Inhibition of Agrobacterium tumefaciens Growth and Biofilm Formation by Tannic Acid

Agrobacterium tumefaciens underlies the pathogenesis of crown gall disease and is characterized by tumor-like gall formation on the stems and roots of a wide variety of economically important plant species. The bacterium initiates infection by colonizing and forming biofilms on plant surfaces, and thus, novel compounds are required to prevent its growth and biofilm formation. In this study, we investigated the ability of tannic acid, which is ubiquitously present in woody plants, to specifically inhibit the growth and biofilm formation of A. tumefaciens. Tannic acid showed antibacterial activity and significantly reduced the biofilm formation on polystyrene and on the roots of Raphanus sativus as determined by 3D bright-field and scanning electron microscopy (SEM) images. Furthermore, tannic acid dose-dependently reduced the virulence features of A. tumefaciens, which are swimming motility, exopolysaccharide production, protease production, and cell surface hydrophobicity. Transcriptional analysis of cells (Abs600 nm = 1.0) incubated with tannic acid for 24 h at 30 °C showed tannic acid most significantly downregulated the exoR gene, which is required for adhesion to surfaces. Tannic acid at 100 or 200 µg/mL limited the iron supply to A. tumefaciens and similarly reduced the biofilm formation to that performed by 0.1 mM EDTA. Notably, tannic acid did not significantly affect R. sativus germination even at 400 µg/mL. The findings of this study suggest that tannic acid has the potential to prevent growth and biofilm formation by A. tumefaciens and thus infections resulting from A. tumefaciens colonization.


Introduction
Biofilm formation on plant surfaces is of considerable importance, as in some cases, biofilms can promote plant growth, whereas in others, they can lead to infections [1,2]. Agrobacterium tumefaciens is a soil-borne, Gram-negative bacterium that thrives in the rhizosphere and readily forms biofilms and thus colonizes plant surfaces [3]. A. tumefaciens exerts its pathogenicity by transferring T-DNA from its tumor-inducing plasmid to host plant cells [4][5][6]. Studies have established that various strains of the genus A. tumefaciens are responsible for the neoplastic condition of plants referred to as crown gall [7]. The host range of A. tumefaciens is extensive, and it has been reported to cause crown gall disease in 643 plants belonging to 331 genera [8]. Not surprisingly, many of these host plants, including those in the Vitaceae and Rosaceae families and the genus Juglans, are economically valuable [7], and thus, A. tumefaciens infections result in huge financial losses due to reduced crop production [9,10].
A. tumefaciens is a pervasive component of soil microbiota [11] that colonizes wounded parts of plants. Amino acids, sugars, and organic acids released from wounds act as chemoattractants for the bacterium, which reaches wounds by chemotaxis and attaches firmly to cellulose fibrils [3]. Infection commences with the horizontal transmission of T-DNA present in Ti-plasmid to host cells. A. tumefaciens efficiently locomotes using multiple flagella positioned around a single pole [12]. The VirA/VirG system of A. tumefaciens, Figure 1. Tannic acid is composed of a central glucose with ten surrounding galloyl units (A). Biofilm formation of A. tumefaciens on polystyrene (B). Gross images and biofilm morphology on 6-well polystyrene plates (C). The normalized ratio of biofilm (Abs570 nm) to cell growth (Abs620 nm) shows tannic acid reduces biofilm formation at ≥ 150 μg/mL (D). Cells were incubated in LB containing tannic acid at the concentrations shown for 48 h at 30 °C under static conditions. Two independent bacterial colonies were used in triplicates (n = 6) and experiments were repeated twice. Asterisks (*) denote significant differences (p ≤ 0.05 by the two-tailed t-test) between treated groups and nontreated controls.
Nonetheless, the effects of tannic acid on A. tumefaciens growth and biofilms have not been explored, which prompted us to assess the ability of tannic acid to inhibit A. tumefaciens planktonic growth and biofilm formation, the major virulent traits of A. tumefaciens (swimming motility, EPS production, protease activity, and cell surface hydrophobicity), and its surface colonization of roots. In addition, we examined the effects of tannic acid on the expressions of 11 biofilm and virulence-related genes in A. tumefaciens.

Chemicals and Bacterial Culture
Tannic acid (Product Code 403040) was purchased from Sigma-Aldrich (St. Louis, MO, USA). A. tumefaciens strain GV2260 was used for all experiments. A single colony of . Gross images and biofilm morphology on 6-well polystyrene plates (C). The normalized ratio of biofilm (Abs570 nm) to cell growth (Abs620 nm) shows tannic acid reduces biofilm formation at ≥150 µg/mL (D). Cells were incubated in LB containing tannic acid at the concentrations shown for 48 h at 30 • C under static conditions. Two independent bacterial colonies were used in triplicates (n = 6) and experiments were repeated twice. Asterisks (*) denote significant differences (p ≤ 0.05 by the two-tailed t-test) between treated groups and nontreated controls.
Nonetheless, the effects of tannic acid on A. tumefaciens growth and biofilms have not been explored, which prompted us to assess the ability of tannic acid to inhibit A. tumefaciens planktonic growth and biofilm formation, the major virulent traits of A. tumefaciens (swimming motility, EPS production, protease activity, and cell surface hydrophobicity), and its surface colonization of roots. In addition, we examined the effects of tannic acid on the expressions of 11 biofilm and virulence-related genes in A. tumefaciens.

