Ginger extract against Ralstonia solanacearum -

Ginger extract against Ralstonia solanacearum

The current study aimed to determine the chemical compositions of ginger extract (GE) and to assess the antibacterial activities of GE against the ginger bacterial wilt pathogen Ralstonia solanacearum and to screen their mechanisms of action. The minimum inhibitory concentration and minimum bactericidal concentration of R. solanacearum were 3.91 and 125mg/ml, respectively. The cell membrane permeability and integrity of R. solanacearum were destroyed by GE, resulting in cell content leakage, such as electrolytes, nucleic acids, proteins, extracellular adenosine triphosphate and exopoly saccharides. Further experiments demonstrated that GE delayed or slowed the occurrence of bacterial wilt on ginger. GE has a significant antibacterial effect on R. solanacearum, and the antibacterial effect is concentration dependent. The GE treatments changed the morphology, destroyed membrane permeability and integrity, reduced key enzyme activity and inhibit the synthesis of the virulence factor EPS of R. solanacearum. GE significantly controlled the bacterial wilt of ginger during infection. This research provides insight into the antimicrobial mechanism of GE against R. solanacearum, which will open a new application field for GE.

Materials and Methods

Bacterial strains and fresh ginger rhizomes were collected and cultured in Nutrient Broth (NB, AOBOX) at 30°C with shaking conditions (200 rev/min) for 24h to reach the logarithmic growth phase.

Preparation of GE Rhizomes were cut into small pieces and dried to constant weight at 65°C and ground into power. Then passed through 40-mesh sieve to get powder size of 0.42mm and stored in an air-tight container at 4°C. Then 5 g ginger rhizome powder was extracted using ethanol in a Soxhlet apparatus for 8 h at 85°C. The extract was concentrated by evaporating and then dissolved with 5 ml of 50% ethanol; the mixture was centrifuged at 5000 g for 20min. The supernatant was filtered by 0.22μm membrane and a yellow product was obtained as GE, the mother liquor concentration of GE is 1 g/ml (1 ml 50% ethanol contains the extract extracted from 1 g ginger rhizome powder)

UPLC-MS/MS analysis of GE A total of 0.3 ml of 70% methanol was added to 0.3 ml of GEs and vortexed for 5 min. Then centrifuging at 12,000g for 10 min, and then stored in a sample bottle. The sample extracts were analysed using UPLC-ESI-MS/MS system.

Antimicrobial activity of GE Oxford cup methods were used to investigate the antimicrobial ability of GE. A total of 0.1 ml R. solanacearum (107 CFU per ml) bacterial solution was added to the surface of solid Nutrient Agar (NA, AOBOX) medium plates (60×10mm) and spread evenly with Lshaped glass rod. Sterilized Oxford cups (Ф 6mm) were placed on the agar medium and filled with 200μl of extracted GE, and 1.6% ethanol (same as the final concentration of ethanol in 5MIC GE) and sterile water were used as controls, respectively. Then the plates were incubated at 30°C for 24 h. The antibacterial activity was evaluated by the diameter of transparent circles around oxford cups.

Determination of MIC and minimum bactericidal concentration GE was diluted to a concentration of 1000.00, 500.00, 250.00, 125.00, 62.50, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, and 0 mg/ml with NB medium. Then the 5 ml of R. solanacearum (107 CFU per ml) solution was centrifuged at 5000 g for 3 min, the resulting pellets were re-suspended with different concentrations of GE, then incubating at 30°C (200 revmin−1) for 12 h. The minimum concentration of GE that inhibits the visible bacteria growth is defined as minimum inhibitory concentration (MIC). The minimum concentration of GE that leads to the complete absence of colonies growth on the agar surface is defined as minimum bactericidal concentration.

Cell membrane permeability analysis, relative electric conductivity, the permeability of bacteria membrane is represented by the relative electric conductivity. Ralstonia solanacearum (107 CFU per ml) culture was centrifuged at 5000 g for 3 min. The resulting pellets were re-suspended with 5% of glucose, and they were used as isotonic bacteria. GE at different concentrations (1MIC, 2MIC, 3MIC, 4MIC and 5MIC) was added to 5% glucose and the electric conductivities of the mixtures were marked as L1. Then the isotonic bacteria solutions were added into different concentrations of GE and the control (5% glucose), respectively. After being mixed completely, the samples were incubated at 30°C for 5 h, and then the conductivities were measured and marked as L2. The conductivity of isotonic bacteria treated with boiling water for 5 min was marked as L0. The permeability of bacteria membrane is calculated according to the formula,

the relative electric conductivity (%) = (L2−L1)/L0×100.

