SONG Ying-lian,LIU Hong-wu,YANG Yi-hong,HE Jing-jing,YANG Bin-xin,YANG Lin-li,ZHOU Xiang,LIU Li-wei,WANG Pei-yi,YANG Song

National Key Laboratory of Green Pesticide/Key Laboratory of Green Pesticide and Agricultural Bioengineering,Ministry of Education/Center for R&D of Fine Chemicals,Guizhou University,Guiyang 550025,P.R.China

Abstract

Natural products have long been a crucial source of,or provided inspiration for new agrochemical discovery.Naturally occurring 18β-glycyrrhetinic acid shows broad-spectrum bioactivities and is a potential skeleton for novel drug discovery.To extend the utility of 18β-glycyrrhetinic acid for agricultural uses,a series of novel 18β-glycyrrhetinic acid amide derivatives were prepared and evaluated for their antibacterial potency.Notably,compound 5k showed good antibacterial activity in vitro against Xanthomonas oryzae pv.oryzae (Xoo,EC50=3.64 mg L–1),and excellent protective activity (54.68%) against Xoo in vivo.Compound 5k induced excessive production and accumulation of reactive oxygen species in the tested pathogens,resulting in damaging the bacterial cell envelope.More interestingly,compound 5k could increase the activities of plant defense enzymes including catalase,superoxide dismutase,peroxidase,and phenylalanine ammonia lyase.Taken together,these enjoyable results suggested that designed compounds derived from 18β-glycyrrhetinic acid showed potential for controlling intractable plant bacterial diseases by disturbing the balance of the phytopathogen’s redox system and activating the plant defense system.

Keywords: 18β-glycyrrhetinic acid,antibacterial activities,defense enzyme activity,reactive oxygen species

1.Introduction

Plant pathogenic bacteria,which can cause numerous diseases for higher plants,and resulting in severely reducing the production and quality (Yangetal.2019).For instance,Pseudomonassyringaepv.actinidiae(Psa),Xanthomonasaxonopodispv.citri(Xac),andXanthomonasoryzaepv.oryzae(Xoo),as main pathogenic bacterium,have destructive effects on crops including kiwifruit,citrus and rice,respectively (Riganoetal.2007; Mansfieldetal.2012).Particularly,abominableXoocan infect rice to cause the bacterial leaf blight disease,and this typical disease widely distributes in many rice-growing areas of the world (Huangetal.1997).Currently,chemosynthetic pesticides (thiodiazole copper,TC; bismerthiazol,BT) and antibiotics (oxytetracycline and streptomycin) are the most powerful tools for controlling plant bacterial diseases.However,these traditional bactericides have potential risk to the environment,and even exist negative effects for the human beings through directly or indirectly transferring pesticide degradant originating from food,garden stuff,and drinking water (Yoonetal.2006; Bassiletal.2007; Iwaietal.2007; Sanbornetal.2007; Vidaetal.2007).More importantly,gradually increasing drug-resistant issues further restrict these pesticides’ control effect (Xuetal.2010; Zhuetal.2013; Panetal.2018).Therefore,development and excavation of new agrochemicals with high-efficiency,low-risk,broad-spectrum,and no crossresistance for controlling the abominable plant bacterial disease is an arduous task.

Fortunately,natural products hold numerous advantages,including structural diversification and versatility in bioactivities,and subsequently contribute to drug discovery for the pharmaceutical and agrochemical markets (Bakeretal.2007).Therefore,natural products-based drug discovery remains the most viable strategy for development of new drugs,at least for now.Glycyrrhetinic acid (GA) is isolated from the herbaceous plantGlycyrrhizauralensisFisch.ex DC.(liquorice root) or synthesized by hydrolysis of glycyrrhizic acid.In general,GA can be divided into the optical isomers 18α-GA and 18β-GA (Fig.1).Furthermore,as illustrated in Fig.2,previous studies indicate that 18β-GA is a dominant bioactive ingredient and shows diverse bioactivities,including antitumor,antiviral (such as compound 1),anti-inflammatory,and antibacterial activities (such as compound 2),as well as other therapeutic functions (liver protection,neuroprotection,and hyperglycemia therapy) (Isbrucker and Burdock 2006; Bodetetal.2008; Kalaiarasi and Pugalendi 2009; Wangetal.2011; Yamaguchietal.2012; Chenetal.2014; Wuetal.2015; Caietal.2016; Huang G Letal.2016; Huang L Retal.2016; Heetal.2018; Schmidetal.2018; Zígoloetal.2018; Chenetal.2020; Markovetal.2020; Lietal.2020; Yangetal.2020).Therefore,GA,as an important type of pentacyclic triterpenoid acid,has been widely investigated in the field of medicine,but to date has been rarely studied for agrochemical applications.

