Plant material
Glycyrrhiza uralensis seeds were collected from wild G. uralensis plants in Urad front flag (40°72′N; 108°65′E), Inner Mongolia, China, in September 2019. Healthy seeds were selected and stored in kraft paper bag at 4 °C until use.
Fungal phytopathogens
The five fungal phytopathogens used in this study, Phyllosticta sp., Fusarium acuminatum, Botrytis cinerea Pers., Fusarium oxysporum Sacc and Scutellariae botrytis, were all provided by Associate Professor Cuiyun Zeng, Gansu University of Chinese Medicine. They were isolated from Dioscorea oppositifolia L., Anemarrhena asphodeloides, Radix Scutellariae, Cucumis sativus L. and Angelica sinensis, respectively, and maintained as 50% (v/v) glycerol at − 70 °C for further use.
Isolation of endophytic bacteria
Glycyrrhiza uralensis seeds were steeped with 85% (v/v) H2SO4 for 45 min, sterilized with 0.1% (v/v) H2O2 for 10 min, rinsed in distilled water for 3 times and soaked in distilled water for more than 3 h at room temperature (Zhang et al. 2020b). Seeds were surface sterilized by stepwise washing in 75% (v/v) ethanol for 5 min, 30 s wash in 1% (v/v) H2O2, 10 s wash in 5% (v/v) NaClO and subsequent rinsing in sterile distilled water for ten times. To evaluate the success of sterilization, 100 μl of the water from the last rinse was inoculated on NA medium (nutrient agar medium; beef extract 3.0 g, proteose peptone 10.0 g, NaCl 5.0 g, agar 20 g, d.H2O 1 l, pH 7.2) at 28 °C for 2–7 days. No microbial growth on NA medium indicated that surface sterilization was efficient. The surface-sterilized seeds were ground in a mortar and diluted up to 10 ml in sterile water. Tissue particles were left for 30 min at 4 °C, and the obtained suspension (200 µl) was spread onto NA medium. After culturing for 2–7 days at 28 °C, single colonies were picked and purified by repeated streaking and microscopic examination. The pure cultures were maintained in NA medium slants at 4 °C and as 50% (v/v) glycerol at − 80 °C for further use.
In vitro antagonistic activity
Dual culture assay
In vitro antagonistic activity of bacterial isolates was evaluated by dual culture method on PDA medium (potato dextrose agar medium; potato infusion 200 g, dextrose 20 g, agar 20 g, d.H2O 1 l). A 6-mm mycelial plug was removed from an actively growing plate of each fungal phytopathogen and placed on the center of a fresh PDA medium plate (90 mm in diameter). Approximately a 1.5 cm from the plug, a 2-day-old bacterial isolate was streaked on opposite sides of the mycelial plug. Control group plates inoculated only with each fungal phytopathogen. Dual culture plates and control plates were incubated at 28 °C up to 5–7 days. The percentage of inhibition was calculated by following formula:
$${\text{Inhibition }}\left( \% \right) \, = \, \left( {R_{1} - R_{2} } \right) \, / \, R_{1} \times \, 100$$
where R1 is the radius of control plate and R2 is the radius of dual culture plate. The experiment was repeated 3 times with 3 independent replications.
A bacterial isolate with the highest percentage of inhibition against 5 fungal phytopathogens was evaluated using the method described above and selected for all subsequent studies. The experiment was repeated 3 times with 3 independent replications.
Detached root assay against F. acuminatum
Angelica sinensis is an herbaceous perennial plant with high medicinal value. Root rot, caused by fungi pathogens such as Fusarium spp., is a ubiquitous disease that seriously harms the output and quality of A. sinensis (Mi et al. 2017). The bacterial isolate FT2 showed the strongest antagonistic activity in the dual culture assay, and thus, detached root assay was performed to evaluate the antagonistic activity of FT2 against A. sinensis root rot caused by F. acuminatum.