Chemicals and Bacterial Culture
Tannic acid (Product Code 403040) was purchased from Sigma-Aldrich (St. Louis, MO, USA). A. tumefaciens strain GV2260 was used for all experiments. A single colony of A. tumefaciens was inoculated in LB medium and incubated for 24 h at 250 rpm and 30 • C. Shake cultures were prepared under similar growth conditions in flasks. Experiments were performed using at least two independent cultures (prepared from two independent colonies) in triplicate.

Evaluation of Biofilm Inhibition
The antibiofilm susceptibility of A. tumefaciens against tannic acid was analyzed using a crystal violet assay as previously described [29]. Briefly, after culturing for 24 h, A. tumefaciens was diluted with LB at 1:50. Tannic acid was then added in the concentration range 10-400 µg/mL, and 300 µL of these mixtures was pipetted into a 96-well plate and incubated at 30 • C for 48 h. The plate was then stained with 0.1% crystal violet for 20 min, washed with sterile water, and air-dried. Crystal violet retained by biofilms was dissolved with 300 µL of 95% ethanol, and biofilm formation was quantified by measuring absorbance (λmax) at 570 nm [29].

Growth Assessments of Tannic Acid Treated A. tumefaciens
Time-dependent growth of A. tumefaciens in the presence of tannic acid was used to investigate the ability of tannic acid to inhibit planktonic cell growth following our earlier described method [30]. After culturing for 24 h, A. tumefaciens was diluted with LB at Biomedicines 2022, 10, 1619 4 of 17 1:100, and tannic acid was added to final concentrations of 10, 20, 25, 50, 75, 100, 150, 200, or 400 µg/mL. A 300 µL aliquot of each of these cultures was then added to a 96-well plate and incubated at 30 • C for 24 h under static conditions. OD values were recorded at 620 nm every 2 h during this 24 h period, and growth curves were plotted. To determine colony-forming units (CFUs), a 24 h culture of A. tumefaciens was diluted with LB (1:100), and tannic acid was added (at 10-400 µg/mL) and cultured at 250 rpm for 24 h at 30 • C. Cultures were then diluted and 100 µL of each dilution was spread onto LB agar plates, which were incubated for two days at 30 • C. Colonies were then counted.

Growth and Biofilm Inhibition under Iron-Limiting Conditions
To check the role of ten galloyl groups of tannic acid in creating iron-limiting conditions and thus inhibition of growth and biofilms, 100 and 200 µg/mL of tannic acid were used with various concentrations of an iron source (FeSO 4 ·7H 2 O). An earlier method [31] was used with modifications. The well-known iron chelator EDTA was first tested to know its threshold concentration that reduces the growth and biofilm of A. tumefaciens in LB. Various concentrations of EDTA (0.01, 0.02, 0.05, 0.1, 0.2, 0.5, and 1 mM) were checked on growth and biofilm formation of A. tumefaciens in 96-well plates under the same incubation conditions as described earlier (Sections 2.2 and 2.3). Biofilm inhibitory concentration of EDTA was then tested with varying doses (0.01-1 mM) of Fe 2+ (FeSO 4 ·7H 2 O) in 96-well plates. In a similar manner, two concentrations of tannic acid (100 and 200 µg/mL) were tested in two different experiments with varying concentrations of FeSO 4 ·7H 2 O (0.01-1 mM), and the growth and biofilm formation were checked after 48 h at 30 • C.

Swimming Motility Assay
Swimming motilities of A. tumefaciens grown in the presence of various doses of tannic acid were investigated using motility agar plates. The agar constituted 1% peptone, 0.5% NaCl, and 0.25% agarose. Before pouring into Petri plates, the agar was thoroughly mixed with different concentrations of tannic acid. Aliquots (1 µL) of 24 h cultures were placed at the centers of agar plates and incubated at 30 • C for 72 h. Swimming diameters were recorded at every 48 h and 72 h during incubation [30].

Estimation of EPS Released under Tannic Acid Stress
Amounts of EPS released by A. tumefaciens cells were estimated using a standard phenol-sulfuric acid assay, as previously described [32]. A. tumefaciens cells (OD600 nm = 0.5) were grown in the presence or absence of tannic acid at different concentrations in 1.5 mL microfuge tubes at 250 rpm for 24 h at 30 • C. After incubation, tubes were centrifuged for 10 min at 13,000 rpm, and supernatants were collected and diluted with chilled ethanol at 1:3. The tubes were then allowed to stand at 4 • C overnight, and precipitates obtained were dissolved in 1 mL of distilled water. Phenol (5%) and concentrated sulfuric acid in the ratio of 1:5 was added to the dissolved EPS and incubated for 30 min at room temperature. Absorbances were recorded at 490 nm.