Leakage of DNA The content of DNA released by the bacteria was detected according to Dong et al. (2020). Ralstonia solanacearum (107 CFU per ml) was centrifuged at 5000 g for 3 min. After washing with phosphate buffered solution (PBS, 0.1 M, pH 7.2), the pellet was re-suspended in GE (1MIC, 2MIC, 3MIC, 4MIC and 5MIC), and cultured in 30°C at 200 revmin−1 for 5 h. The treated bacterial suspension was centrifuged at 5000 g for 10 min, 3 ml supernatant was collected and detected by spectrophotometry at 260nm.

Protein content of enzyme extracts was determined according to Coomassie brilliant blue method. The 100μl supernatant was collected and detected by spectrophotometry at 595nm.

The cell membrane integrity of GE treated bacterial was determined by fluorescent probe PI (propidium iodide) analysis. The R. solanacearum (5 ml) was centrifuged at 5000 g for 5 min and the obtained pellet was resuspended in 5 ml GE (1 MIC and 5MIC), respectively. After being cultivated for 5 h at 30°C at 200 rev min−1, the bacterial suspension was centrifuged at 5000 g for 10 min and the precipitated bacteria were washed with PBS (0.1 M, pH 7.2). And 1 ml of cell suspension solution (107 CFU per ml) was stained with 3 μl PI (5 mM) in the dark for 20 min. Then prepared samples were further analysed using fluorescence microscopy (Ni-U, NIKON) with an excitation wavelength of 546 nm.

Cellular SDH and AKP activity The R. solanacearum (5 ml) was centrifuged at 5000 g for 5 min and the obtained pellet was re-suspended in 5 ml GE (1MIC, 2MIC, 3MIC, 4MIC and 5MIC), then culturing at 30°C for 5 h and centrifuged at 5000g, respectively. The pellet was re-suspended in 500μl muramidase (10 mg/ ml) and 500μl TE (Tris-EDTA) buffer for 20min at 30°C. Then, the samples were ultrasound in the ice bath for 5 min (200W, interval 1.1 s), and centrifuged at 8000 g for 5 min. The supernatant was collected to determine SDH and ALP activity using assay kit by measuring the absorbance at 600 and 660nm, respectively

The R. solanacearum (5 ml) was centrifuged and the obtained pellet was re-suspended in 5 ml GE (1MIC, 2MIC, 3MIC, 4MIC and 5MIC), then incubated at 30°C for 5 h. The supernatants obtained by centrifuge were used as the extracellular components. And the pellet was incubated collected, divided and then centrifuged as mentioned above, with the supernatants used as the intracellular components. The concentrations of intracellular and extracellular ATP and EPS were measured by the ATP and EPS assay kit by measuring absorbance values at 636nm using microplate spectrophotometer. Scanning electron microscopy analysis For scanning electron microscope (SEM) analysis, R. solanacearum (5 ml) was prepared by the same method as mentioned above and re-suspended in 5 ml GE (1 MIC and 5MIC), respectively. After being cultivated for 5 h at 30°C at 200 rev/min, the bacterial suspension was centrifuged at 5000 g for 10 min and the precipitated bacteria were washed by PBS (0.1 M, pH 7.2). Then cells were fixed with 2.5% glutaraldehyde for 12h at 4°C, then post-fixed in 1% osmium tetroxide at 4°C for 2 h. After being dehydrated in a series of ethanol solutions and permeated in white resin, the cells were dried by vacuum freeze dryer for 24h and coated with gold. The morphologies of R. solanacearum cells were observed using SEM.

Controlling ginger bacterial wilt by GE The uniformly grown 6-week-old ginger seedlings were selected to survey the effects of GE on ginger bacterial wilt. A total of 20ml of 1MIC GE solution were irrigated evenly into ginger root soil, without GE solution was set as the blank control. Then 1 ml R. solanacearum (107 CFU per ml) bacterial solution was injected into the ginger rhizomes with syringe. Twelve ginger seedlings were carried out for each treatment, and each treatment was replicated three times. The inoculated ginger seedlings were cultivated under greenhouse at a temperature of 30°C, relative humidity of 85% and light period of 12 h. Following the inoculation of R. solanacearum, the disease occurrence of the ginger seedlings and disease index were investigated at 3days since the observation of first bacterial wilt plant. Bacterial wilt disease classification of ginger plants was evaluated.