Fusarium acuminatum was activated on PDA medium and cultured in the dark at 22 °C for 7 days. The spores were washed with sterile water and made into a suspension of 106 cfu/ml (Mi et al. 2017). The bacterial isolate FT2 was incubated in NA liquid medium at 28 °C for 48 h. The resultant bacterial culture was centrifuged (10 min at 9000 rpm) in sterile distilled water and then re-suspended in sterile distilled water to a final OD600 of 1.0, which is approximately to a final concentration of 108 cfu/ml.
For surface sterilization, the healthy roots of A. sinensis were washed with running water for 30 min, soaked in 70% (v/v) ethanol for 30 s and then in 3% (v/v) sodium hypochlorite for 3 min and washed with sterile distilled water for 5 times. The surface-treated roots were cut into 5 mm slices with a sterile scalpel and placed on plates containing filter paper moistened with 1 ml sterilized water (Mi et al. 2017). Five treatments were included: (i) Control, roots slices were inoculated with sterilized water only; (ii) Fa only, roots slices were inoculated with F. acuminatum only; (iii) FT2 only, roots slices were inoculated with the bacterial isolate FT2 only; (iv) Fa + FT2, roots slices were inoculated with a mixture of the bacterial isolate FT2 and F. acuminatum; and (v) Fa + Pre-FT2, roots slices were pre-inoculated with the bacterial isolate FT2, followed by inoculation of F. acuminatum after 24 h of incubation. A 40 µl spore suspension or bacterial suspension was applied to the roots slices, and control was treated with the same amount of distilled water and then incubation at 22 °C, 50% RH for 3 days. There were 4 slices per plate and 3 plates for each treatment. The assay was performed with 3 replicates.
Disease severity was recorded on a 0–5 visual scale according to Tian et al. (2021), which 0 = no symptoms, 1 = less than 20% root slice area rotted, 2 = 21%–40% root slice area rotted, 3 = 41%–60% root slice area rotted, 4 = 61%–80% root slice area rotted, and 5 = more than 80% root slice area rotted. The disease index (DI) and biocontrol effect (BE%) were calculated by following formula:
$${\text{DI }}\left( \% \right) \, = \sum \left( {ab} \right)/AB \, \times \, 100$$
where a represents the number of root slices with the same disease severity scale, b represents the disease severity scale, A represents the total number of root slices, and B represents the highest disease severity scale.
$${\text{BE }}\left( \% \right) \, = \, \left( {{\text{PT}} - {\text{BT}}} \right) \, /{\text{ PT }} \times \, 100$$
where PT represents the disease index for the pathogen treatment and BT represents the disease index for the bacterial isolate plus pathogen treatment.
Identification of endophytic bacteria
Phenotypic and physiological characterization
The bacterial isolate from overnight raised culture in NA medium was streaked in LB medium (Luria–Bertani medium; tryptone 10 g, yeast extract 5 g, NaCl 10 g, agar 20.0 g, d.H2O 1 l, pH 7.4) and incubated for 2–4 days at 28 °C. Colony morphology was analyzed based on their shape, color and other characteristics (form, margin and transparency) on LB medium.
Growth tests for pH range were carried out by using NA medium adjusting the pH to 2, 5, 7, 9, 11 with 1 mol/l HCl or 1 mol/l KOH after sterilization. In order to study the temperature growth range, the overnight culture was spotted on the NA medium plates and incubated at different temperatures (4, 10, 15, 20, 28, 37 and 42 °C) for 2–4 days and then observed for positive growth. NaCl tolerance test for growth was performed using NA medium supplemented with different concentrations of NaCl (0, 3, 5, 7, 10 and 15%, w/v) and incubated at 28 °C for 2–4 days. Furthermore, the utilization of sole carbon sources, such as D-mannitol, D-galactose, sucrose, D-maltose, D-glucose, D-fructose, D-xylose and D-sorbitol, and the utilization of sole nitrogen sources, such as glutamine, histidine, urea, glycine and ammonium sulfate, were analyzed as the described method (Abo-Elyousr et al. 2021). All the experiments were performed in triplicate.