Cell Surface Hydrophobicity Assay
A. tumefaciens cells (OD600 nm = 0.5) were grown for 24 h in the presence of various concentrations of tannic acid in 1.5 mL microfuge tubes at 30 • C and 250 rpm. Tubes were then centrifuged for 10 min at 13,000 rpm. The pellets obtained were washed three times and resuspended in 1 mL of sterile PBS, and OD was measured at 600 nm (denoted A 0 ). Xylene (250 µL) was added to suspensions, vortexed vigorously, and left undisturbed for 30 min. Aqueous phases were then carefully removed, and absorbances (Ai) were measured at 600 nm. Percent hydrophobicities were then calculated using the following formula [33]: Percent cell surface hydrophobicity (% CSH) = (A 0 − Ai)/Ai × 100

Effect of Tannic Acid on Protease Production
Bacterial exoprotease production was quantified as previously described [34] in the presence or absence of tannic acid. A. tumefaciens was grown with 25, 50, 75, 100, 150, or 200 µg/mL of tannic acid at 250 rpm for 24 h at 30 • C. Microfuge tubes were centrifuged for 10 min at 13,000 rpm, and supernatant (100 µL) was collected in fresh microfuge tubes, treated with 100 µL of azocasein for 30 min at 37 • C, and then with 600 µL of trichloroacetic acid (10%) to halt proteolysis. The tubes were then cooled at −20 • C for 30 min, and 700 µL of 1 M NaOH was added. Absorbances were recorded at 440 nm.

Visualization of A. tumefaciens Biofilms on Nitrocellulose Membranes and R. sativus Roots by Scanning Electron Microscopy (SEM)
The biofilms of A. tumefaciens were formed on 3 × 3 mm nitrocellulose membranes in a 96-well plate. Bacterial culture diluted with LB (1:50) and treated with 200 µg/mL of tannic acid was pipetted into wells and allowed to grow for 48 h at 30 • C. To investigate biofilm development on roots and the effects of tannic acid, surface-sterilized seeds of R. sativus were placed on soft agar plates (0.7% agar + 0.86 g/L Murashige and Skoog plant growth medium) and grown for four days. At least five seedlings from control and treated groups were placed in 6-well plates containing 200 µg/mL of tannic acid in inoculum of A. tumefaciens (1:50 dilution) and incubated at 30 • C for 48 h. Roots were then gently rinsed once with PBS. Before SEM imaging, samples (nitrocellulose membranes and roots) were processed as follows: (i) they were fixed in a mixture of 2.5% glutaraldehyde and 2% paraformaldehyde for 30 min at room temperature and overnight at 4 • C, and (ii) samples were dehydrated using an ethanol series (30%, 50%, 70%, 90%, and 100% for 10 min each), (iii) preserved in isoamyl acetate, (iv) critical point dried and sputter coated with gold or platinum, and (v) visualized at different magnifications using a S-4200 Hitachi Field Emission (FE) SEM microscope (Hitachi Systems Ltd., Tokyo, Japan) at 15 kV.

Potato Disc Antitumor Assay
An earlier described method for potato disc tumor formation was followed [35] with slight modifications. Potato discs were infected by A. tumefaciens and checked after 3 weeks (incubation at 25 • C) for the development of tumors. Potatoes were surface cleaned with tap water thoroughly to remove all the dirt, followed by surface cleaning with ethanol (70%). Potatoes were cut to obtain flat cubes. Thick slices were made using the sterile knife and immersed in NaOCl (6%) for 15 min. Potato discs (1 cm in diameter) were cut from the middle part of cubes using a sterile cork borer. Discs were rinsed thrice in sterile distilled water. For infection, A. tumefaciens culture (OD600 nm = 1.0) was exposed to tannic acid (200 µg/mL) in PBS and vortexed. A 50 µL of the cell suspension was added to the surface of each potato disc and placed in 6-well tissue culture plates. Plates were sealed and incubated at 25 • C for three weeks in the dark. The numbers of A. tumefaciens induced tumors that appeared on potato surfaces were counted, and percent tumor induction was calculated.
Tumor induction (%) = Number o f tumors in treated samples Untreated control × 100

Effects of Tannic Acid on Seed Germination
R. sativus seeds were rinsed with distilled water three times and once with 95% ethanol and surface disinfected by emersion in 3% sodium hypochlorite for 10 min. Seeds (n = 10) were then placed on agar plates containing different concentrations of tannic acid (25-400 µg/mL), 0.7% agar, and 0.86 g/L Murashige and Skoog plant growth medium [30]. Plates were then incubated for four days at 25 • C, and percent seed germinations were calculated with respect to nontreated controls.

RNA Extraction and Gene Expression Analysis by qRT-PCR
To determine whether tannic acid treatment affected the expression levels of genes related to biofilm, stress, motility, and virulence, A. tumefaciens was grown in the presence or absence of tannic acid. A. tumefaciens culture in 25 mL LB having an OD of 1.0 at 600 nm was added with 200 µg/mL of tannic acid and kept at 250 rpm for 24 h at 30 • C, then 700 µL of RNase inhibitor (RNAlater, Ambion, TX, USA) was added with gentle mixing [23]. Cultures were maintained under chilled conditions in an ice bath to prevent RNA degradation and harvested by centrifugation at 13,000 rpm for 10 min at 4 • C. RNAs from cell pellets were extracted using the Qiagen RNeasy mini kit (Valencia, CA, USA), and RNA purities were checked using a nanodrop spectrophotometer (Cytiva NanoVue Plus Spectrophotometer, Fisher Scientific, Loughborough, UK). Quantitative reverse transcription real time polymerase chain reaction (qRT-PCR) was carried out to assess the expressions of genes responsible for biofilm formation (celA, cheA, exoR, phoB), virulence (chvE, chvG), motility (flgE, fliR, motA), and stress response (clpB, dnaK). Gene functions and primer sequences are given in Supplementary Table S1. The 16S rRNA gene was used as the housekeeping gene, and the reaction was carried out using SYBR green master mix and the ABI StepOne Real-Time PCR System, Applied Biosystems, Foster City, CA, USA [23].

