Introduction

The development of human agricultural civilization has a history of nearly 10,000 years [1]. However, a sharply increased demand in food production has attracted unprecedented attention as the growing world population makes it an important responsibility to protect crops from phytopathogenic bacteria [2,3,4]. Phytopathogenic bacteria cause enormous yield loss in various crops worldwide every year, particularly Xanthomonas oryzae PV. Oryzae (Xoo), Xanthomonas axonopodis PV. citri (Xac), and Pectobacterium atrosepticum (Pa) [5,6,27,28]. In addition, in the process of agribactericides discovery, the number of screening new active compounds increased significantly. Searching for active lead compounds from approved drugs and then carrying out structural modification or derivatization has been proved to be a successful way to find agribactericides with new action modes [29,30,31]. However, the discovery of lead compound remains a major challenge, The number of compounds rose from 52,500 in 1995 to 140,000 in 2005 to discover a new agrochemical lead compound [16, 32]. Thus, the lead compound is a prerequisite for the discovery of agrochemicals.

To this extent, we screened 700 approved drugs against Xoo, Xac and Pa. Among them, the structure-activity relationship and structural similarity analysis of active drugs classify potent agri-bactericides into 8 lead series: salicylanilides, cationic nitrogen-containing drugs, azole antifungals, N-containing group, hydroxyquinolines, piperazine, kinase inhibitor, and miscellaneous groups.

Materials and methods

Bacterial Strains and growth conditions

Xoo ACCC 11602 and Pa ACCC 19901 were purchased from the Agricultural Culture Collection of China (ACCC). Xac was provided by Professor Song Yang’s research group from Guizhou University. The bacteria were experienced the 16 S ribosome gene series alignment, the comparison results are provided in the Supporting Information.

The above strains containing 30% glycerol were frozen at – 80 °C in the laboratory. The frozen strains were taken out, scribed on nutrient broth (NB) solid media, culturing at 28 °C until a single colony grew. Then, a single colony was picked from the solid media to the nutrient broth (NB) media and cultured to the logarithmic growth phase at 28 °C on a shaker incubator at 180 rotations per min (rpm). The strain in the logarithmic growth phase was diluted with nutrient broth (NB) media to about 106 CFU ml−1 for later use.

The nutrient broth (NB) media: 3.0 g of beef extract, 5.0 g of peptone, 1.0 g of yeast extract, 10.0 g of sucrose, 8.0 g of sodium chloride, 1 L of distilled water, pH = 7.0 − 7.2.

Chemicals and compounds

All drugs or compounds were purchased from commercial suppliers and available without purification (unless stated otherwise). The above-tested drugs were dissolved in DMSO at concentrations of 100,000 μg ml−1 and stored at −4 °C or −20 °C. Then, to a 2 ml tube, 998 μl of nutrient broth (NB) media, 2 μl of the compounds dissolved in DMSO were added so that the final concentration is 200 μg ml−1 for later use.

