2.1. Microbial strains
P. chlororaphis subsp. aurantiaca ST-TJ4 was isolated from the poplar rhizosphere in Tianjin, China. It is preserved at the China Center for Type Culture Collection (CCTCC) under accession number M2020435. The pathogenic bacterium A. tumefaciens PX-1 was obtained from cherry blossom crown galls in Xuzhou, China. Both strains, ST-TJ4 and PX-1, were provided by our laboratory[5, 20]. The strain ST-TJ4 was routinely grown on King‘s B (KB) medium, while strain PX-1 was cultured on Luria-Bertani medium (LB). Both strains were stored at − 80°C.
2.2. Antibacterial activity assay of VOCs produced by strain ST-TJ4
The effect of VOCs produced by P. chlororaphis subsp. aurantiaca ST-TJ4 on A. tumefaciens strain PX-1 was tested using a dual-culture assay[21]. A Petri dish was filled with KB medium seeded with P. chlororaphis subsp. aurantiaca ST-TJ4, and another dish was filled with LB medium inoculated with an overnight culture of A. tumefaciens strain PX-1 (OD600 = 0.5). The two open plates were then joined together and tightly sealed with parafilm. For the controls, KB-filled Petri dishes were not seeded with P. chlororaphis subsp. aurantiaca ST-TJ4. After 48 h, A. tumefaciens colonies were removed from the plates, measured, suspended in sterilized water, diluted and spread on LB agar medium. The CFU/mL of A. tumefaciens strain PX-1 was calculated after incubation at 28°C for two days.
To evaluate the antibacterial activity of VOCs produced by the different concentrations of P. chlororaphis subsp. aurantiaca ST-TJ4, water-suspended cells of P. chlororaphis subsp. aurantiaca ST-TJ4 (1 × 107 CFU/ml) were prepared as described above and spread (10, 50, 100 and 200 µL) onto one plate containing KB agar medium, but not on the control plate, and A. tumefaciens strain PX-1 was spot inoculated (10 µL) onto another plate containing LB agar medium. The CFU/mL of A. tumefaciens strain PX-1 was calculated after two days as described above in this section. These experiments had three replicates and were repeated twice.
2.3 Scanning and transmission electron microscopy observations
After exposure to VOCs of P. chlororaphis subsp. aurantiaca ST-TJ4 at 28°C for 48 h, the external morphological changes of A. tumefaciens PX-1 were observed by scanning electron microscopy (SEM), and the ultrastructural changes of cells were observed by transmission electron microscopy (TEM).
For the preparation of A. tumefaciens strain PX-1 cells for SEM and TEM, the cells were collected directly into Eppendorf tubes, washed three times with sterile water and fixed with 2% glutaraldehyde for 30 min at 4°C. Fixed cells were rinsed three times for 10 minutes with 100 mM phosphate buffer, postfixed for 3 h in 1% osmium tetroxide, and dehydrated through a stepwise ethanol gradient (30%, 50%, 80%, 90%, and 100%) for 15 minutes at each concentration[12]. Later, the samples were coated with gold, and electron micrographs were obtained using SEM (Quanta 200, FEI, USA).
Similarly, for TEM, the samples dehydrated by ethanol were embedded and solidified, sliced by an ultrathin microtome and stained with 3% uranium‒lead citrate. Ultrastructural changes in the cells were observed using TEM (JEM-1400, JEOL, Japan)[19]. These experiments had three replicates and were repeated twice.
2.4 Swimming and chemotaxis traits assay
The swimming motility assay was performed according to previously described methods [18, 22] with minor modifications. Briefly, 2 µL of A. tumefaciens strain PX-1 (OD600 = 0.5) cultured overnight was inoculated in ATGN medium (K2HPO4 10.5 g, KH2PO4 4.5 g, MgSO4 0.2 g, FeSO4 5 mg, CaCl2 10 mg, MnCl2 2 mg, (NH4)2SO4 15 mM, glucose 5 g, agar 3 g, H2O 1000 mL; pH 7.0) and tightly sealed with a Petri dish filled with KB seeded with P. chlororaphis subsp. aurantiaca ST-TJ4. In the controls, the KB-filled Petri dish was not seeded with P. chlororaphis subsp. aurantiaca ST-TJ4. After 48 h of incubation, the swimming diameter of A. tumefaciens strain PX-1 was recorded.
The chemotaxis assay was performed as described by Zhou et al.[23]. In short, 2 µL of A. tumefaciens strain PX-1 (OD600 = 0.5) cultured overnight was inoculated on one side of the ATGN medium (without glucose), and one strip of filter paper with glucose was placed 4 cm away from the bacterial solution. Another dish was filled with KB seeded with P. chlororaphis subsp. aurantiaca ST-TJ4. In the controls, the KB-filled Petri dish was not seeded with P. chlororaphis subsp. aurantiaca ST-TJ4. The two open plates were then joined together and tightly sealed with parafilm. Treatment without strain ST-TJ4 was performed as a control, and a blank control without filter paper with glucose was used to demonstrate the glucose chemotaxis of strain PX-1. These experiments had three replicates and were repeated twice.
