Changes in plant nutrient conditions have affected the disease severities of various crops such as tomato plants (Dordas, 2008; Tripathi et al., 2022). Foliar application of potassium nitrate (KNO₃) reduced lesion size on Alternaria solani-inoculated tomato leaves (Blachinski et al., 1996). However, increasing nitrate and ammonium nitrogen nutrition led to increased disease susceptibility of tomato plants to bacterial speck and bacterial wilt caused by Pseudomonas syringae pv. tomato and R. solanacearum, respectively (Ding et al., 2021). Interestingly, ammonium-nitrogen increased tomato crown and root rot by Fusarium oxysporum f. sp. radicis-lycopersici infection, whilst nitrate-nitrogen ameliorated the disease severity (Duffy and Dé fago, 1999). CaCl₂ supplement in nutrient solution decreased disease severity of bacterial canker by Clavibacter michiganense subsp. michiganense and bacterial wilt by R. solanacearum in tomato plants (Berry et al., 1988; Yamazaki and Hoshina, 1995). Enhanced plant immunity, such as elevated H₂ O₂ levels and increased peroxidase and polyphenol oxidase activities, was involved in the CaCl₂-mediated suppression of tomato bacterial wilt (Jiang et al., 2013). Micronutrients such as boron, manganese and zinc could also altered disease development of several tomato plants. Boron levels in the nutrient solution was negatively correlated with tomato bacterial wilt severity (Jiang et al., 2016). Increased MnSO₄ concentration in the nutrient solution reduced tomato black leaf mold caused by Pseudocercospora fuligena (Heine et al., 2011). Powdery mildew of tomato plants by a natural inoculation was suppressed by foliar spraying manganese (Mn-EDTA) and zinc (Zn-EDTA), each alone or in combination (Bakeer et al., 2012). Early blight in tomato plants inoculated with Alternaria solani was ameliorated by ZnSO₄ supplement (Awan et al., 2019). Interestingly, zinc (Zn-EDTA)-amended hydroponic nutrient solution alone could not reduce tomato Fusarium crown and root rot by F. oxysporum f. sp. radicis-lycopersici, but the zinc improved biocontrol activity by Pseudomonas fluorescens (Duffy and Dé fago, 1997). Micronutrient iron also has could alter disease severity in tomato plants. Foliar spraying Fe-EDTA reduced powdery mildew of tomato plants (Bakeer et al., 2012). Limited iron supplement in the hydroponic cultures accelerated wilting of a resistant tomato cultivar against Verticillium dahlia infection, indicating the critical role of iron in plant immunity (Macur et al., 1991). However, information on the micronutrients including iron for bacterial disease management was hardly discovered in tomato plants.

In this study, the effect of exogenous iron (FeCl₂) on in vitro growth of R. pseudosolanacearum and on bacterial wilt progression in detached tomato leaves were investigated.