Statistical Analysis
Two independent bacterial colonies were used for experiments in triplicates, and results are presented as means ± standard deviations. Values of p ≤ 0.05 were considered significant as per Student's t-test. All statistical analyses were performed, and graphs were plotted on SigmaPlot Windows Version 14.0 (Build 14.0.0.124), Systat Software, Inc., Chicago, IL, USA. Asterisks in figures indicate significant changes versus nontreated controls.

Tannic Acid Inhibited Biofilm Formation by A. tumefaciens
Tannic acid ( Figure 1A) in the range 10-400 µg/mL dose-dependently and significantly inhibited A. tumefaciens biofilm formation ( Figure 1B). Tannic acid at 100 µg/mL inhibited biofilm formation by 86.6%, and this inhibition increased further on increasing tannic acid concentration (e.g., to 92.7% at 200 µg/mL). Bright-field microscopy ( Figure 1C) showed slight biofilm formation at 100 µg/mL and little evidence of biofilm formation at higher concentrations. The normalized data of biofilm (570 nm) to growth (620 nm) first increased up to 100 µg/mL, which then decreased sharply at 150 µg/mL and further reduced at 200 µg/mL ( Figure 1D). This suggests that apart from bacteriostatic/bactericidal activity, tannic acid also interferes with the biofilm formation of A. tumefaciens and reduces it. Micrographs of biofilms were recreated as three-dimensional LUT mesh plots ( Figure 2). Colors and color intensities of biofilms produced with or without tannic acid showed slight biofilm formation at 50-100 µg/mL but almost no biofilm formation at 200 µg/mL. Biofilm quantification and their visualization on polystyrene confirmed the inhibitory potential of tannic acid.

Tannic Acid Compromised the Growth and Viability of A. tumefaciens
Tannic acid has antibacterial activity against several clinical pathogens like S. aureus, P. aeruginosa, Escherichia coli, Streptococcus pyogenes, Enterococcus faecalis, and Listeria innocua [19]. We found tannic acid dose-dependently delayed the growth of A. tumefaciens ( Figure 3A). At concentrations from 50 to 150 µg/mL and incubation for 8 h, tannic acid had similar inhibitory effects on A. tumefaciens growth. In addition, tannic acid dosedependently reduced cell viabilities ( Figure 3B). At 75 µg/mL, tannic acid remarkably inhibited cell viability (> 70%), and at 200 µg/mL, few cells survived.
increased up to 100 μg/mL, which then decreased sharply at 150 μg/mL and further reduced at 200 μg/mL ( Figure 1D). This suggests that apart from bacteriostatic/bactericidal activity, tannic acid also interferes with the biofilm formation of A. tumefaciens and reduces it. Micrographs of biofilms were recreated as three-dimensional LUT mesh plots ( Figure  2). Colors and color intensities of biofilms produced with or without tannic acid showed slight biofilm formation at 50-100 μg/mL but almost no biofilm formation at 200 μg/mL. Biofilm quantification and their visualization on polystyrene confirmed the inhibitory potential of tannic acid.

Tannic Acid Compromised the Growth and Viability of A. tumefaciens
Tannic acid has antibacterial activity against several clinical pathogens like S. aureus, P. aeruginosa, Escherichia coli, Streptococcus pyogenes, Enterococcus faecalis, and Listeria innocua [19]. We found tannic acid dose-dependently delayed the growth of A. tumefaciens ( Figure 3A). At concentrations from 50 to 150 μg/mL and incubation for 8 h, tannic acid had similar inhibitory effects on A. tumefaciens growth. In addition, tannic acid dose-dependently reduced cell viabilities ( Figure 3B). At 75 μg/mL, tannic acid remarkably inhibited cell viability (> 70%), and at 200 μg/mL, few cells survived.

External Iron Supply Restored the Growth and Biofilm of A. tumefaciens
The EDTA (1 mM) significantly reduced the biofilm formation and growth of A. tumefaciens growing in LB with no supply of external iron (Fe2SO4•7H2O) ( Figure 4A). When Fe2SO4•7H2O (0.01-1 mM) was supplied with 1 mM EDTA in LB, the growth and biofilm formation started to resume at 0.02 mM Fe2SO4•7H2O and increased in an iron concentration-dependent manner ( Figure 4A). This suggests that iron was one of the limiting factors for the growth and biofilm of A. tumefaciens. Similarly, when tannic acid (100 and 200 Figure 3. Growth inhibition of A. tumefaciens by tannic acid cultured in LB for 24 h at 30 • C. Growth curves (A) and percent cell viability (B) after exposure to tannic acid. Two independent bacterial colonies were used in triplicates (n = 6) and experiments were repeated twice. '*' denotes significant difference between nontreated and treated A. tumefaciens. p ≤ 0.05 as determined by the two-tailed t-test.