Biochemical characterization
Urease activity and lipase activity were assayed according to method reported by Guenoun et al. (2018). Peptone and coagulation of milk (20%, w/v skimmed milk), liquefaction of gelatin (20%, w/v) and production of H2S were examined as described previously (Chen et al. 2021a). Indole test was performed according to the previous description (Avinash and Rai 2014). Citrate utilization, starch hydrolysis and oxidase activity were also carried out. Other biochemical characteristics were done by following Bergey’s Manual of Determinative Bacteriology (Holt et al. 1994). All tests were carried out in triplicate.
Genotypic characterization and identification
DNA extraction and PCR conditions
Genomic DNA was extracted using the genomic DNA extraction kit from Shanghai Sangon Biotech. 16S rRNA sequence of the extracted DNA was amplified by using the universal primer 27F (AGTTTGATCMTGGCTCAG) and 1492R (GGTTACCTTGTTACGACTT). Final PCR reaction was carried out in 50 μl reaction mixture containing 20–100 ng DNA template, 0.5 U Taq DNA polymerase, 5 μl 10 × buffer, 2 μl dNTP Mix (10 mM) and 2 μl of each primer (10 μM). Amplification of the 16S rRNA genes was performed in a thermal cycler according to the following steps: 94 °C initial denaturation for 4 min, followed by 30 cycles of denaturation at 94 °C for 45 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min and a final extension at 72 °C for 10 min. The PCR product was analyzed by gel electrophoresis on 1.0% (w/v) agarose gel.
16S rRNA sequencing and phylogenetic analysis
The PCR product electrophoresis band cut the desired DNA target electrophoresis band, and the PCR product was directly sequenced with PCR primers. The 16S rDNA sequence was aligned on the ribosome database, and the obtained sequence was submitted to the National Centre for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov). The BLAST program (http://www.ncbi.nlm.nih.gov/BLAST) on the NCBI database was used to compare the obtained sequence with the sequence of the reference strain stored in the public database. The computer software package ClustalW was used for sequence alignment,
and the maximum compound likelihood model was used to analyze by the neighbor-joining method, and finally, the tree constructions were performed with the MEGA 7.0 version software.
Screening for plant growth-promoting traits
Plant growth-promoting traits of the isolated strain FT2 were studied through the following standard tests. All tests were performed in triplicate.
IAA production
The production of IAA was examined by Salkowski’s reagent. The test bacterial strain was inoculated into 250 ml NA liquid medium containing L-tryptophan (0.5 mg/ml) and cultured at 28 °C and 180 r/min for 3 days. After the bacterial culture was centrifuged at 9000 rpm for 10 min, 2 ml of the supernatant was mixed with 4 ml Salkowski’s reagent (0.5 mM FeCl3 in 50 ml H2SO4) and incubated at room temperature in dark condition for 30 min. The appearance of the red color indicated the production of IAA, which was quantified using an ultraviolet spectrophotometer at 530 nm. Un-inoculated medium was used as control. A calibration curve was established using pure IAA and expressed as milligram per liter.
Siderophore production
The production of siderophore was qualitatively estimated on Chrom Azurol S (CAS) agar medium. The bacterial strain was spotted on CAS agar medium and incubated at 28 °C for 2–3 days. Appearance of yellow-orange halos around the colonies was considered to be a positive test for siderophore production.
Nitrogen fixation
The Ashby nitrogen-free solid medium was inoculated with strain grown on NA medium for 2 days and cultured in a constant temperature incubator at 28 °C for 1–2 days. The strain could grow on selective medium and proved that it had the ability to fix nitrogen.
Phosphate solubilization
A two-day-old bacterial strain was spot inoculated on inorganic phosphorus medium containing 0.05% (NH4)2SO4, 0.03% NaCl, 0.03% MgSO4·7H2O, 0.003% MnSO4, 0.03% KCl, 0.003% FeSO4·7H2O, 0.5% Ca3(PO4)2, 1% glucose and 2% agar and organic phosphorus medium containing 0.05% (NH4)2SO4, 0.03% NaCl, 0.03% MgSO4·7H2O, 0.003% MnSO4, 0.03% KCl, 0.003% FeSO4·7H2O, 1% glucose, 0.5% CaCO3, 0.02% lecithin and 2% agar. The formation of a clear zone around the bacterial colonies indicated the ability to solubilize phosphates (inorganic/organic P).