R. pseudosolanacearum strain GMI1000 (formerly known as R. solanacearum GMI1000), causing tomato bacterial wilt, was isolated from tomato plants in French Guiana in 1978 (Deberdt et al., 2014; Tano et al., 2021). The bacterial strain was cultured in 4 ml of casamino acid-peptone-glucose (CPG) liquid medium in a 15-ml conical tube at 30°C overnight (Hong et al., 2016). The bacterial culture was centrifuged, and the bacterial pellet was resuspended with sterile water to adjust 0.15 at 600 nm using a spectrophotometer for a bacterial suspension (10⁸ cfu/ml) (Hong et al., 2013). Different concentrations (5, 10, 20, 50, 100, 200, and 500 µM) of FeCl₂ were added to 4 ml of the CPG broth in 15-ml conical tubes. The bacteria (10⁵ cfu/ml) were initially inoculated in the CPG broth supplemented with the FeCl₂, and cultured at 30°C with shaking until the OD value was reached 0.25±0.02 in the mock-treated control culture. Relative bacterial growths by FeCl₂ treatments were expressed as percentage (%) compared to the mock-treated control culture. Independent experiments were performed four times, and each experiment contained four biological replicates. In vitro bacterial population was investigated after the liquid culture of R. pseudosolanacearum GMI1000 (Rp-GMI1000) in the absence or presence of FeCl₂ (Fig. 1A). Increasing concentrations of FeCl₂ suppressed the in vitro bacterial growth in dose-dependent manners. Fifty µM of FeCl₂ began to reduce the bacterial growth (ca. 85.7%) compared to the mock-treated cultures, and the higher FeCl₂ concentrations 100 and 200 µM more inhibited the bacterial growth to ca. 80.5 and 71.3%, respectively. Iron-reduced mycelial growth of the phytopathogenic fungi was shown, indicating the in vitro antifungal activity of exogenous iron. The mycelium dry weight of F. oxysporum were significantly reduced by more than 200 µ g/ml of FeCl₃ (Hashem, 1995). Mycelial growth of six fungal species infecting grapevine, Botrytis cinerea, Eutypa lata, Phaeomoniella chlamydospora, Phaeoacremonium aleophilum, Diplodia seriata, and Neofusicoccum parvum, decreased on the media supplemented with FeSO₄ (5 mM) (Fleurat-Lessard et al., 2011). However, direct antibacterial activity of iron has rarely demonstrated against phytopathogenic bacteria including R. pseudosolanacearum. Treatment with 50-200 µM of FeCl₂ concentrations may suppress R. pseudosolanacearum by direct contact with the bacteria in this study, and it can be applied in the hydroponic cultures for many crops vulnerable to bacterial wilt. Both elevated bacterial inoculum levels and mechanical injury were involved in the increased bacterial wilt of tomato plants (Singh et al., 2014). The efficiency of the antimicrobial activity of FeCl₂ against the increasing bacterial population of R. pseudosolanacearum more than 10⁵ cfu/ml should be evaluated in a further study because 50-200 µM of FeCl₂ in our current study limited the only bacterial numbers initially inoculated with 10⁵ cfu/ml as an inoculum density.

Tomato seeds (cv. Cupirang) were sown in pots containing commercial soil mixtures and grown in a walk-in plant growth room under environmental conditions previously described in our study (Hong et al., 2018b). Third true leaves in the five-week-old plants were detached and used for chemical treatments and bacterial inoculation (Jo et al., 2020). We investigated phenotypic changes of the detached tomato leaves by dipping the petioles in the different concentrations of FeCl₂ solutions because the excess iron in the ambient environment can lead to toxicity in diverse plant species (Ahammed et al., 2020; Li et al., 2016; Turhadi et al., 2019). Different concentrations (5, 10, 20, 50, 100, and 200 µM) of FeCl₂ were prepared in distilled water. Distilled water was used as a mock treatment. The petiole of the detached tomato leaves was dipped in 10 ml of each solution in 50-ml conical tube. Fresh weight (FW) of the detached tomato leaves was measured before the petiole dipping in the FeCl₂ solutions and then further measured at 3 and 5 days after the petiole dipping. Relative FW (%) of the FeCl₂-treated leaves was expressed as a percentage compared to untreated leaves before the petiole dipping. Independent experiments were performed four times, and each experiment contained four biological replicates. Effects of FeCl₂ applications on FW of the detached tomato leaves were investigated in the absence of the bacterial population (Fig. 1B, C). The distinct phenotypic change was not observed in the detached tomato leaves treated with increasing concentrations (2-200 µM) of FeCl₂ at 3 and 5 days after petiole dipping, as shown in Fig. 1B. FW were investigated in the detached leaves at 3 and 5 d after petiole dipping (Fig. 1C). FW of the detached leaves were not different by the different concentrations of FeCl₂ treatment at 3 and 5 days. These indicate that 2-200 µM of FeCl₂ directly transported into the detached leaves through petioles did not cause a phytotoxic effect. Metal stresses to plants can depend on plant organs and growth stages (John et al., 2009; Souri et al., 2019). Therefore, we cannot exclude the possibility that treatment with FeCl₂ (2-200 µM) in soil or hydroponic cultures can lead to damage in tomato roots. The FeCl₂ concentrations can be re-adjusted to apply to the soil or hydroponic cultures of different growth stages of tomato plants.