External Iron Supply Restored the Growth and Biofilm of A. tumefaciens
The EDTA (1 mM) significantly reduced the biofilm formation and growth of A. tumefaciens growing in LB with no supply of external iron (Fe 2 SO 4 ·7H 2 O) ( Figure 4A). When Fe 2 SO 4 ·7H 2 O (0.01-1 mM) was supplied with 1 mM EDTA in LB, the growth and biofilm formation started to resume at 0.02 mM Fe 2 SO 4 ·7H 2 O and increased in an iron concentration-dependent manner ( Figure 4A). This suggests that iron was one of the limiting factors for the growth and biofilm of A. tumefaciens. Similarly, when tannic acid (100 and 200 µg/mL) was used instead of EDTA (1 mM), it substantially reduced the biofilm in the absence of external iron supply ( Figure 4B,C). The Fe 2 SO 4 ·7H 2 O restored the biofilm formation at 0.01 mM in the presence of 100 µg/mL tannic acid ( Figure 4B), while 0.02 mM Fe 2 SO 4 ·7H 2 O was required to mitigate the effect of 200 µg/mL tannic acid ( Figure 4C). At ≥0.05 mM Fe 2 SO 4 ·7H 2 O, the biofilm of A. tumefaciens was restored completely and was found equivalent to nontreated group; however, the growth increased further than the nontreated control until 1 mM Fe 2 SO 4 ·7H 2 O.  EPS benefits embedded bacterial cells by providing a protective shield that prevents antimicrobial ingress and enhances the structural stabilities and integrities of biofilms [36]. We examined the effect of tannic acid on EPS production by A. tumefaciens using a phenol-sulfuric acid assay ( Figure 5A). Marginal EPS increases of 17% and 19% were observed at tannic acid concentrations of 20 and 25 μg/mL, presumably in response to tannic  (panel (B)), and 200 µg/mL tannic acid (panel (C)). Bar for 'none' shows biofilm formation without EDTA or tannic acid or Fe 2 SO 4 ·7H 2 O. Biofilm (570 nm) bars marked with ' †' show biofilm in the presence of an iron source. Two independent bacterial colonies were used in triplicates (n = 6), and each experiment was repeated at least twice. Asterisks denote significant differences between 'none' and treated A. tumefaciens at p ≤ 0.05 by the two-tailed t-test.

Effects of Tannic Acid on Virulence Properties: EPS, Protease Activity, and Cell Surface Hydrophobicity (CSH)
EPS benefits embedded bacterial cells by providing a protective shield that prevents antimicrobial ingress and enhances the structural stabilities and integrities of biofilms [36]. We examined the effect of tannic acid on EPS production by A. tumefaciens using a phenolsulfuric acid assay ( Figure 5A). Marginal EPS increases of 17% and 19% were observed at tannic acid concentrations of 20 and 25 µg/mL, presumably in response to tannic acidinduced stress [37]. However, at concentrations above 50 µg/mL, tannic acid significantly and dose-dependently reduced EPS production. For example, at 100 and 200 µg/mL, tannic acid reduced EPS production by 41% and 90%, respectively. containing peptone agar (1% peptone, 0.5% NaCl, 0.25% agarose). Swim diameters were recorded after incubation for 48 or 72 h (E). For EPS production, protease activity, and %CSH, two independent bacterial colonies were used in triplicates (n = 6) and experiments were repeated twice. For swimming motility, three independent colonies were used (n = 3), and experiment was repeated twice. Asterisks denote significant differences between nontreated and treated A. tumefaciens. p ≤ 0.05 by the two-tailed t-test.
The extracellular release of proteases is another important virulence factor, as this release plays an important role in the spread of infection and can cause irreparable host damage [38]. We found that at 50 to 400 μg/mL tannic acid dose-dependently reduced protease activity ( Figure 5B), and that at 200 and 400 μg/mL, it caused reductions of 77.3% and 91.3%, respectively.
The CSH is also an important virulence factor, as it influences the abilities of cells to adhere to biotic and abiotic surfaces. Tannic acid decreased %CSH significantly at concentrations ≥ 150 μg/mL ( Figure 5C), after which its effect plateaued.

Swimming Motility of A. tumefaciens Exposed to Tannic Acid
Swimming motility plays a central role in bacterial biofilm formation and in the pathogeneses of resulting diseases [12]. A. tumefaciens GV2260 demonstrated swimming motility on agar plates in the absence of tannic acid; maximum swim diameters of nontreated controls were 4.7 and 8 cm after incubation for 48 h and 72 h, respectively. However, tannic acid treatments concentration-dependently reduced swimming motility ( Figure 5D,E), and its effect was greatest at 100 μg/mL, at which swim diameters were 2.4 and 4.5 cm after incubation for 48 h and 72 h, respectively. At concentrations of 150 and 200 μg/mL Concentration-dependent effect of tannic acid on the swimming motility of A. tumefaciens (D) after incubation for 48-72 h tannic acid containing peptone agar (1% peptone, 0.5% NaCl, 0.25% agarose). Swim diameters were recorded after incubation for 48 or 72 h (E). For EPS production, protease activity, and %CSH, two independent bacterial colonies were used in triplicates (n = 6) and experiments were repeated twice. For swimming motility, three independent colonies were used (n = 3), and experiment was repeated twice. Asterisks denote significant differences between nontreated and treated A. tumefaciens. p ≤ 0.05 by the two-tailed t-test.
The extracellular release of proteases is another important virulence factor, as this release plays an important role in the spread of infection and can cause irreparable host damage [38]. We found that at 50 to 400 µg/mL tannic acid dose-dependently reduced protease activity ( Figure 5B), and that at 200 and 400 µg/mL, it caused reductions of 77.3% and 91.3%, respectively.
The CSH is also an important virulence factor, as it influences the abilities of cells to adhere to biotic and abiotic surfaces. Tannic acid decreased %CSH significantly at concentrations ≥150 µg/mL ( Figure 5C), after which its effect plateaued.