Potassium solubilization
Potassium solubilization was tested by spotting the strain on potassium solubilization agar medium containing 0.5% sucrose, 0.2% Na2HPO4, 0.05% MgSO4·7H2O, 0.0005% FeCl3, 0.01% CaCO3, 0.1% potassium feldspar powder and 1.8% agar. After culturing at 28 °C for 5–7 days, a transparent hydrolysis circle around the bacteria means the result was positive.
NH3 production
The bacterial strain was inoculated into 100 ml of peptone water (peptic digest 10 g, NaCl 5 g, d.H2O 1 l, pH 7.6) and cultured at 28 °C for 2 days, and then, 0.5 ml of Nessler’s reagent (K2HgI4 and NaOH) was added. The color change of peptone water (from brown to yellow) indicated the production of NH3.
ACC deaminase production
The ability of the bacterial strain to utilize ACC as the sole nitrogen source was analyzed. The strain was inoculated on Dworkin and Foster (DF) salt minimal medium, which contained 4 g KH2P04, 6 g Na2HPO4, 0.2 g MgSO4·7H2O, 0.1 g FeSO4·7H2O, 10 μg H3BO3, 10 μg MnSO4, 70 μg ZnSO4, 50 μg CuSO4, 10 μg MoO3, 2 g glucose–gluconic acid, 2 g citric acid, 2 g (NH4)2SO4 and 20 g agar. The presence of bacterial growth on the media was deemed positive for ACC deaminase production.
Extracellular enzyme production
Strain was tested further for its ability to produce the following enzymes:
Protease
Protease activity was assayed by spotting the bacterial strain on Skim Milk Agar medium (skim milk powder 15 g, agar 20 g, d.H2O 1 l). After 2 days incubation at 28 °C, a clear zone around the colony indicated positive result (Abdel-Kareem et al. 2021).
Cellulase
Cellulase activity was determined using CMC agar medium (CMC-Na 10 g, KNO3 2 g, MgSO4·7H2O 0.5 g, FeSO4·7H2O 0.01 g, NaCl 0.5 g, K2HPO4 1 g, agar 20 g, ddH2O 1 l). After culturing at 28 °C for 2 days, the plates were stained with 5 ml of 0.1% (w/v) Congo red for 1 h at room temperature. Then, the plates were washed twice with 1 M NaCl for 10 min. A clear hydrolysis zone around the colony was deemed positive for cellulase production.
Biocontrol of Cucumber Fusarium Wilt
Cucumber (Cucumis sativus L.) is an important vegetable crop. However, the growth of this crop is often threatened by Cucumber Fusarium Wilt, which is a common fungal disease caused by Fusarium spp. (Zhao et al. 2012). A pot experiment was designed to assess the biocontrol efficiency of strain FT2 against Cucumber Fusarium Wilt caused by F. oxysporum Sacc.
Preparation of cucumber seedlings and spore suspension
One-month-old cucumber seedlings of susceptible cultivar Jinchun No. 4 were used in this experiment. Cucumber seedlings with similar size were transplanted to pots (12.5 × 8.5 cm) filled with a mixture of sterilized sand, coconut fiber, perlite and peat (2:1:1:1, w/w). These pots, each containing one transplant, were placed in a climate chamber at 25 °C, with 50% RH, and a photoperiod of 12 /12 h (light/dark). Seedlings were watered when needed.
Fusarium oxysporum Sacc was grown on PDA medium at 25 °C for 7 days, and the grown mycelium was washed with 10 ml sterile water to create spore suspension. Spore suspension was calculated by using a hemocytometer and adjusted for 107 spores/ml by serial dilutions with sterile water.