Petioles of the detached tomato leaves were placed in the 10-ml solutions of different FeCl₂ concentrations and inoculated by bacterial suspension of R. pseudosolanacearum to make 10⁵ and 10⁷ cfu/ml as inoculum densities as described in our previous studies (Hong et al., 2013, 2018a). The bacterial wilt was evaluated by measuring the FW of the detached tomato leaves at 3 and 5 days after the bacterial inoculation. The FW of the detached tomato leaves at 3 and 5 dpi was compared to that of the leaves prior to the petiole dipping. Wilting symptoms of the detached leaves inoculated by the two inoculum densities (10⁵ and 10⁷ cfu/ml) of Rp-GMI1000 with or without FeCl₂ treatments were demonstrated as relative fresh weights (%) (Fig. 2). Independent experiments were performed three times, and each experiment contained four biological replicates. Treatment with FeCl₂ ameliorated the bacterial wilt of the detached leaves, and the reduced bacterial wilt was FeCl₂ concentration-and the bacterium inoculum density-dependent. By the low inoculum density (10⁵ cfu/ml), the reduced bacterial wilt was found in the leaves treated with more than 2 µM of FeCl₂ at 3 dpi. The bacterial wilt suppression was more evident in the leaves treated with 100 and 200 µM of FeCl₂ compared to the mock-treated control at 3 dpi. The detached leaves in the mock-treated control were more wilted at 5 dpi compared to 3 dpi. Increasing FeCl₂ also reduced bacterial wilt at 5 dpi. The bacterial wilt was also suppressed in the leaves inoculated by the high inoculum density (10⁷ cfu/ml) treated with different concentrations of FeCl₂. In vitro antibacterial activity was shown by more than 50 µM of FeCl₂ in this study, but the disease reduction in the detached leaves inoculated by Rp-GMI1000 (10⁵ cfu/ml) could be achieved by 10-20 µM of FeCl₂. It suggests that suppressed bacterial wilt of the detached tomato leaves treated with FeCl₂ can be mediated by enhanced plant immunity together with direct antibacterial activity. Iron homeostasis has been closely associated with activating defences in several plants under pathogen threats, such as wheat leaves inoculated with Blumeria graminis f. sp. tritici (Aznar et al., 2015; Liu et al., 2007). Interestingly, iron homeostasis was important to regulate production of reactive oxygen species (ROS) in plant root development and leaf cell death (Reyt et al., 2015; Tewari et al., 2013). In our study, accumulation of ROS and lipid peroxidation were found in the petiole tissues of leaves inoculated by Rp-GMI1000 (Hong et al., 2013). Tomato leaf tissues directly syringe-infiltrated with R. solanacearum showed increased hydrogen peroxide generation in the bacterial dose-dependent manner (Flores-Cruz and Allen, 2009). Recently, hydrogen peroxide production and lipid peroxidation in the R. solanacearum-inoculated tomato leaves were significantly ameliorated during the silica nanoparticles-mediated disease control of tomato bacterial wilt (Wang et al., 2022). But hydrogen peroxide production in tomato leaves during the early phase of bacterial wilt was faster in high boron-treated protected plants (Jiang et al., 2016). The involvement of exogenous FeCl₂ in redox regulation remains elucidated for understanding iron-mediated management of tomato bacterial wilt disease.

In conclusion, FeCl₂ of more than 50 µM showed antibacterial activity against R. pseudosolanacearum. No adverse effect was found in the detached tomato leaves treated with FeCl₂. The bacterial wilt of the detached tomato leaves was ameliorated by the FeCl₂ application, and disease control efficacy was dependent on the FeCl₂ concentration and bacterial inoculum density. Introducing microelement iron in tomato production fields can provide protection for tomato plants against bacterial wilt. Efficient FeCl₂ application methods should be further evaluated in different growth stages of tomato plants in greenhouses for stable fruit production.