Preparation of nematodes, their secreted metabolites and extracts
C. elegans N2 was obtained from the CGC (Caenorhabditis Genetics Center), and two soil-dwelling bacterial-feeding nematodes Mesorhabditis sp. and Acrobeloides sp. were obtained from the alluvial soil from Banqiao Town, Nanjing City, Jiangsu Province, China (Liu et al. 2018).
The nematodes were cultivated at 20 °C on freshly prepared nematode growth medium (NGM) (3 g NaCl, 2.5 g peptone, 17 g agar and 975 mL H2O were autoclaved; when cooled to 55 °C, supplied with 25 mL 1 M KPO4 buffer (pH 6.0), 1 mL 1 M CaCl2, 1 mL 1 M MgSO4 and 1 mL 5 mg/mL cholesterol in ethanol, which were filtered through 0.22-μm filter). The worms were usually fed with Escherichia coli OP50, a standard food for bacterial-feeding nematodes in laboratory, or other three IAA-producing bacteria when required. After reached the desired stage, the worms were harvested from the petri plates with M9 buffer (5 g NaCl, 3 g KH2PO4, 6 g Na2HPO4, 1 mL 1 M MgSO4, H2O to 1 L autoclaved) and collected by the modified Baermann funnel method (Liu et al. 2008). To remove the medium and bacteria from the nematode cuticle, the worms were washed and centrifuged three times with M9 buffer. They were then placed in M9 buffer for 30 min at 22 °C at 250 rpm to digest the bacteria in their guts. After washing three times by sterile water, the worms were collected for further experiments. All the operations were ensured sterility.
For collecting nematodes-secreted metabolites, the worms need to be incubated in sterile water for 1 hour at 22 °C at 250 rpm with a density of ~15000 worms/mL. Then, the metabolites were collected by gentle centrifugation, filtered through 0.22-μm filter, lyophilized and stored at -80 °C. The metabolites secreted by one worm in 1 hour is defined as 1 worm equivalent (WE) (Srinivasan et al. 2008). For collecting nematodes extracts, the worms were incubated in sterile water for 1 hour at 22 °C at 250 rpm and then broken up by Ultrasonic Cell Crusher (XO-1000D) with 60 % duty ratio (working 3s with rest 3s) for 45 min. Then, the extracts were collected by centrifugation, filtered through 0.22-μm filter, lyophilized and stored at -80 °C.
Bacteria and culture conditions
Three IAA-producing bacteria used in this study were Bacillus amyloliquefaciens JX1, Arthrobacter pascens ZZ21 and Arthrobacter chlorophenolicus L4 from our laboratory (Table 1). B. amyloliquefaciens JX1 was isolated from the same alluvial soil with the two soil-dwelling nematodes (Yu et al. 2015). A. pascens ZZ21 was isolated from the forest soil from Zijin Mountain in Nanjing City, Jiangsu Province, China (Li et al. 2018). A. chlorophenolicus L4 was isolated from the red soil from the Red Soil Ecological Station of Chinese Academy of Sciences. E. coli OP50 was obtained from the CGC. These three IAA-producing bacteria were inoculated into mineral liquid medium (5 g glucose, 2 g (NH4)2SO4, 0.5 g NaH2PO4, 0.5 g K2HPO4, 0.2 g MgSO4·7H2O and 0.1 g CaCl2·2H2O with 1 L H2O at pH 7.0) at 1% (V/V) for 3 days. 200 mg/L tryptophan was added to the medium to stimulate bacterial IAA synthesis.
Table 1 Descriptions of the IAA-producing bacteria used in the study
Bacteria
|
Gram strain
|
Accession number
|
B. amyloliquefaciens JX1
|
G+
|
JX424611
|
A. pascens ZZ21
|
G+
|
KF515608
|
A. chlorophenolicus L4
|
G+
|
JQ277449
|
Detection of the effect of bacterial-feeding nematodes and their metabolites on bacterial IAA synthesis
Bacterial IAA production affected by C. elegans
The nematode C. elegans and three IAA-producing bacteria (B. amyloliquefaciens JX1, A. pascens ZZ21 and A. chlorophenolicus L4) were employed to investigate the effect of nematodes on bacterial IAA synthesis. C. elegans, fed with the corresponding IAA-producing bacteria, were added to medium at 80 worms/mL (the amount of nematodes added was based on the experimental results of Jiang et al. (2016)). After that, the medium were moved to the incubator set at 22 ℃ for 7 days (the suitable incubating temperature and time for the growth of nematodes). The groups without nematodes were controls. The concentration of IAA in the medium was detected on day1, 2, 3, 5 and 7.
Bacterial IAA production affected by C. elegans-secreted metabolites and extracts
The nematodes C. elegans and three IAA-producing bacteria mentioned above were employed to investigate the effect of nematodes-secreted metabolites on bacterial IAA synthesis. C. elegans-secreted metabolites were added to medium at 2000 WE/mL (equivalent to the metabolites that secreted by 80 worms for 1 day). Then, the medium were moved to the incubator set at 30 °C for 3 days. The groups without C. elegans-secreted metabolites were controls. The concentration of IAA in the medium was detected every day.
In order to facilitate the enrichment of nematode metabolites, another ultrasonic fragmentation method was used to collect the metabolites (called extracts). Based on the results of above experiments, C. elegans and A. pascens ZZ21 were selected to investigate the effect of nematodes extracts on bacterial IAA synthesis (see results). The extracts of C. elegans were added to medium 2000 WE/mL. After that, the medium were moved to the incubator set at 30 °C for 3 days. The groups without C. elegans extracts were controls. The concentration of IAA in the medium was detected every day.
Bacterial IAA production affected by soil-dwelling bacterial-feeding nematodes-secreted metabolites
Besides, we selected two soil-dwelling bacterial-feeding nematodes (Mesorhabditis sp. and Acrobeloides sp.) and two IAA-producing bacteria (B. amyloliquefaciens JX1 and A. pascens ZZ21) to investigate the effect of soil-dwelling nematodes-secreted metabolites on bacterial IAA synthesis. Soil-dwelling bacterial-feeding nematodes-secreted metabolites were added to medium at 2000 WE/mL. Subsequently, the medium were moved to the incubator set at 30 °C for 3 days. The groups without nematodes-secreted metabolites were controls. The concentration of IAA in the medium was measured every day.
Quantification of IAA Levels
IAA levels in the supernatants of samples were measured spectrophotometrically (Gordon and Weber 1951). The supernatants were equal-ratio mixed with Salkowski reagent (50 mL 35% HClO4 combined with 1 mL 0.5 M FeCl3). After incubating in darkness for 30 min at room temperature for color development, the absorbance was measured at 530 nm by spectrophotometer. The concentration of IAA was calculated by comparing the absorbance with a standard curve constructed with the known concentrations of IAA. Meanwhile, the OD600 was determined at each test time.
Statistical analysis
Data were analyzed by SPSS 22.0 statistical software. One-way analysis of variance (ANOVA) was performed to analyze the abilities of bacteria to synthesize IAA under different treatments. Duncan’ test was used to assess the significant differences among the means (P<0.05). All figures were performed using Origin.