Snake venoms and purified toxins as biotechnological tools to control Ralstonia solanacearum

The objective of this work was to evaluate the in vitro antibacterial activity of snake venoms and purified toxins on the phytopathogenic bacterium Ralstonia solanacearum. The evaluations were performed with 17 crude venoms (13 from Bothrops, 3 from Crotalus, and 1 from Lachesis) and seven toxins (1 from Bothrops and 6 from Crotalus). Antibacterial activity was assessed in MB1 medium containing solubilized treatments (1 μL mL-1). A total of 100 μL bacterial suspension (8.4 x 109 CFU mL-1) was used. After incubation at 28°C, the number of bacterial colonies at 24, 48, and 72 hours after inoculation was evaluated. SDS-PAGE gel at 15% was used to analyze the protein patterns of the samples, using 5 μg protein of each sample in the assay. Furthermore, the minimum inhibitory concentration (MIC) and lethal concentration (LC50) values were determined by the Probit method. Venoms and toxins were able to reduce more than 90% of R. solanacearum growth. These results were either equivalent to those of the positive control chloramphenicol or even better. While MIC values ranged from 4.0 to 271.5 μg mL-1, LC50 ranged from 28.5 μg mL-1 to 4.38 mg mL-1. Ten crude venoms (7 from Bothrops and 3 from Crotalus) and two purified toxins (gyroxin and crotamine) are promising approaches to control the phytopathogenic bacterium R. solanacearum.