Experimental design
In this experiment, cucumber seedlings with fully expanded true leaves were used. The experiment was adopted a completely randomized designs, with 4 treatments and four replicates. The 4 treatments were as follows: (i) Untreated control (UC), plants were inoculated with distilled water only; (ii) Pathogen control (PC), plants were inoculated with pathogen only; (iii) Pathogen + chemical fungicide (PF), plants were inoculated with pathogen and the chemical fungicide; and (iv) Pathogen + FT2 (PT), plants were inoculated with pathogen and bacterial isolate FT2. 50 ml of F. oxysporum spore suspension was pre-inoculated in PC, PF and PT treatments. Two days after the pathogen incubation, 50 ml of FT2 bacterial suspension (108 cfu/ml) or the chemical fungicide (carbendazim, 1.25 g/l) was applied to the roots by drenching, and untreated control plants were treated with the same amount of distilled water (Zhang et al. 2021). During the inoculation, plant roots were slightly injured by a sterile needle.
Disease assessment
The development of Fusarium wilt on seedlings was evaluated 30 days after pathogen inoculation. The wilting development of each cucumber plant was rated on a scale of 4 (Chen et al. 2012): 0 = whole plant was healthy; 1 = less than 25% of leaves wilted; 2 = 26–50% of leaves wilted; 3 = more than 50% of leaves wilted; and 4 = whole plant died. Disease severity index (DSI) and biocontrol effect (BE) were calculated using the following formula (Chen et al. 2012):
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Disease severity index (%) = [∑ (scale × number of plants with the same scale) / (total number of plants × highest scale)] × 100
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Biocontrol effect (%) = (disease severity index of pathogen control—disease severity index of the treatment) / disease severity index of pathogen control × 100
Growth parameters determination
Destructive sampling was carried out after disease assessment. The plants under different treatments were cleaned with tap water and absorbed surface water with filter paper, and the plant height, stem diameter, leaf number and fresh weight were measured. The plant height was measured with a ruler, and the stem diameter was measured with a vernier caliper. An electronic scale was used to measure the fresh weight.
Defense-related enzyme activities determination
At the end of the experiment, healthy leaves with different treatments were collected to determine the defense-related enzyme activities. Phenylalanine ammonia-lyase (PAL) activity was assayed by following the method described by Liu et al. (2017) with some modifications. The reaction mixture containing 0.02 M l-phenylalanine 1 ml, 0.1 M borate buffer (pH 8.8) 2 ml and enzyme extract 0.1 ml was incubated at 30℃ for 30 min. 200 μl 6 M HCl was added to terminate the reaction, and the absorbance of the solution was measured at 290 nm. One unit represented the conversion of 1 μmol L-phenylalanine to cinnamic acid for 1 g of fresh weight per minute.
Polyphenol oxidase (PPO) activity was measured as described by Feng et al. (2021) with a slight modification. The reaction consisted 0.1 ml enzyme extract, 3.9 ml 0.05 mM phosphate buffer (pH 5.5) and 1 ml 0.1 M catechol. 2 ml 20% (w/v) TCA was added to terminate the reaction, and the absorbance of solution was measured at 525 nm. One unit of PPO activity was presented as the change of 0.01 in absorbance at 525 nm for 1 g fresh weight per minute.
Peroxidase (POD) activity was measured as described by Fang et al. (2018) with some modification. The reaction mixture containing 19 μl 50 mM guaiacol, 50 ml 0.2 M sodium phosphate buffer (pH 6.0), 28 μl 30% (v/v) H2O2 and 1 ml enzyme extract. One unit of enzyme activity was defined by a change in absorbance of 0.01 at 470 nm for 1 g of fresh weight per minute.
Plant growth promotion
Glycyrrhiza uralensis, as a perennial herb of the genus Leguminosae, is a commonly used Chinese herbal medicine. The strain FT2 was evaluated for its growth promotion effect on G. uralensis by the following experiments.
