods of the day, when both stomata closure and plant defenses would impede successful infection [63].