Introduction
Through large-scale production systems, conventional agriculture plays an important role to attend to the growing food demand. However, food production can be affected by several factors, including pathogen attacks on host plants. Overall, it is estimated that, for many crops, potential loss caused by pathogens can reach over 30% of agricultural production worldwide (Yuliar et al., 2015;Rodrigues et al., 2020).
Phytobacteria can cause damage to several crops of economic interest and are responsible for important losses globally. Ralstonia solanacearum, one of the most important plant pathogenic bacterium, is responsible for important diseases in different crops, such as bacterial wilt, brown rot, and Moko disease in potato, tomato, and banana (Baptista et al., 2007;Peeters et al., 2013). In some cases, even with management measures, crop production may be seriously affected by R. solanacearum infection. The pathogen induces rapid and destructive damage to host tissues. This bacterium is a soil-borne pathogen with a large host variety, it penetrates the plants through their roots, reaching the xylem vessels where its multiplication occurs (Yadeta & Thomma, 2013;Dalsing et al., 2015).
Chemical products have been used for disease control, but, despite their efficiency, they are highly expensive and can cause damage to the environment and human health (Kwak et al., 2015). Thus, innovative products able to effectively control crop diseases with minimal impact on environmental and human populations are needed. In this scenario, natural/ synthetic antimicrobial molecules, as well as animal and vegetal biodiversity emerge as an immeasurable source of compounds with potential to control R. solanacearum strains. Some studies have shown that the essential oil extracted from Lantana camara and epsilon-poly-L-lysine (EPL), an antimicrobial peptide (AMP), inhibited bacteria growth (Cespedes et al., 2015;Mohamed et al., 2019;Rodrigues et al., 2020). These results can provide new antimicrobial substances, which are extremely important due to the current widespread of bacterial resistance (Datta et al., 2015).
Because of the relevance of R. solanacearum and its impact on crop production, new solutions have been searched for the improvement of crop productivity, cost and toxicity of chemical products, growing number of antibiotic-resistant bacteria, as well as results showing antibacterial effects of snake venom compounds, and new biotechnological tools to control plant diseases.
The objective of this work was to evaluate the in vitro antibacterial activity of snake venoms and purified toxins on the phytopathogenic bacterium R. solanacearum.
The protein profiles of the venoms and purified toxins were evaluated electrophoretically under reducing conditions using 15% polyacrylamide gels containing sodium dodecyl sulfate (SDS-PAGE) (Laemmli, 1970). For the analyses, total protein concentration was determined using bicinchoninic acid (BCA) method (Smith et al., 1985), and final concentration was adjusted to 5 μg for each sample in 20 μL final volume.
An electrophoretic run was carried out (100 V, 180 min), and gels were stained with 0.5% G-250 Coomassie Blue solution. Amersham ECL Rainbow Marker -Full Range kit (GE Healthcare, Amersham, Buckinghamshire, UK) was used as a molecular weight marker.
The bioactive potential of venoms and toxins was evaluated against the phytopathogenic bacterium Ralstonia solanacearum after solubilizing venoms and toxins in phosphate-buffered saline (PBS), pH 7.4, to 2 mg mL -1 final concentration.
Bacterial cultures were grown in liquid MB1 medium (medium 523) (Kado & Heskett, 1970) for 12 hours under agitation (100 rpm, 28°C). Bacterial growth was monitored using a spectrophotometer until it reached approximately 0.5 for A 540 . For serial dilution preparations, 1 mL of each bacterial suspension was added to 9 mL of sterile mineral water. After homogenization, 1 mL was transferred from tube 1 to tube 2 that contained 9 mL of sterile mineral water. This procedure was repeated through tube 10.
Petri dishes (80 mm diameter) containing solid MB1 medium were prepared, and 100 μL of each bacterial suspension dilution were deposited onto each plate. Plates were incubated at 28°C for 24 hours in a bacteriological oven, while serial dilutions were stored at 4°C. Subsequently, the number of colony forming units (CFU mL -1 ) was determined using the formula: CFU = NC x 10 tube / aliquot (mL), in which NC is the number of colonies, and 10 tube is the selected dilution tube used in the assay. Each plate containing 30-300 colonies was selected, and the respective dilution tube was used for antibacterial assay (adapted from Kass, 1956).
Hereafter, the plates for preliminary screenings were prepared with semi-solid MB1 medium containing the solubilized treatments (1 μL of venom or toxin per mL of culture medium). Samples were added to MB1 medium after autoclaving and when agar cooled down to 40°C. After solidification, 100 μL of the selected bacterial dilution tube (8.4x10 9 CFU mL -1 ) were deposited and scattered using Drigalski's spatula. The plates were incubated in a bacteriological oven at 28°C, and the number of bacterial colonies were evaluated at 24, 48, and 72 hours after inoculation. Chloramphenicol (0.5 mg mL -1 ) and PBS were used as positive and negative controls, respectively. All treatments were carried out in triplicate.
To determine the minimum inhibitory concentration (MIC) and lethal concentration (LC 50 ) values, venoms and toxins were solubilized in PBS pH 7.4, with a final concentration of 2 mg mL -1 and 0.6 mg mL -1 for venoms and toxins, respectively. MIC test was carried out only when effective results were evident, as observed across the treatments. Graphical representation of sample concentrations in relation to the inhibition percentage allowed of the LC 50 determination by Probit analysis.
MIC was determined using Probit analysis to evaluate the percentage of bacterial colonies that did not survive in the applied concentrations of venoms and toxins. Seven different venom concentrations (31.25 µg mL -1 -2 mg mL -1 ) and toxins (9.37 µg mL -1 -0.6 mg mL -1 ) were prepared and used in the analyses. Chloramphenicol and PBS were also evaluated as positive and negative controls, respectively. Antibacterial assays were carried out as previously described. As a means of standardizing the evaluations, all dilutions were performed immediately prior to assembling the experiments. In the evaluation of antibacterial activity, the LC 50 value corresponds to the concentration responsible for the inhibition of 50% of the number of colonies, and the MIC is considered to be the concentration that inhibits 1% of bacterial growth. For the screening tests, a completely randomized design was considered in a factorial arrangement with three replicates, to test 24 single concentration treatments and two controls (PBS and chloramphenicol). Data were subjected to the analysis of variance, and the means were compared using the Tukey's test, at 1% probability. Statistical analyses were performed using the Genes software (Cruz, 2016).