In vitro antibacterial assay

Antibacterial activities of target drugs and compounds were tested against three phytopathogenic bacteria (Xoo, Xac, and Pa) using the turbidimetric method [3 and Fig. 4, the first azole series we investigated was 1-(phenylethyl)imidazole derivatives (lead series 6), active drugs of this lead series contained fenticonazole nitrate, miconazole, econazole, butoconazole nitrate (MIC90 ranged from 3.12 to 12.5 μg ml−1). The preliminary structure-activity relationships indicated that the substitution of benzyl contributed to increaseing the antibacterial activity, introduction of oxygen and sulfur atoms to form ethers could cause a more potent antibacterial effect. Among the 1-(phenylethyl)imidazole derivatives, the position of the halogen substituent on the benzene ring seemed to greatly improve the antibacterial activity, especially with 2,6-dichloro-substituted. Miconazole and econazole, a broad-spectrum imidazole fungicide, inhibit synthesis in fungal cell membranes and RNA, the screening and further confirmation revealed that miconazole and econazole were found to exhibit a considerable activity against Xoo (MIC90 = 12.5 μg ml−1). Nitroimidazoles and benzimidazoles are our second lead series of azoles (lead series 7), with triclabendazole being the standout for antibacterial activity(the MIC value was 6.25 μg ml−1 against Xoo and Xac). Interestingly, our screening identified the third azole series (lead series 8), simple-structured thiazolinones exhibit strong antibacterial activity. The substituent of thiazolinone affects the activity of drugs, methyl and chlorine decreased the activity 4-time (5-chloro-2-methylisothiazol-3(2H)-one), as compared to the unsubstituted thiazol-3-one. In addition, the introduction of a long chain into the nitrogen atom of thiazolinone does not indicate an increase or decrease in activity compared with thiazol-3-one. Therefore, nitrogen may not be the key factor affecting the anti-agribacterial activity. The benzothiazoles, 1,2-benzisothiazol-3(2H)-one, 2-methyl-1,2-benzothiazol-3(2H)-one, and 6-fluoro-1,2-benzisothiazol-3(2H)-one, show considerable activities, especially 6-fluoro-1,2-benzisothiazol-3(2H)-one with the substitution of fluorine on its phenyl rings, which may be accountable for the higher activity as a functional group.

Table 3 In vitro antibacterial activities (Inhibition rate/%) of the azole antifungals drugs against phytopathogenic bacteria
Full size table
Fig. 4
figure 4

The MICs of azole antifungals drugs (lead series 6–8) against phytopathogenic bacteria

Full size image

Hydroxyquinolines

Hydroxyquinolines are established to own wealthy biological activities and can be used as herbicides, disinfectants, preservatives, chemical intermediates, etc, that determines their wide application within the field of medication. Our research group previously conducted research on 8-hydroxyquinoline as metal chelators against agricultural fungi, and the results showed that this kind of compounds has excellent antifungal activity, revealing great potential as agricultural fungicides [41].

As shown in Table 4 and Fig. 5, the results of the screening experiments indicated that the quinoline derivatives with 8-hydroxyl group exhibited increased antibacterial activity at primary screening of 100 μg ml−1, compared with other positions of hydroxyl substitution, such as the 2, 5, 6 hydroxyl groups. With 8-hydroxyquinoline (lead series 9) as the skeleton, different group substitutions even have different antibacterial effects. When the NO2 on the 5-position is substituted, the activity of 8-hydroxyquinoline against three pathogenic bacteria is greatly improved, with the increased bactericide result against Xoo, Xac, Pa by 4, 16 and 8 times respectively (the MICs are 0.39, 6.25, 1.56). Substitution of Cl and Br at the 5-position produces a similar effect either. However, CH3 substitution did not appear to have a positive effect, even reduced activity against Xoo. In addition, 8-hydroxyquinoline bears two groups substituents at the 5 and 7 positions have less potential, especially 5,7-dibromo-8-hydroxyquinoline. Studies have shown that the ability of the 8-hydroxyquinoline scaffold to chelate divalent ions make this molecule an important fragment to interact with metalloproteins in microorganisms as targets, which may be the main reason for its antibacterial activities.

Table 4 In vitro antibacterial activities (Inhibition rate/%) of the hydroxyquinolines against phytopathogenic bacteria
Full size table
Fig. 5
figure 5

The MICs of Hydroxyquinoline (lead series 9) against phytopathogenic bacteria

Full size image

It is worth mentioning that the commercialized chloroquinadol, as one of the main components of the clinically used drug Ke**gbao, is well known for its anti-Candida albicans effect. In fact, our experiments show that its in vitro antibacterial activity against Xoo is even better than that against Candida albicans, with MIC of 0.39 μg ml−1 against Xoo and 0.12 μg ml−1 against Ca (The data were measured by us simultaneously). Overall, our repurposing of the commercially available drugs, 8-hydroxyquinoline, endows it with a broader application, is warrant further investigation within the area of controling phytopathogenic pathogens.