Fig.1 Structures of 18α- and 18β-glycyrrhetinic acid.

In our previous work,a series of 18β-GA ester derivatives were prepared and their antibacterial potency was evaluated.Notably,compound 3 displayed outstanding anti-Xooactivity (EC50=3.81 mg L–1).Further bioassays suggested that the derivative compounds possessed the ability to induce bacterial cell apoptosis and disrupt pathogenic bacterial cell morphology (Xiangetal.2019).As the primary active pharmacophore,amide units exist ubiquitously in many bioactive molecules,including fluopyram,tolfenpyrad,propanil,fenhexamid,isoflucypram,and compound 4 (Molina-Torresetal.2004).Therefore,from the perspective of novel agrochemical development,GA is a potential lead skeleton to alleviate the shortage of new bactericides.

In this study,we further explored the potential agricultural applications of 18β-GA.A series of 18β-GA amide derivatives (Fig.2) with an isopropanolamine moiety were prepared and their antibacterial potency was assessed.The structure-activity relationship (SAR) of target compounds was summarized to guide further efforts to design antibacterial agents with greater potency.In addition,the antibacterial behavior of target compounds was investigated by means of a series of biochemical assays.

2.Materials and methods

2.1.Instruments and chemicals

The NMR data (1H,13C and19F NMR) of target compounds were determined on a JEOL-500 NMR Spectrometer (JEOL Ltd.,Japan) or a Bruker Biospin AG-400 Spectrometer (Bruker Optics,Switzerland),meanwhile,TMS was used as an internal standard,and CDCl3was used as the solvent.In addition,the chemical shift (δ) was expressed in parts per million and the following abbreviations were utilized in expressing multiplicity: s=singlet,d=doublet,t=triplet,q=quartet and m=multiplet.The HRMS spectra were analyzed by Waters Xevo G2-S QTOF MS (Waters MS Technologies,Manchester,UK) apparatus.The melting points were detected by the SGW?X-4B Melting Point Apparatus (Shanghai Yidian Physical Optical Instrument Co.,Ltd.).OD values were detected using a Cytation?5 Multimode Readers (BioTek Instruments,Inc.,USA).TEM experiments were recorded on an FEI Talos F200C Microscope (Thermo Fisher Scientific,Massachusetts,USA).Fluorescence intensity and images were obtained with a FluoroMax?-4P Fluorescence Spectrophotometer (HORIBA Scientific,Paris,France) and an Olympus BX53 Microscope (Olympus,Japan),respectively.In addition,18β-GA was purchased by Bide Pharmatech Co.,Ltd.(Shanghai,China).All the chemicals were purchased from commercial sources.

2.2.Synthetic procedures of the target compounds

All title compounds were skillfully prepared through adopting previously reported methods (Zhouetal.2012).The details of the synthetic procedures and data (1H,13C,19F NMR data and HRMS data) of corresponding compounds were provided in the supporting information.

2.3.Experimental section

The experimental methods (such as antibacterial bioassaysinvitroandinvivo,and morphological changes ofXoocells),were investigated by our previously reported methods (Wangetal.2019; Zhouetal.2020,2021; Yangetal.2021).

2.4.Reactive oxygen species (ROS) detection assay

The experimental methods of ROS detection assay were conducted by our published protocol (Xiangetal.2020).Briefly,a ROS Assay Kit (GENMED Scientific Inc.,USA) was carried out to detect accumulation of ROS.First and foremost,Xoocells (OD595=0.6–0.8) were harvested by centrifugation (6 000 r min–1,5 min,4°C),and washed with phosphate buffer saline (PBS) buffer (10 mmol L–1,pH 7.2).Then,theseXoocells were re-suspended in PBS buffer (10 mmol L–1,pH 7.2),and subsequently treated with 5k at different concentrations (6.25,12.5,and 25.0 mg L–1) or dimethyl sulfoxide (DMSO) as the control sample for 12 h at 28°C.After that,these samples were centrifuged,and washed with PBS for three times.Finally,300 μL sample was incubated with 2.5 μL Genmed staining solution for 20 min at 28°C,further measured by FluoroMax?-4P Fluorescence Spectrophotometer and captured by an Olympus BX53 Microscope,respectively.