Swimming Motility of A. tumefaciens Exposed to Tannic Acid
Swimming motility plays a central role in bacterial biofilm formation and in the pathogeneses of resulting diseases [12]. A. tumefaciens GV2260 demonstrated swimming motility on agar plates in the absence of tannic acid; maximum swim diameters of nontreated controls were 4.7 and 8 cm after incubation for 48 h and 72 h, respectively. However, tannic acid treatments concentration-dependently reduced swimming motility ( Figure 5D,E), and its effect was greatest at 100 µg/mL, at which swim diameters were 2.4 and 4.5 cm after incubation for 48 h and 72 h, respectively. At concentrations of 150 and 200 µg/mL incubation with tannic acid for 48 h completely inhibited swimming motility.

Tannic Acid Inhibited Biofilm Formation on Nitrocellulose and on the Roots of R. sativus
Biofilms on nitrocellulose membranes and plant roots in the presence and absence of tannic acid were visualized by SEM. Comparisons of micrographs of biofilms formed in the absence (Figure 6A-C) and presence of tannic acid ( Figure 6D-F) showed that biofilm formation on nitrocellulose membranes was severely inhibited by tannic acid at 200 µg/mL. In the absence of tannic acid, biofilms were dense, well-structured, and uniform on nitrocellulose, but only a few scattered cells of A. tumefaciens were found in the presence of tannic acid. However, despite the obvious inhibition of biofilm formation by tannic acid, the only morphological difference noticed was a slight reduction in cell length.

Tannic Acid Inhibited Biofilm Formation on Nitrocellulose and on the Roots of R. sativus
Biofilms on nitrocellulose membranes and plant roots in the presence and absence of tannic acid were visualized by SEM. Comparisons of micrographs of biofilms formed in the absence (Figure 6A-C) and presence of tannic acid ( Figure 6D-F) showed that biofilm formation on nitrocellulose membranes was severely inhibited by tannic acid at 200 μg/mL. In the absence of tannic acid, biofilms were dense, well-structured, and uniform on nitrocellulose, but only a few scattered cells of A. tumefaciens were found in the presence of tannic acid. However, despite the obvious inhibition of biofilm formation by tannic acid, the only morphological difference noticed was a slight reduction in cell length.

Influence of Tannic Acid on Tumor Induction by A. tumefaciens and Seed Germination
Tannic acid (200 μg/mL) exposed cells of A. tumefaciens (OD600 nm = 1.0) lost their ability to induce tumors on potato slices. The average reduction in tumor formation was 78% by tannic acid over nontreated control (Figure 7). Tannic acid was examined to determine any impact on R. sativus seed germination (Figure 8). R. sativus seeds germinated Biofilm formation on the roots of R. sativus was examined after incubation for 48 h with A. tumefaciens in the presence or absence of tannic acid. SEM images provided insight into the extent of colonization of root surfaces ( Figure 6G-L). Roots incubated in the absence of tannic acid showed extensive colonization by the pathogen on root hairs and surrounding areas ( Figure 6G-I). On the other hand, barely any colonization was observed on the root surfaces of seedlings incubated in the presence of tannic acid at 200 µg/mL ( Figure 6J-L).

Influence of Tannic Acid on Tumor Induction by A. tumefaciens and Seed Germination
Tannic acid (200 µg/mL) exposed cells of A. tumefaciens (OD600 nm = 1.0) lost their ability to induce tumors on potato slices. The average reduction in tumor formation was 78% by tannic acid over nontreated control (Figure 7). Tannic acid was examined to determine any impact on R. sativus seed germination (Figure 8). R. sativus seeds germinated on soft agar plates ( Figure 8A) containing MS plant growth solution and tannic acid (25-400 µg/mL) were checked after four days of incubation. Notably, tannic acid did not inhibit germination at any of the concentrations examined ( Figure 8B). on soft agar plates ( Figure 8A) containing MS plant growth solution and tannic acid (25-400 μg/mL) were checked after four days of incubation. Notably, tannic acid did not inhibit germination at any of the concentrations examined ( Figure 8B).  Representative pictures of potato discs with tumors marked with blue arrows are shown on the top. Two independent bacterial colonies were used in triplicates (n = 6), and experiment was repeated twice. '*' shows a significant difference at p ≤ 0.05 by the two-tailed t-test.

Effect of Tannic Acid on A. tumefaciens Gene Expressions
Certain bacterial genes influence characteristics such as biofilm formation (celA, cheA, exoR, phoB), stress response (clpB, dnaK), motility (flgE, fliR, motA), and virulence (chvE, chvG), and thus, we examined the effects of tannic acid at 200 μg/mL on the expressions of these genes (Figure 9). qRT-PCR showed that exoR, which is required for biofilm formation [39], was downregulated by >15-fold by tannic acid. Downregulation of this gene has been proposed to be a cause of compromised surface adherence to plant and abiotic surfaces [40]. With the exceptions of dnaK and chvE, which were slightly downregulated, tannic acid had no effect on the expression of other genes tested.