Results and discussion
The evaluated protein patterns of venoms showed that the presence of proteins ranged mainly between 12 and 76 kDa (Figure 1 A and B), while most of the isolated toxins had a molecular weight smaller than 20 kDa (Figure 1 C). These protein patterns are similar to those of others snake venoms, as well as compounds isolated from them (Torres et al., 2010;Nunes et al., 2011).
The antibacterial activity assays of snake venoms and toxins against colonies of R. solanacearum were subjected to analysis of variance and a significant reduction in colonies was observed ( Table 2). Out of the 24 venoms and toxins evaluated in the present study, 12 showed antibacterial activity against R. solanacearum (Figure 2). Seven Bothrops venoms (B. atrox, B. insularis, B. leucurus, B. brazili, B. moojeni, B. neuwiedi, and B. pauloensis), three Crotalus (C. durissus terrificus, C. durissus cascavella, and C. atrox), and two toxins (gyroxin, crotamine) showed highly significant antibacterial activity, with a bacterial growth inhibition level of 100%, similarly to the positive control chloramphenicol. PLA2-CB and B. jararacussu venom also showed significant activity, with 52 and 38% of bacterial growth inhibition, respectively, while other venoms such as those of Lachesis muta and B. urutu, and BthTX-I, did not differ from the negative control used in the tests. Thus, ten venoms and two toxins were selected for the MIC and LC 50 analyses.
Pesq. agropec. bras., Brasília, v.55, e01756, 2020 DOI: 10.1590/S1678-3921.pab2020.v55.01756 Figure 2. Ralstonia solanacearum colony averages when challenged with snake venoms and toxins. Chloramphenicol and phosphate-buffered saline (PBS) were used as positive and negative controls, respectively. Means of columns followed by equal letters do not differ by the Tukey's test, at 1% probability.  (Table 3). While LC 50 values ranged from 28.50 µg mL -1 to 4.39 mg mL -1 , MIC values ranged from 0.4 to 271.5 µg mL -1 . Antibacterial activity was also described for L-amino acid oxidase (LAAO) purified from C. durissus cascavella venom, against Xanthomonas axonopodis pv. passiflorae and Staphylococcus mutans, with LC 50 of 35 µg mL -1 and 12.3 µg mL -1 , respectively (Toyama et al., 2006). Other studies indicated that the venoms of C. adamanteus, Daboia russelli russelli, A. halis, Pseudechis australis, B. candidus, and P. guttata showed activity against different pathogenic bacteria, with higher activity against S. aureus, and MIC values ranged from 20.0-40.0 µg mL -1 (Samy et al., 2007). Moreover, a PLA 2 purified from Vipera russellii venom and the VRV-PL-VII-A fraction obtained from D. pulchella russelli venom showed activity against Escherichia coli, Klebsiella pneumoniae, and Salmonella paratyphi (Sudharshan & Dhananjaya, 2015). Furthermore, BmLec, a protein purified from the venom of Bothrops moojeni, was able to reduce 15% of the bacterial growth of X. axonopodis pv. passiflorae (Barbosa et al., 2010). A venom fraction of C. durissus terrificus showed antibacterial activity against the phytopathogenic pathogens X. axonopodis pv passiflorae and Clavibacter michiganensis michiganensis (Rádis-Batista et al., 2005).   In the present study, most of the antibacterial activity was found in venoms of the Bothrops genus (Figures 2 A, 2 B, 3 A, and 3 B). This activity may be explained by the high variability in the composition of venoms, which can be responsible for a local damage that possibly deactivates the bacterial wall and initiates an irrecoverable process, hindering the genetic synthesis that allow of bacteria replication, which justifies the minimal or none bacterial growth (Gutiérrez et al., 2017;Malange et al., 2019).
The present work provides new information on plant bacterial study, presenting substances with potential uses in biotechnological processes to improve pathogen control. Therefore, ten crude venoms and two purified toxins against R. solanacearum were herein selected based on their antibacterial activity. Further studies Pesq. agropec. bras., Brasília, v.55, e01756, 2020 DOI: 10.1590/S1678-3921.pab2020.v55.01756 should clarify the mechanisms involved in this activity and future applications.

Conclusions
1. Ten crude snake venoms show antibacterial activity against Ralstonia solanacearum -seven from Bothrops spp. and three from Crotalus spp. are able to inhibit more than 90% of the bacteria growth in vitro, with special attention to Bothrops insularis and Crotalus atrox.
2. Two snake toxins -gyroxin and crotamine -, isolated from Crotalus durissus terrificus, are able to inhibit the in vitro growth of R. solanacearum.

Acknowledgments
To Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, through CFF -grant no. 485047/2013-6), to Financiadora de Estudos e Projetos (Finep), to Fundação Rondônia de Amparo ao Desenvolvimento das Ações Científicas e Tecnológicas e de Pesquisa do Estado de Rondônia (Fapero), to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes, finance code 001), for financial support and for postgraduate fellowships to Rita de Cássia Alves, Tamiris Chaves Freire, and Aline Souza Fonseca ; to Domingos Sávio G. Silva and Antônio M. Marques, for technical support; to Amy Nicole Grabner for the English review of the manuscript; and to Program for Technological Development in Tools for Health-PDTIS-Fiocruz, for the permission to use of its facilities. The funders had no role in study design, data collection and analyses, decision to publish, or preparation of the manuscript.