2.5.Defense enzyme activity assay

The defense enzyme activities of title compounds-treated plant samples were tested according to the published protocol by Jiangetal.(2020).In this study,we sampled and assessed the activities of defense enzymes (e.g.,catalase (CAT),phenylalnine ammonia lyase (PAL),superoxide dismutase (SOD),and peroxidase (POD)) for rice after inoculated withXoocells at different times.Briefly,rice leaves were pre-treated with title compound for 24 h.Then,these leaves were inoculated withXoocells for 1,3,5,7,and 9 days,and further selected to determine the activities of defense enzymes.Finally,defense enzyme activities including CAT,SOD,POD,and PAL,were determined and calculated according to the corresponding enzymes assay kits (Solarbio Life Sciences,China).

3.Results

3.1.Synthesis

Novel 18β-GA amide derivatives 5a–5w were skillfully synthesized by four steps and are illustrated in Fig.3.Briefly,the intermediate 4 was prepared by a series of reactions comprising esterification,condensation,and oxidation.The target compounds 5a–5w were generated by means of a ring-opening reaction of the epoxy framework with diverseN-containing scaffolds.The structures of the target compounds were verified by NMR spectroscopy and high-resolution mass spectrometry (HRMS).All data (1H NMR,13C NMR,19F NMR,and HRMS) and the synthetic procedures of target compounds were provided in the Appendices A–F.

3.2.Antibacterial bioassay

Preliminary results of anti-Xoo,anti-Xac,and anti-Psa bioassay in vitroPreliminary bioassay results (Table 1) revealed that most target compounds exhibited good antibacterial potency againstXooandXac.However,these compounds showed no anti-Psapotency at the concentrations of 50 and 100 mg L–1.Therefore,the EC50values of the target compounds towardsXooandXacwere further assessed by using serial dilution at the concentrations of 50,25,12.5,6.25,and 3.125 mg L–1.The corresponding EC50values were summarized in Table 2.

Table 1 Preliminary inhibitory effects of target compounds 5a–5w against Xoo,Xac,and Psa1)