N-containing group

As shown in Table 5 and Fig. 6, N-containing group drugs were screened as lead series 10. The pharmacophore of these compounds includes amines, ureas and guanidines. Amines are nitrogenous aliphatic or heterocyclic substances with biological functions. **njunan is a precursor in the synthesis of Junduqing, a broad-spectrum bactericide which was successfully developed by China Shandong Chemical Development Center in 1989. It has been used for various crops to control agricultural diseases caused by fungi, bacteria and viruses for many years. **njunan has good antibacterial activity against three phytopathogenic bacteria in this screening experiment. In order to investigate the impact of amino groups on antibacterial activity, the activity of commercial fatty amines was tested, but these fatty amines have no antibacterial activity. which shows that the exposed amino group is not the active center. **njunan to a reasonable improvement of activity only when the bilateral amino groups are connected by a long aliphatic chain. In addition, compounds with urea and guanidine groups such as triclocarban and chlorhexidine acetate have been widely used in the field of medical sterilization and disinfection, which have broad-spectrum antimicrobial activity and are harmless in direct contact with the human skin. These N-containing groups as the hydrophilic head of these molecules contain strong positive charges and adsorb negatively charged bacterial cell membranes by electrostatic interaction. Our results suggest that these drugs have equal effect against plant bacteria.

Table 5 In vitro antibacterial activities (Inhibition rate/%) of the N-containing group against phytopathogenic bacteria
Full size table
Fig. 6
figure 6

The MICs of N-containing group (lead series 10) against phytopathogenic bacteria

Full size image

Piperazine

As shown in Table 6 and Fig. 7, the category discussion of lead series 11 was based on the presence of a central heterocyclic ring system containing at least one nitrogen atom (piperazine and piperidine groups). From the structure-activity point of view, the nature and position of the electron donating functional groups on the piperazine and piperidine core may contribute to the antibacterial activity. It is worth mentioning that Penfluridol, a commercial long-acting antipsychotic indicated for the maintenance treatment of chronic schizophrenia, has high antibacterial activity against Xoo and Xac with the MICs of 3.12 μg ml−1, providing a basis again for the strategy of drug-repurposing.

Table 6 In vitro antibacterial activities (Inhibition rate/%) of the piperazine against phytopathogenic bacteria
Full size table
Fig. 7
figure 7

The MICs of piperazine (lead series 11) against phytopathogenic bacteria

Full size image

Kinase inhibitors

Kinase inhibitors attracted much attention for a long time, owing to their significant role in the field of anti-tumor. However, bacterial growth processes are also affected by signal pathways. Hence, many studies have focused on the application of kinase inhibitors to the antibacterial field recently. For instance, Philipp Le found the anti-cancer drug Sorafenib showed high anti-bacterial activity against MRSA strains and did not induce in vitro resistance.

As shown in Table 7 and Fig. 8, among this established series (lead series 12), 4,4' -(dithiodicarbonothioyl)dimorpholine (JX06) is well known as a PDK inhibitor, which usually binds covalently to cysteine residues in an irreversible manner resulting in antitumor activity. In this study, we screened the 53 key kinase inhibitors led to the discovery of JX06 as a outstanding hit effectively killing the two specific plant pathogenic strains at concentrations of micromoles per milliliter. The MICs of JX06 were 6.25 and 12.5 μg ml−1 against Xoo and Xac respectively. Besides, Perifosine also has a similar effect (the MICs of 6.25 and 25 μg ml−1 against Xoo and Xac respectively), which may be the result of the combined effect of cation membranes permeability and certain signaling pathway regulation. Taken together, these results support the potential application of these kinase inhibitors with antibacterial activity for bacterial disease control in plants.