Effect of Tannic Acid on A. tumefaciens Gene Expressions
Certain bacterial genes influence characteristics such as biofilm formation (celA, cheA, exoR, phoB), stress response (clpB, dnaK), motility (flgE, fliR, motA), and virulence (chvE, chvG), and thus, we examined the effects of tannic acid at 200 µg/mL on the expressions of these genes (Figure 9). qRT-PCR showed that exoR, which is required for biofilm formation [39], was downregulated by >15-fold by tannic acid. Downregulation of this gene has been proposed to be a cause of compromised surface adherence to plant and abiotic surfaces [40]. With the exceptions of dnaK and chvE, which were slightly downregulated, tannic acid had no effect on the expression of other genes tested.

Discussion
A. tumefaciens resides in the top layer of soil and can adhere to biotic and abiotic surfaces [3,30]. The transition from sessile to surface-attached A. tumefaciens is determined by adhesins and flagellar motility [40]. Tannic acid is ubiquitously present in woody and herbaceous plants and possesses antimutagenic and antitumor properties and antibacterial and antiviral activities [19,41], which has generated interest in the extraction of polyphenols with unique antimicrobial properties from natural sources. Many pathogens de- Figure 9. qRT-PCR analysis of the expressions of genes after treating A. tumefaciens with tannic acid at 200 µg/mL. The asterisk denotes a significant difference between nontreated and treated A. tumefaciens. Two independent bacterial colonies were used in duplicates (n = 4), and experiment was repeated twice. p ≤ 0.05 by the two-tailed t-test.

Discussion
A. tumefaciens resides in the top layer of soil and can adhere to biotic and abiotic surfaces [3,30]. The transition from sessile to surface-attached A. tumefaciens is determined by adhesins and flagellar motility [40]. Tannic acid is ubiquitously present in woody and herbaceous plants and possesses antimutagenic and antitumor properties and antibacterial and antiviral activities [19,41], which has generated interest in the extraction of polyphenols with unique antimicrobial properties from natural sources. Many pathogens develop resistance against conventional treatments when their planktonic forms are targeted [16], and thus, biofilm targeting is viewed as an attractive potential treatment option [42]. We investigated whether tannic acid at concentrations from 10 to 400 µg/mL could effectively inhibit A. tumefaciens biofilm formation and explored the effects of tannic acid on its biofilmforming capacity, root colonizing ability, and virulence attributes. We found that tannic acid markedly reduced A. tumefaciens biofilm formation at 100 µg/mL ( Figure 1B,C) and reduced the viability of A. tumefaciens cells (Figure 3). In addition, tannic acid dramatically inhibited protease secretion and EPS production by A. tumefaciens and its surface hydrophobicity ( Figure 5A-C), cell motility ( Figure 5D,E), and abilities to adhere to polystyrene or nitrocellulose (Figures 2 and 6) and root surfaces ( Figure 6G-L). This was possibly caused by hydrophobic and H-bonding interactions between tannic acid and bacterial proteins [19]; as has been reported, tannic acid acts as a multidentate ligand due to interactions between its aromatic rings and phenolic groups and protein surfaces [43]. Furthermore, inhibition of the NorA efflux pump by tannic acid has been proposed to be responsible for its antimicrobial effects on Staphylococcus aureus and Enterococcus faecalis [28]. NorA confers resistance against ciprofloxacin in S. aureus [44], and the AcrAB efflux pump has a similar function in A. tumefaciens and provides resistance against ciprofloxacin and other drugs [45]. Thus, it is possible that tannic acid targets AcrAB or related efflux pumps in A. tumefaciens.
Tannic acid significantly inhibited biofilm formation by A. tumefaciens (Figure 1), and previously was found to inhibit S. aureus biofilm formation in the presence of transglycosylase IsaA [46]. Tannic acid also effectively inhibited biofouling by P. aeruginosa [47]. These antibiofilm effects may be due to the high antioxidative capacity of tannic acid, as previous studies have shown that antioxidants inhibit biofilm formation by reducing the expressions of genes responsible for oxidative stress [48]. Furthermore, the EPS of A. tumefaciens accounts for up to 20% of its dry weight and importantly protects the bacterium from environmental stresses [49], and in the current study, tannic acid significantly and dose-dependently decreased the EPS secretion by A. tumefaciens from a concentration of 50 µg/mL ( Figure 5A).
A. tumefaciens can sense chemical signals released from plant wounds and uses its swimming motility to reach these sites [12]. Swarming and twitching motilities have not been reported for this genus, and thus, swimming motility is believed to be the only means of locomotion as reported [12] and ourselves [30]. In a previous study, tannic acid inhibited the swimming motility of C. violaceum (a quorum-sensing-positive bacteria) at concentrations of ≥30 µg/mL [50], and in the present study, tannic acid at 25 µg/mL reduced the swimming motility of A. tumefaciens ( Figure 5D,E). Tannic acid also impaired the other virulence traits of A. tumefaciens, namely protease activity and cell surface hydrophobicity ( Figure 5B,C), by dose-dependently decreasing them. Likewise, tannic acid has been reported to inhibit the productions of quorum-sensing (QS) molecules and to attenuate biofilm formation by E. coli [51] and Aeromonas hydrophila [52].
The 3D images of A. tumefaciens biofilms on polystyrene ( Figure 2) showed tannic acid dose-dependently reduced biofilm thicknesses, and this was in line with the near absence of biofilm formation on nitrocellulose membranes at a tannic acid concentration of 200 µg/mL as determined by SEM ( Figure 6D-F). Plausibly, this may have been due to the inhibitory effects of tannic acid on adherence to associated genes and EPS production. Furthermore, it has also been proposed that metal ion chelation by tannic acid might cause cells to burst and thus prevent biofilm formation [53]. The ten galloyl groups of tannic acid chelate iron [54], and this process has been shown to be correlated with the antioxidant activity of tannic acid and thus to the prevention of biofilm formation [48,55]. This iron-chelating strategy has also been proposed as a means of inhibiting the activities of diverse pathogens [56]. Heindl et al. [31] suggested the possible role of manganese and iron in the surface attachment and biofilm formation of A. tumefaciens. They used ethylenediamine-di-o-hydroxyphenyl acetic acid (EDDHA) or 2,2 -dipyridyl (DIP) at 20-1000 µM for iron chelation, and the relative biofilm formation was inhibited dose-dependently; however, the addition of 11-1100 µM FeSO 4 resumed the biofilm formation under the constant 200 µM concentration of either EDDHA or DIP [31]. Iron is crucially required for A. tumefaciens infection [57]; therefore, we assessed its role in the biofilm formation of A. tumefaciens. In our study, tannic acid at 100 and 200 µg/mL acted as the iron chelator similar to the EDTA (1 mM) (Figure 4). Tannic acid limited the iron supply required for the growth and biofilm formation of A. tumefaciens, and external supplication of iron by 0.05 mM Fe 2 SO 4 ·7H 2 O completely restored the biofilmforming potential of A. tumefaciens ( Figure 4B,C) growing with 100 or 200 µg/mL tannic acid. This suggests that tannic acid strongly chelates the iron and makes it unavailable for A. tumefaciens growth and significantly reduces its biofilm formation.
Tannic acid when present in the medium also inhibits the plant genetic transformation by A. tumefaciens. For example, varying levels of tannins released by roots of sorghum genotypes [58] have been suggested to be responsible for low A. tumefaciens inoculation efficiencies [59], which concurs with our SEM observations that tannic acid inhibited biofilm formation on the roots of R. sativus ( Figure 6G-L) and inhibition of tumor formation by A. tumefaciens on potato slices (Figure 7). This reduction can be corroborated by a recent study by Torres et al. [60], where tannic acid was applied with aminoethylcysteine (chemical treatment) or Pseudomonas protegens CHA0 (biological treatment) to tomato roots infected with A. tumefaciens C58 derivative C107-KmR RifR. These chemical or biological combinations significantly reduced the LogCFU g −1 of A. tumefaciens C58 in root colonization and relative abundance [60].
In the present study, qRT-PCR revealed slight downregulation of the dnaK gene, which oversees bacterial stress response to tannic acid [61]. The VirA/VirG two-component system of A. tumefaciens is a prerequisite for wounded plant infection and tumor genesis, and cheV protein (encoded by the chvE gene) causes a sugar-induced increase in the expression of vir genes [13]. Furthermore, chvE mutation can create an imbalance between sugar utilization and consumption, which suggests the gene plays a major role in the control of A. tumefaciens virulence [62]. He et al. [62] reported that tannic acid downregulated the expression of chvE (responsible for sugar-induced increase in virulence), which suggested tannic acid reduced virulence. In addition, tannic acid targeting may be dependent on strain type; for example, tannic acid significantly repressed biofilm formations by methicillin-sensitive or resistant S. aureus by targeting the ica operon and several other regulatory genes [23], and we found tannic acid significantly downregulated the biofilm regulating the periplasmic regulator exoR gene by >15-fold ( Figure 9). This gene appears to play a pivotal role in biofilm formation since exoR mutants failed to produce biofilms on polystyrene [39,63,64]. In addition, these mutants lost flagellar motility [63], which explained the reduced swimming motility of A. tumefaciens in the absence of any downregulation of genes directly governing motility. Moreover, ExoR may have multiple interacting partner proteins in periplasm and thus control other regulatory pathways [63].