2.5 Root colonization and biofilm formation assays
For the root colonization assay, one-week-old sterile Arabidopsis thaliana plants were used for testing. The A. tumefaciens strain PX-1 cell suspensions with and without exposure to the VOCs of P. chlororaphis subsp. aurantiaca ST-TJ4 were prepared as described in Section 2.2 and diluted to a concentration of 1 × 107 CFU/mL with sterilized water. Later, the cells were incubated in a 5 mL cell suspension of strain PX-1 at room temperature and, after 12 h, rinsed with sterile water three times and blotted lightly. The samples were treated according to the method described in Section 2.3 and observed by SEM (Quanta 200, FEI, USA).
For the biofilm formation assay, divided Petri plates were used. The biocontrol P. chlororaphis subsp. aurantiaca strain ST-TJ4 was spot inoculated onto one compartment of a divided Petri plate containing KB agar medium, while in the other compartment, a small culture dish was placed in the culture dish on the other side. In the small culture dish, 1 mL of LB medium was added, 1% A. tumefaciens was inoculated, and the culture was sealed and kept at 28 ℃. The control was not coated with strain ST-TJ4. The plates were sealed with parafilm and placed at 28°C for 48 h. Biofilm estimation was performed using the crystal violet staining method[24]. After cultivation, the bacterial solution was removed, and the planktonic bacteria were gently washed with 0.1 M phosphate-buffered saline (PBS, pH 7.2). Biofilms were dried at room temperature, fixed with 1 mL of methanol, stained with 1 mL of 0.1% crystal violet, washed with PBS to remove excess dye, and quantified after solubilization of the dye with ethanol by reading the absorbance of the microplates at 570 nm. These experiments had three replicates and were repeated twice.
2.6 Gene expression
A. tumefaciens strain PX-1 cultivated as described above without VOCs of strain ST-TJ4 served as the negative control. Cells were harvested by centrifugation at 4°C and 12000 rpm and then washed with precooled sterile PBS. RNA was extracted using an RNA extraction kit (Nanjing Jiancheng Bioengineering Research Institute, China), and quantitative real-time PCR (qRT‒PCR) was performed with the reference gene
lepA (atu0241) set as the internal control
[25]. cDNA samples were prepared using HiScript II Q Select RT Supermix for qPCR (Yisheng, China). The entire reaction was conducted on an ABI 7500 (Applied Biosystems, Waltham, MA, USA) using 1.0 µL of cDNA diluted 1:10 as the template. The primers are listed in Supplementary Table 1. Experimental consumables, including 96-well 0.2 mL semiskirted PCR plates, were obtained from NEST Biotechnology Co. Ltd. (Wuxi, China). The 2
−ΔΔCT method was used for relative quantification of changes in gene expression
[26]. These experiments had three replicates and were repeated twice.
2.7 Rose infection assay
A. tumefaciens strain PX-1 cell suspensions with and without exposure to the VOCs of
P. chlororaphis subsp.
aurantiaca ST-TJ4 were prepared as described in Section
2.2 and diluted to a concentration of 1 × 10
7 CFU/mL with sterilized water. Stems of potted rose plants were inoculated with bacterial suspensions using a needle as previously described
[27]. The control was inoculated with sterile water. Photographs were taken after inoculation for one week. These experiments had three replicates and were repeated twice.
2.8 Antibacterial activity assay of pure VOCs
In previous research in our laboratory, we identified the main components in the ST-TJ4 volatile spectrum by GC/MS analysis, which were 1-undecene, 2-undecone, 1-nonanol, 2-heptanone, and vinyl decanoate[12]. Here, the effects of the five pure chemicals on the growth of strain PX-1 were evaluated in separate Petri plates. Sterile filter paper discs containing different volumes (10, 30, and 60 µL) of each pure VOC purchased from Nanjing Zebra Trading Co., Ltd. (Nanjing, China) were placed in one compartment of divided plates, while the other compartment containing LB agar medium was spot inoculated (10 µL) with the cell suspension of A. tumefaciens PX-1. The control was not subjected to any treatment. The plates were sealed with parafilm and incubated at 28°C. After 48 h, the colony counts of A. tumefaciens strain PX-1 (CFU/mL) on LB agar medium were determined as described in Section 2.2. The results, expressed as the inhibition rate compared with the control, were photographed. These experiments had three replicates and were repeated twice.
2.9 MICs of pure volatile organic compounds against A. tumefaciens PX-1
The minimum inhibitory concentration (MIC) of 2-undecone, 1-nonanol and 2-heptanone was determined using the microdilution method with an inoculum of 1–5 × 105 CFU/mL [28]. Growth analysis was performed by inoculating 0.1% overnight cultures of A. tumefaciens strain PX-1 into LB medium supplemented with different concentrations of 2-undecone, 1-nonanol and 2-heptanone[29]. After incubation at 28°C for 24 h, growth was determined based on the absorbances at 600 nm. These experiments had three replicates and were repeated twice.
2.10 Statistical analysis
All experimental results are expressed as the mean ± standard deviation (± SD). One-way analysis (ANOVA) or an independent samples t test was used to determine significant differences (p < 0.05). Graphs were generated using GraphPad Prism 8.0 (GraphPad Software, Inc., United States).