Scanning electron microscope (SEM) analysis, R. solanacearum (5 ml) was prepared by the same method as mentioned above and re-suspended in 5 ml GE (1 MIC and 5MIC), respectively. After being cultivated for 5 h at 30°C at 200 rev/min, the bacterial suspension was centrifuged at 5000 g for 10 min and the precipitated bacteria were washed by PBS (0.1 M, pH 7.2). Then cells were fixed with 2.5% glutaraldehyde for 12h at 4°C, then post-fixed in 1% osmium tetroxide at 4°C for 2 h. After being dehydrated in a series of ethanol solutions and permeated in white resin, the cells were dried by vacuum freeze dryer for 24h and coated with gold. The morphologies of R. solanacearum cells were observed using SEM. Controlling ginger bacterial wilt by GE The uniformly grown 6-week-old ginger seedlings were selected to survey the effects of GE on ginger bacterial wilt. A total of 20ml of 1MIC GE solution were irrigated evenly into ginger root soil, without GE solution was set as the blank control. Then 1 ml R. solanacearum (107 CFU per ml) bacterial solution was injected into the ginger rhizomes with syringe. Twelve ginger seedlings were carried out for each treatment, and each treatment was replicated three times. The inoculated ginger seedlings were cultivated under greenhouse at a temperature of 30°C, relative humidity of 85% and light period of 12 h. Following the inoculation of R. solanacearum, the disease occurrence of the ginger seedlings and disease index were investigated at 3days since the observation of first bacterial wilt plant. The disease incidence, disease index and control efficacy were calculated using Equations as follows: In addition, the ginger rhizomes were cut in half and wounded (2mm deep and 2mm wide) with a sterile nail at the equator, then 5 μl R. solanacearum (107 CFU per ml) bacterial solution was added to wound, without R. solanacearum solution was set as the blank control. The treated ginger rhizomes were put in plastic boxes with sterile water to maintain a high relative humidity and stored at 28°C.

Results

UPLC-MS/MS analysis of GE The chemical compositions of GEs were analysed by UPLC and MS/MS, and a total of 393 compounds that were classified into 26 components were identified, including sesquiterpenoids (3.35%), monoterpenoids (0.39%), chalcones (0.57%), phenolic acids (46.29%), flavonoid (0.81%), flavonols (0.41%), quinones (3.93%), lignans (1.84%), triterpene (1.41%), alkaloids (17.67%), coumarins (0.25%), isoflavones (2.04%), plumerane (0.19%), stilbene (0.25%) and others (20.28%). The analysis of GEs showed that phenolic acids and alkaloids were major components.

In this study, the antibacterial effect of GE against R. solanacearum was examined. The inhibition zones were observed around the Oxford cup when GE (1000.00mg/ml) infiltrated the medium and the average diameter of the inhibition zones was 23.26±0.16mm. While 1.6% ethanol and sterile water had no inhibitory activity on R. solanacearum, no inhibition zones were detected when 1.6% ethanol and sterile water were added. The MIC of GE against R. solanacearum was 3.91mg/ml and the MBC was 125mg/ml, which demonstrated the high inhibitory activity of GE against R. solanacearum.

The permeability of the cell membrane was determined by measuring the relative electric conductivity and cell constituents (DNA and protein). Compared to the control (0 MIC), after the addition of 1MIC, 2MIC, 3MIC, 4MIC and 5MIC GE for 5 h, the relative electric conductivity and the proteins and DNA release increased with the increased GE concentration. The relative electrical conductivity increased by 27.7%, 94.7%, 167.3%, 599.6% and 1507.0% with the addition of 1MIC, 2MIC, 3MIC, 4MIC and 5MIC GE, respectively. The protein release increased by 69.1%, 87.2%, 103.6%, 122.7% and 258.2% with the addition of 1MIC, 2MIC, 3MIC, 4MIC and 5MIC GE, respectively. Correspondingly, the DNA release increased by 40.9%, 125.0%, 174.6%, 273.2% and 557.7%.