Conclusions
This study demonstrates the polyphenol tannic acid efficiently prevents planktonic growth and biofilm formation of A. tumefaciens on abiotic (nitrocellulose and polystyrene) and plant root surfaces and suppresses virulence traits such as EPS secretion, protease activity, cell surface hydrophobicity, and swimming motility. Tannic acid remarkably inhibited biofilm formation on the surfaces of R. sativus roots without affecting R. sativus seed germination. One of the possible mechanisms of antibacterial and subsequent biofilm inhibition could be the chelation of iron by tannic acid due to the presence of galloyl groups. The other mechanism could be decreased expressions of dnaK and chvE, but significant downregulation of exoR, which codes for periplasmic succinoglycan-regulatory protein (a prerequisite of A. tumefaciens motility). This study improves the understanding of the interaction between tannic acid and A. tumefaciens. The results from this in vitro study are promising and pave the way for further research to assess the antipathogenic applications of tannic acid in agricultural settings with other plant species. Though pure tannic acid may inhibit the growth of other nontarget microbes; however, the possibility is low since some plants also release exudates having different tannins and still recruit beneficial microbes to the rhizosphere. Based on the results obtained, we propose that tannic acid be assessed for use in anti-Agrobacterium formulations under field conditions. Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/biomedicines10071619/s1, Table S1: Biofilm and virulence related genes of A. tumefaciens with their primer sequences used for qRT-PCR.