Seed germination experiment
Glycyrrhiza uralensis seeds were steeped with 85% H2SO4 for 2.5 h, soaked in 0.1% (v/v) H2O2 for 10 min, washed 10 times with sterile distilled water and air-dried. Seeds were soaked in the bacterial suspension of strain FT2 for 6 h (control group seeds were soaked in sterile distilled water for the same period of time), placed in Petri dishes (diameter 90 mm) containing filter paper moistened with sterilized water and incubated at room temperature. Treated seeds were watered regularly until germination was completed. The number of germinated seeds was recorded every 24 h from the 2nd day, and the germination number remaining unchanged for 2 consecutive days was determined as the time germination was completed. Each treatment was replicated 4 times with 30 seeds constituting one replication in the germination experiment. Germination rate, germination energy, germination index and seedling vigor index were calculated using the following formula:
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Germination rate (%) = n/N × 100, where n is the number of germinated seeds and N represents the total number of tested seeds
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Germination energy (%) = (the number of seeds germinated that reach the highest peak) / (the number of seeds at the beginning of the experiment) × 100
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Germination index = ∑(Gt/Dt), where Gt is the number of seeds germinated at time t, and Dt represents the corresponding germination to date
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Seeding vigor index = seedling length (cm) × germination index
Seedling growth experiment
This used the same procedure above for seed surface disinfection. The seeds were immersed in sterile water for 6 h and evenly sown in boxes (12 × 12 × 6 cm) filled with 630 g high-pressure sterilized and fully dried sand medium collected from native desert regions which do not contain any nutrients and pre-irrigated with 100 ml of distilled water. After growth for 20 days, seedlings were inoculated with 50 ml of bacterial suspension of strain FT2. Sterile water was used as control. Each treatment had 5 repeats with 20 seeds per repeat. Boxes were positioned in a climate chamber (28 °C, 12/12 h light/dark, RH 50%). Seven days after the inoculation, growth parameters of seedlings, enzyme activities, available nutrient contents and bacterial count in rhizosphere soil were analyzed.
Growth parameters determination
The stem and root lengths were measured by a ruler, and the stem and root diameters were measured by a vernier caliper. After placing them in a 55 °C oven for 48 h, their dry weights were also recorded using an analytical balance.
Soil enzyme activity determination
The rhizosphere soil samples were sieved (1-mm) before analyzed. The activities of soil urease, phosphatase, saccharase and catalase were separately measured.
Saccharase activity was assessed by Guan (1986) technique, with slight changes. Briefly, 5 g of soil was mixed with 15 ml 8% (w/v) sucrose, 5 ml phosphate buffer (pH 5.5) and 0.25 ml toluene. The mixture was incubated at 37 °C for 24 h and then measured on a spectrophotometer at 508 nm. The results were expressed as mg of glucose released by 1 g of soil in 24 h.
Soil urease activity was assayed according to the method by Guan (1986), and the results were expressed as mg of released NH3-N by 1 kg of soil per hour at 37 °C.
Catalase activity was determined using the technique of Guan (1986) with slight changes. Briefly, 5 g of soil with 40 ml distilled water and 5 ml 0.3% H2O2 (v/v) was added into centrifuge tubes and then shaken for 30 min. The filtrate was titrated with 0.1 mol/l KMnO4. The results were expressed as 0.1 mol/l KMnO4/100 g/hrs.
Acid phosphatase activity was assessed with minimal changes, according to Guan (1986). Briefly, 5 g of soil was incubated in acetate buffer (pH 5.0) at 37 °C for 12 h. 5 ml buffer solution and a 2 ml filtrate were transferred into a 50-ml volumetric flask and then diluted to 50 ml with distilled water, and the phenol released at 600 nm was measured by a spectrophotometer. Acid phosphatase activity was expressed as mg hydrolyzed phenol by 1 kg soil per hour at 37 °C.
Soil available nutrient content and bacterial count determination
The available nitrogen was determined by the alkali diffusion method, and available phosphorus was measured by sodium bicarbonate extraction–molybdenum blue method (Chen et al. 2021b). The rhizosphere soil bacterial count was determined by the dilution plate method of Lin (2010).
Statistical analysis
SPSS 26.0 was used for statistical analysis of the data. Differences among means were tested using the least significant difference (LSD) test, P < 0.05 representing a significant difference. Mean values and standard errors (SE) were shown in figures and tables. Bar diagrams were prepared using Microsoft Excel.