Fluorescence micrographs of R. solanacearum cells treated with different concentrations of GE (0MIC, 1MIC and 5MIC) for 3 h. However, the PI fluorescence intensity of R. solanacearum cells increased with increased concentrations of GE, suggesting that cell membrane integrity was damaged. With the addition of 1MIC and 5MIC GE, 5.2% and 81.4% of cells, respectively, demonstrated red fluorescence.

The activities of SDH declined by 21.7%, 39.1%, 60.8%, 71.7% and 87.0% with the addition of 1MIC, 2MIC, 3MIC, 4MIC and 5MIC GE, respectively. The activities of AKP decreased by 20.8%, 27.7%, 43.3%, 55.8% and 72.7%, respectively. These results indicated that GE could inhibit the activity of the endoenzymes, which are important for the growth of bacteria; as the concentration increased, the inhibition became stronger.

Determination of intracellular and extracellular ATP and EPS content compared with the control (0MIC), different concentrations of GE (1MIC, 2MIC, 3MIC, 4MIC and 5MIC) decreased intracellular ATP and EPS content but increased extracellular ATP and EPS content. Compared with 0 MIC, the intracellular ATP content of R. solanacearum treated with 1MIC, 2MIC, 3MIC, 4MIC and 5MIC GE declined by 9.6%, 14.0%, 34.5%, 40.7% and 46.7%, respective and the extracellular ATP content increased 5.0-, 13.0-, 39.0-, 61.3- and 76.6-fold, respectively. The intracellular EPS content of R. solanacearum treated with 1MIC, 2MIC, 3MIC, 4MIC and 5MIC GE decreased by 6.9%, 13.6%, 18.3%, 32.2% and 45.6%, respectively and the extracellular EPS content increased 88.4%, 183.7%, 202.3%, 260.5% and 272.1%, respectively. Furthermore, the decreased concentration of intracellular EPS is much greater than the increased concentration of extracellular EPS.

Scanning electron microscopy analysis of ultrastructure changes in R. solanacearum cells SEM was used to observe the ultrastructure changes in R. solanacearum cells with and without the addition of GE (0MIC, 1MIC and 5MIC). With the addition of 1MIC GE, the membrane surfaces showed folds and holes, and cells are stacked and adhered. With the addition of 5MIC GE, R. solanacearum cells showed obvious morphological changes. The SEM observation confirmed that GE induced severe physical damage and considerable morphological alterations to R. solanacearum. The alterations may be due to GE-enhanced cell permeability and destabilization of membrane integrity.

Control efficacy of GE on bacterial wilt on ginger

The results were recorded as disease incidence, disease index and control efficacy after the inoculation of R. solanacearum. The disease incidence and disease index in the control and GE-treated plants were 15.8, 11.0, 50.8, 99.2 and 0, 9.2, 19.2 and 65.8 at 5, 10, 15 and 20days after inoculation, respectively. The control efficiency of GE on ginger bacterial wilt was 0%, 30.8%, 69.7% and 10.2% at 5, 10, 15 and 20days after inoculation, respectively. The emergence of wilting symptoms was markedly delayed, and disease development was slower in GE-treated plants than in control plants. However, in vitro inoculation of the rhizome with directly R. solanacearum showed that ginger rhizomes had no obvious decay symptoms after 5days as compared with control.

Overall, this study found that GE has a significant antibacterial effect on R. solanacearum, and the antibacterial effect is concentration-dependent. GE can exert inhibitory effects on R. solanacearum in several ways. First, the GE treatments changed the morphology of R. solanacearum cells, with some of the cell walls becoming blurred and cytoplasmic content leakage. Secondly, the relative electric conductivity, losses of DNA and proteins of the suspension, and the fraction of R. solanacearum cells with PI fluorescence increased with increasing treatment time and concentration of GE, which verified that GE has a destructive effect on R. solanacearum membrane permeability and integrity. In addition, GE could lower key enzyme activity and effectively inhibit the synthesis of the virulence factor EPS. Furthermore, pot experiments demonstrated that GE can significantly control the bacterial wilt of ginger during infection.

Reference:

Zhang, L., Qin, M., Yin, J., Liu, X., Zhou, J., Zhu, Y. and Liu, Y., 2022. Antibacterial activity and mechanism of ginger extract against Ralstonia solanacearum. Journal of Applied Microbiology, 133(4), pp.2642-2654.