Table 2 EC50 values of target compounds against Xoo and Xac in vitro

Structure–activity relationship analysisGiven these enjoyable results,part of the target compounds demonstrated excellent antibacterial activities and had EC50values less than 10 mg L–1.Therefore,we analyzed the effect of diverse nitrogen-containing units on the antibacterial capabilities.The corresponding results were summarized as follows: (1) Compared with compound 5a,which contained a piperazine group (anti-Xoo: EC50=48.6 mg L–1; anti-Xac: EC50＞100 mg L–1),most target compounds containing aN-substituted group displayed improved antibacterial potency,such as when the H atom at theN-position of the piperazine ring was replaced with –CH3(compound 5b,anti-Xoo: EC50=5.06 mg L–1; anti-Xac: EC50=9.56 mg L–1),–CH2CH3(compound 5c,anti-Xoo: EC50=4.69 mg L–1; anti-Xac: EC50=6.29 mg L–1),–CH(CH3)2(compound 5d,anti-Xoo: EC50=32.8 mg L–1; anti-Xac: EC50=17.2 mg L–1),and –C(CH3)3(compound 5e,anti-Xoo: EC50=31.1 mg L–1; anti-Xac: EC50=37.2 mg L–1).Thus,weak electrondonating groups (such as an alkyl group) or a small steric hindrance group at theN-position of the piperazine ring were beneficial to enhance antibacterial activity.(2) Introduction of phenyl or electron-withdrawing groups at the para-position of the piperazine ring were unfavorable to increase antibacterial potency; for instance,compound 5f (phenyl,EC50＞100 mg L–1forXooandXac),compound 5g (acetyl,anti-Xoo: EC50=53.3 mg L–1; anti-Xac: EC50＞100 mg L–1),and compound 5h (formyl,EC50＞100 mg L–1forXooandXac).(3) Compared with compound 5i (piperidyl,anti-Xoo: EC50=28.1 mg L–1; anti-Xac: EC50=58.2 mg L–1),placing an alkyl group on the piperidine ring recovered the anti-Xacactivity; for example,compound 5j (2-CH3,anti-Xoo: EC50=4.62 mg L–1; anti-Xac: EC50=61.2 mg L–1),compound 5k (3-CH3,anti-Xoo: EC50=3.64 mg L–1; anti-Xac: EC50=20.5 mg L–1),and compound 5l (4-CH3,anti-Xoo: EC50=11.0 mg L–1; anti-Xac: EC50=26.5 mg L–1).(4) Introduction of a hydroxyl group (compound 5m) to the piperidine ring sharply decreased anti-Xooactivity (EC50=46.4 mg L–1) and slightly increased anti-Xacactivity (EC50=18.4 mg L–1).However,adding a methylene group between the hydroxyl group and the piperidine ring recovered the antibacterial potency (compound 5n,anti-Xoo: EC50=5.65 mg L–1; anti-Xac: EC50=8.83 mg L–1).(5) With introduction of an ester group into the piperidine ring,compound 5o displayed moderate anti-Xooactivity (EC50=18.3 mg L–1) and weak anti-Xacactivity (EC50＞100 mg L–1).(6) Transformation of the piperidyl ring into a morpholinyl group (compound 5p) significantly decreased antibacterial activities (EC50＞100 mg L–1forXooandXac).(7) Notably,replacing the piperidine ring with a pyrrole ring (compound 5q) decreased anti-Xooactivity (EC50=39.7 mg L–1) and enhanced anti-Xacactivity (EC50=14.2 mg L–1).(8) Introduction of aliphatic amines into the target compounds (5r and 5s) significantly decreased antibacterial activities (EC50＞100 mg L–1forXooandXac).(9) Compared with aromatic amines (compound 5t,anti-Xoo: EC50=41.6 mg L–1; anti-Xac: EC50＞100 mg L–1),introduction of an electron-withdrawing group into the benzene ring,or introduction of a thiophene ring,sharply decreased antibacterial potency; for example,compound 5u (4-fluoro aniline group,EC50＞100 mg L–1forXooandXac),compound 5v (aniline group,EC50＞100 mg L–1forXooandXac),and compound 5w (thiophene ring,EC50＞100 mg L–1forXooandXac).Overall,target compounds containing piperazine and piperidine groups displayed high antibacterial potency.In particular,compound 5k with a 3-CH3piperidine group exhibited the strongest antibacterial potency towardsXoo,and compound 5c with a 1-ethylpiperazine-yl group displayed the strongest anti-Xacactivity.

Based on the aforementioned results,to verify whether the presence of the acetyl group preserved the bioactivities of the target compounds,the acetyl group of GA derivatives was removed,yielding compounds 6a and 6b (Fig.4).Bioassay results (Table 3) showed that the anti-Xoopotency of target compounds sharply decreased after removal of the acetyl group (6a,EC50=10.2 mg L–1; 6b,EC50=10.9 mg L–1),which were weaker than that of parent compounds 5c (EC50=4.69 mg L–1) and 5k (EC50=3.64 mg L–1).More interestingly,their anti-Xacactivities (6a,EC50=4.16 mg L-1; 6b,EC50=5.16 mg L–1) were increased,which were higher than those of compounds 5c (EC50=6.29 mg L–1) and 5k (EC50=20.5 mg L–1).In brief,the SAR analysis of title compounds was summarized in Fig.5.

Table 3 EC50 values of the modified molecules against Xoo and Xac in vitro

Fig.4 Synthesis for the modified molecules 6a and 6b.

Fig.5 Description diagram of structure–activity relationship (SAR) analysis.Xoo,Xanthomonas oryzae pv.oryzae; Xac,Xanthomonas axonopodis pv.citri.

3.3.In vivo bioassay for rice bacterial leaf blight

To access theinvivoanti-Xooactivity of compound 5k,a pot experiment was conducted to evaluate its potential application under a controllable greenhouse environment.The protocol of the anti-Xoobioassay followed that of our previous study (Wangetal.2020).The corresponding results were showed in Table 4 and Fig.6.Regarding curative efficiency,compound 5k displayed good control efficiency (50.54%),which was superior to that of BT (35.23%) and TC (40.75%).Compared with the curative efficiency,the protective efficiency of compound 5k exhibited stronger control efficiency (54.68%),which was superior to that of BT (36.19%) and TC (37.27%).

