Origin of strains
Strain JTB8–2 was isolated from the rhizosphere soil of Egyptian broomrape in Jimusar County, Xinjiang Province, China, and was identified as B. velezensis. The strain was also deposited in the Guangdong Microbial Culture Collection Center (No. GDMCC 60755). This strain was grown on Nutrient Agar (NA) medium (3 g beef paste, 7 g peptone, 5 g NaCl, and 17 g agar in 1 L distilled water, pH 7.0) for routine culturing.
The purified single colony was inoculated into NB medium (3 g beef paste, 7 g peptone, 5 g NaCl in 1L distilled water, pH 7.0), and the bacterial culture was obtained through incubation at 130 revolutions per minute (rpm) at 25 ℃ for 24 h. The bacterial culture was transferred into two 500 mL-Erlenmeyer flasks containing 200 mL NB medium, and fermentation broth was obtained by oscillating culture at 28 ºC and 180 rpm for 24 h.
The experimental treatments included strain JTB8-2 fermentation broth, culture medium NB and blank control, with 3 replicates per treatment and 3 pots per replicate. Plastic basin specifications were 25 cm in diameter and 18 cm in height, with holes at the bottom. Tomato cultivar HS1015 was planted in hole trays and transplanted when tomato seedlings had 4 ~ 5 leaves. Potting soil was medium loam soil (Alkaline hydrolyzed nitrogen: 82.682 mg/kg; Available phosphorus: 42.824 mg/kg; Available potassium: 289.522 mg/kg; pH: 7.86) which taken from the field of the Anning Ditch test site in Urumqi, Xinjiang. About 200 g of fine soil was put into a 600 ml plastic bottle and then added 50 mg (approximately 10,000 seeds) O. aegyptiaca seeds, the mixture was shaken well and sprinkled evenly on the tomato roots. Covered with soil and then poured water. B. velezensis JTB8-2 fermentation broth at a concentration of 5×108 CFU/mL and culture medium NB were diluted 25fold, 50-fold, 100-fold respectively, and then irrigated with 1 L/pot the next day, once every 15 d, 3 consecutive times, and water as blank control. Tomato root soil was removed 60 d after transplantation, and the parasitism number in each pot was investigated. The fresh weight was taken, and then the collected broomrape was put into the oven to dry at 60°C.
The field test
Field experiments were carried out on processing tomato in Shuanghe Village, Qingyang Lake Township, Jimusar County, Changji Prefecture, Xinjiang (E: 89°1' 35"; N: 44°2' 3"). The soil was medium loam soil (Alkaline hydrolyzed nitrogen: 80.627 mg/kg; Available phosphorus: 41.033 mg/kg; Available potassium: 237.655 mg/kg; pH: 7.88). The experimental treatments included JTB8–2 agent 0.8 L/105m2, 1.6 L/105m2, 3.2 L/105m2 and water, with three replicates per treatment. Each replicate was randomly arranged in the field. The fermentation broth concentration of the strain was 5×108 CFU/mL by turbidimetric method. The first drip irrigation, 15 d after tomato seedlings were transplanted, came from an electric sprayer, and 40 L of bacterial liquid was applied every time, applied once every 20 d on 3 consecutive times. The first field investigation was carried out 25 d after the third application. 30 m2 area was select randomly from each 105 m2 area respectively, andthe number of O. aegyptiaca, tomato plants, and broomrape parasites were investigated and fresh weight was taken. The collected broomrape was placed into an oven to dry at 60°C, and the biomass was collected.
Parasitism rate (%) = number of parasitized tomato plants/number of investigated tomato plants×100
The measured yield area of each plot was 4.5 m2. Yield increasing effects of bacterial treatments were evaluated by determining fruit weight of plant (kg/plant), weight of 100 fruits (kg/100) and fruit weight of plot (kg).