Table 7 In vitro antibacterial activities (Inhibition rate/%) of the kinase inhibitors against phytopathogenic bacteria
Full size table
Fig. 8
figure 8

The MICs of kinase inhibitors (lead series 12) against phytopathogenic bacteria

Full size image

Miscellaneous groups

As shown in Table 8 and Fig. 9, the last series (lead series 13) is some chemically dispersed drugs. Drugs which are conducted in this screen category are quinine, sulfa anti-inflammatory, nucleoside anticancer, cephalosporin antimicrobial and S-containing drugs which include thioether, mercaptan, disulfide drugs. Highly active anti-agribacterial drugs identified in the screening are listed by class. It was also attracted that the derivative of pyrithione (Zinc pyrithione, Sodium omadine, Copper pyritione, and Pyrion disulfide) has reasonable anti-phytopathogenic bacteria activity. In previous reports, Zinc pyrithione passed the increase in cellular zinc levels, decrease in lipase expression, and inhibition of mitochondrial function against M. restricta[42]; Bithionol exhibits bactericidal activity against both antibiotic-resistant S. aureus with its ability to pass through and embed in bacterial membranes lipid bilayers [43]. The anti-phytopathogenic bacteria activities of double phenol-containing drugs (Dichlorophen, Triclosan, and Bithionol) might be attributed to the anti-corrosion and weak acidity of the phenolic part. Among them, triclosan and dichlorophen have the strongest antibacterial activity, both drugs contain similar structure, the MIC90 ranged from 3.12 to 25 μg ml−1; Abafungin was found to have potentiality antifungal activity whether the pathogens are growing or resting [44]. The anti-phytopathogenic bacteria activity of these five drugs against Xoo, Xac, and Pa has never been reported and therefore is worth further exploration; Pleuromutilin is a broad-spectrum diterpene antibiotic produced by Pleurotus mutilus. It inhibits bacterial growth by disturbing bacterial protein synthesis. Retapamulin and valnemulin hydrochloride are based on pleuromutilin antibiotics. In this study, retapamulin and valnemulin show excellent anti-phytopathogenic bacterial activities against Xoo with a MIC of 6.25 and 0.78 μg ml−1. Although there is no necessary connection between the activity and structure of this group of drugs, these results provide a approach based structure screening for the repurposing of commercially available drugs, expecting to quicken the discovery of drugs against phytopathogenic bacteria.

Table 8 In vitro antibacterial activities (Inhibition rate/%) of the miscellaneous groups against phytopathogenic bacteria
Full size table
Fig. 9
figure 9

The MICs of miscellaneous groups (lead series 13) against phytopathogenic bacteria

Full size image

Risk

Although drug repurposing provides a rapid and efficient method to screen antibacterial leads from approved drugs, which are making a significant impact on the development of antimicrobial resistance (AMR) [45, 46]. When clinical drugs or other drugs are used as agrichemicals, it may provide new resistant strains and accelerate the development of AMR. The clinical drugs or other antimicrobial agents use in agriculture practice, particularly as agrichemicals used in the field, are one of the causes of the development of AMR. However, this risk is extending to humans through the food chain, the use of antimicrobial agents in food and agriculture has a direct or indirect impact on the development of antimicrobial resistance (AMR) in plant-associated bacteria [47]. For those reasons, alternative antimicrobials are also needed to combat the phenomenon of AMR in clinical settings and agricultural practices, such as in farms and food premises. To reduce or replace the use of common antibiotics, drug repurposing provides new lead compounds from the antibacterial screening of approved drugs, while also paying attention to the risks that exist in drug repurposing.

Conclusion

Our work provides a basis for drug discovery that enables the discovery of agricultural bacterial drugs superior to current traditional methods. Hopefully, we will enable the development of repurposed approved drugs to be effective against phytopathogenic bacteria. In addition to drug repurposing, approved drugs-with known well-documented safety, stability, and toxicological effects-can be used as new lead compounds.