Table 4 In vivo control effiencies of 5k (200 mg L–1) against rice bacterial blight after spraying 14 days

Fig.6 Control efficancies of compounds 5k,bismerthiazol (BT),and thiodiazole copper (TC) towards rice bacterial blight under greenhouse conditions at 200 mg L–1.

3.4.Determination of ROS content and fluorescence imagines

To evaluate intracellular ROS accumulation inXoocells,the fluorescence generated after incubation with a nonfluorescent oxidation-reactive dye,6-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA),was used as an index of ROS accumulation.Compared with the control (0 mg L–1),a gradual increase in fluorescence intensity was observed with increase in concentration (6.25,12.5,and 25 mg L–1) of compound 5k (Fig.7).These findings indicated that intracellular ROS accumulation ofXoocells may be significantly increased by supplementation with the target compound 5k.To confirm these results,CM-H2DCFDA-treatedXoocells were observed with an Olympus BX53 microscope after incubation with compound 5k.As expected,gradual increase in the fluorescence intensity of CMH2DCFDA-treatedXoocells was observed with increase in concentration (6.25,12.5,and 25 mg L–1) of compound 5k (Fig.8).These results suggested that compound 5k might interfere with the balance of the redox system ofXoocells,stimulate excessive ROS generation,and thereby lead to death of the bacterial cells.

Fig.7 The accumulation of reactive oxygen species (ROS) in Xanthomonas oryzae pv.oryzae (Xoo) cells after incubation with various dosages of 5k,respectively.λex (excitation wavelength)=490 nm.

Fig.8 Fluorescence images of Xanthomonas oryzae pv.oryzae (Xoo) cells stained by the nonfluorescent oxidation-responsive dye CM-H2DCFDA after incobation with compound 5k at various dosages of 0 mg L–1 (A),6.25 mg L–1 (B),12.5 mg L–1 (C),and 25.0 mg L–1 (D),respectively.Scale bars are 10 μm.

3.5.Morphological changes of Xoo cells

Reactive oxygen species cause damage to cellular components,including fatty acids and proteins,and may lead to death of bacterial cells (Belenkyetal.2015).We observed the morphological changes ofXoocells after incubation with compound 5k.An intact cell envelope and well-proportionedXoocells were observed in the control group (Fig.9-A).After co-incubation ofXoocells with different doses (6.25 or 12.5 mg L–1) of compound 5k,the cell envelope was wrinkled and disrupted.InXoocells treated with compound 5k at the dosage of 25 mg L–1,pores were observed in the cell envelope.

Fig.9 Morphology study of Xanthomonas oryzae pv.oryzae (Xoo) after incobation with compound 5k at various dosages of 0 mg L–1 (A),6.25 mg L–1 (B),12.5 mg L–1 (C),and 25.0 mg L–1 (D),respectively.Scale bars are 2 μm.

3.6.Determination of defensive enzyme activities

The enzymes CAT,POD,SOD,and PAL,as crucial defensive enzymes in plants,enable host to withstand oxidative stress caused by harsh environmental stimuli,and are involved in defense responses against attack from phytopathogens (Khareetal.2018).The anti-Xoobioassaysinvivoindicated that compound 5k had excellent protective efficiency of 54.68%.Therefore,utilizing knowledge- and experience-based approaches,the effect of compound 5k on plant defense enzyme activity in rice was assessed (Fig.10).

Catalase directly catalyzes H2O2decomposition to yield H2O and O2(Apel and Hirt 2004).The CAT activities of compound 5k-treated samples peaked at day 3 (8 486 U g–1),which was superior to that of TC (7 798 U g–1) and the control (5 007 U g–1) (Fig.10-A).Thereafter,the CAT activities of compound 5k-treated samples remained stable from day 3 to 7.Superoxide dismutase is an antioxidant enzyme that catalyzes the decomposition of superoxide anions into H2O2and O2(Apel and Hirt 2004).The SOD activities of compound 5k-treated samples gradually increased and peaked at 219 U g–1on day 5,which was superior to those of TC (165 U g–1,day 5) and the control (152 U g–1,day 5).Thereafter,in the compound 5k-treated samples,SOD activity decreased to 120 U g–1on day 9 (Fig.10-B).Peroxidase is a peroxide scavenger that eliminates excess H2O2in plants.The POD activities of compound 5k-treated samples gradually increased from day 1 to 3,and slightly decreased on day 5.Interestingly,compound 5k-treated samples attained the highest POD activity of 89 327 U g–1on day 9,which was greater than those of TC and the control (52 871 and 53 361 U g–1,respectively).The trend for POD activities of the compound 5k-treated samples were consistent with a previous report (Heetal.2019).In addition,phenylalanine ammonia lyase is an important participant in the biosynthesis pathway of certain natural plant products originating from the main chain of phenylpropanoids (particularly salicylic acid),and was among the earliest “defense genes” to be identified in plants (Lawton and Lamb 1987; Appertetal.1994; Diallinas and Kanellis 1994; Dixon and Pavia N L 1995; Sanchez-Ballestaetal.2000).The PAL activities of compound 5k-treated samples gradually increased and peaked on day 5 (31 U g–1),which were superior to those of TC (25 U g–1) and the control (21 U g–1).Subsequently,in compound 5k-treated samples,PAL activities declined on days 7 and 9 (27 and 26 U g–1,respectively).