Chemical extraction, isolation and purification
The scaled-up fermentation was carried out in ten 1000 mL-Erlenmeyer flasks. For each flask, 10 mL secondary seed fermentation liquid was inoculated into 200 mL NBmedium, and 9 g macroporous resin (XAD-16) was added to absorb the secreted metabolites. After incubation at 28°C for 5 days, the fermentation broth was discarded, and the macroporous resin was repeatedly washed with distilled water and dried in oven at 28 ºC. Next, the resin was extracted with methanol for 3 times, and the resulting methanol solution was combined and concentrated under vacuum. The concentrate was redissolved in 50% methanol solution, and then extracted with equal-volume dichloromethane for 4 times. The organic layer was evaporated to dryness under vacuum to get a 6.5 g crude extract, which was further fractionated by silica gel Vacuum Liquid Chromatography (VLC) eluting with gradient PE (petroleum ether)–EtOAc solution. All fractions were evaluated for their inhibitory effects on the germination of Egyptian broomrape seeds, and inhibition rate of fractions eluted with 25% and 40% EtOAc were 100%. Thus, the 25% EtOAc fraction (87.68 mg) was purified by reverse phase high performance liquid chromatography (RP HPLC) on a C18 column (Kromasil 100-5-C18; 5 µm; 10 × 250 mm; 40% MeOH in H2O over 28 min; 2 mL/min) to get compound 1 (6.1 mg, tR 9.10 min) and 2 (20 mg, tR 11.99 min). The 40% EtOAc fraction (153.1 mg) was also purified by semipreparative RP HPLC (40% MeOH in H2O over 30.0 min; 2 mL/min) to get compounds 3 (4.5 mg, tR 13.4 min) and 4 (7.0 mg, tR 24.5 min).
The 1H and 13C NMR data were collected on a Bruker Avance 500 MHz NMR spectrometer equipped with a 5-mm triple resonance cryoprobe at 298 K. Chemical shift values (δ) are given in parts per million (ppm) and the coupling constants (J values) are in Hz. Chemical shifts were referenced to the residual solvent peaks.
Seed sterilization: The O. aegyptiaca seeds were disinfected in 75% ethanol for 30 s and then transferred to 3% sodium hypochlorite (effective chlorine) solution for 10 min. After rinsing in sterilized water 3 times, the seeds were dried on sterile filter paper for later use.
Activity determination of crude extract: 100 mg crude extrac was dissolved in methanol and then mother liquor was prepared with add 100 µL distilled water for activity test before column chromatography separation. Whatman filter paper (GF/A) was cut into round paper with a diameter of 14 mm and placed in a 24-well cell culture plate bottom, with 2 pieces for each well. Approximately 50 sterilized and dried seeds were added to each well. Dissolved 1 mg GR24 in 200 mL distilled water. Added 200 µL GR24 (5 µg/mL) solution to each well and then added the crude extract mother liquor, respectively. Crude extracts were assayed at a final concentration from 5 µg/mL to 0.6 µg/mL. Water was set as blank control. After 5 days of shading culture at 25°C, the number of germinated seeds was observed under a microscope. The germination rate and inhibition rate were calculated according to the following formula for evaluate the effect of crude extract. The assay was repeated 4 times for each concentration.
Germination rate (%) = Germination seed number/The total number of seeds×100
Inhibition ratio (%) = (Seed germination rate of blank control-Germination rate of treated seeds)/Seed germination rate of blank control×100
Activity determination of pure compounds: Nitrogen blowing was carried out on the nuclear magnetic tube solution, and the quality of each compound was detected after drying. According to the molecular weight of different compounds, 8 mM mother liquor was prepared by adding sterile water and then diluted to 4 mM, 2 mM and 1 mM. Then, 0.25 mL of the assay solution was mixed with 0.25 mL GR24 solution at a concentration of 5 µg/mL. The pure metabolites were assayed atconcentrations between 4 mM and 0.5 mM. The assay was repeated 4 times for each pure compound.
Data were presented as mean ± SE and analyzed using analysis of variance. For in vitro and in vivo results, completely randomized design (CRD) design was used while field data were analyzed using randomized complete block design (RCBD) design. Statistical software SPSS 16.0 was used. Significant difference in the treatment was measured with LSD test and separated by using lettering.