4.Discussion

4.1.In vitro and in vivo bioassay results

Altogether 25 novel 18β-GA amide derivatives were skillfully prepared,and their antibacterial activities were assayed.Several compounds displayed outstanding antibacterial activities compared to the parent 18β-GA,were EC50values＜10 mg L–1towardsXooandXac.Notably,the introduction of 3-CH3piperidine group at acid group of parent 18β-GA resulted in highly potent derivatives (compound 5k) againstXoowith value of 3.64 mg L–1,which was 7 times higher than commercial BT (132 mg L–1).And when introducing 1-ethylpiperazine-yl group into parent 18β-GA,compound 5c was obtained,and had the strongest anti-Xacactivity with value of 6.29 mg L–1,which were superior to BT (132 mg L–1) and TC (78.3 mg L–1),respectively.Most interestingly,although the hydroxyl group of title compounds was unfavorable for anti-Xooactivity,but was beneficial for anti-Xacactivity.These activity results were the enjoyable evidence to prove our design hypothesis,and consistent with our previous results (Wangetal.2020).In addition,pot experiments demonstrated that compound 5k exhibited good curative efficiency (50.54%) and excellent protective efficiency (54.68%) against rice bacterial blight at the concentration of 200 mg L–1,which were superior to those of BT (35.23 and 36.19%,respectively) and TC (40.75 and 37.27%,respectively).

4.2.ROS accumulation in Xoo triggered by compound 5k

Following the ROS detection assay,treatment with compound 5k substantially increased fluorescence intensity.Therefore,compound 5k could interfere with the redox system balance inXoocells.Moreover,after treatment with compound 5k,someXoocells had incomplete cell envelope,leading to intracellular component leakage.Combining above-mentioned results,compound 5k had a notable capability to kill bacteria through disrupting the balance of the redox system ofXoocells,and damaging the cell envelope.

4.3.Compound 5k-regulated plant defense responses

Encouraged by the excellent protective efficiency of compound 5k,the induce resistance mechanism of plant was preferentially verified by defense enzyme activity assay.The enzymes (CAT,POD,and SOD) act synergistically to equilibrate free radicals at a relatively low concentration,and subsequently alleviate the harm of excess ROS accumulation triggered by phytopathogens (Apel and Hirt 2004).Given the defense enzyme activity results,CAT and SOD of plant could be improved at an early stage after treatment with compound 5k.When free radical was excessive and harmful in rice,POD activity was further increased to eliminate excessive free radicals,and CAT and SOD activities were reduced.Therefore,compound 5k could regulate plant defense responses to control plant diseases.

5.Conclusion

In this study,we further extend the utility of 18β-GA for agricultural uses.Notably,the compound 5k displayed the strongest antibacterial activityinvitroandinvivo.Antibacterial mechanism suggested that compound 5k not only stimulated excess ROS generation and damaged to the cell envelope,but also improved defense enzyme activities to enhance the resistance of rice to bacterial leaf blight.Taken together,the present results demonstrated that the designed compounds derived from natural 18β-GA showed potential for controlling intractable plant bacterial diseases.

Acknowledgements

We greatly appreciate the fundings provided by the National Natural Science Foundation of China (21877021 and 32160661),the Guizhou Provincial S&T Program [(2018)4007],and the Program of Introducing Talents of Discipline to Universities of China (D20023,111 Program).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on https://doi.org/10.1016/j.jia.2022.10.009