Rice (Oryza sativa cv. Nipponbare) seeds were surface-sterilized with 2% (v/v) sodium hypochlorite for 15 min and rinsed with distilled water three times. The sterilized seeds were transferred to seeding tray for germination. After seven days, the germinated seedlings were transplanted into a plastic box (length × width × height, 35 × 25 × 12 cm) containing 7 L nutrient solution prepared based on the receipt from International Rice Research Institute (Yoshida et al. 1976). The nutrient solution was changed every three days. All rice plants were grown for 40 days in a greenhouse at Fujian Agriculture and Forestry University (Fuzhou, China) with a day: night temperature regime of 28 °C (14 h): 22 °C (10 h), 70% relative humidity and a light intensity of 30,000 lx.
Field collected rice striped stem borer (Chilo suppressalis, SSB) larvae were collected from in a field located at the campus of Fujian Agriculture and Forestry University (Fuzhou, China, 119.23 N, 26.08 E). Laboratory-maintained SSB colony was kindly provided by Prof. Yunhe Li from the Institute of Plant Protection, Chinese Academy of Agricultural Sciences. The colony has been maintained in the laboratory for over 20 generations before being used for experiment. The egg masses of lab-reared insects were sterilized by soaking in 2.5% bleach solution for 5 min, and rinsed with distilled water three times. The sterilized eggs were transferred to petri dishes (diameter: 9 cm) for hatching. The neonates (10–15) were transferred into a glass tube (diameter: 2.4 cm; length: 7 cm) supplied with autoclaved artificial diet. The sterilized diet contained all components of a previously described diet (Han et al. 2012) but lacked bactericidal antibiotics that may compromise establishment of bacteria in subsequent experiments. When the larvae reached the third instar, the diet was changed and only 2–3 larvae were allowed in each tube to avoid cannibalization until pupation. The larvae were reared under conditions of 70–80% RH, 26 ± 1 °C, and a photoperiod of 16L: 8D. The pupae were removed from the rearing tubes and kept separately in petri dishes for adult emergence. The newly emerged adults were kept in cages containing rice plants at tillering stage for oviposition under conditions of 85–90% RH, 26 ± 1 °C, and a photoperiod of 16L: 8D. Then, the egg masses were collected, and the rice plants were replaced with fresh plants every day.
To examine the effects of microbes in the oral secretion (OS) of SSB on mediating induced defense responses in rice plants, an antibiotic cocktail (AB) was used to reduce the microbes present in field-collected larvae as far as possible. The AB solution was made up of 0.01 g neomycin sulfate (MP Biomedicals), 0.05 g aureomycin (BioServ), and 0.003 g streptomycin (Sigma) dissolved in 50 ml of sterile MiliQ water as described by Chung et al. (2013). A tube of artificial diet (1 cm × 1 cm × 1 cm) was prepared and treated with 200 μL of AB, and placed in a laminar flow hood until dry. One larva at 3rd instar was placed in a plastic cup (1.5 oz) containing a tube of artificial diet with or without AB solution. Larvae were fed on artificial diet for 2 days, and then the body weight was measured. The AB-treated SSB larvae were used for the subsequent experiments. To verify the effects of AB treatment on removing insect-associated bacteria, we compared the bacterial quantities in the OS collected from SSB pretreated with or with AB. Two microliters of crude OS were freshly collected from SSB larvae treated with AB for 2 days by gently tapping larval heads according to a previously described procedure (Peiffer and Felton 2009; Acevedo et al. 2017), and diluted to 10–3 (μL) with sterilize water. The aliquot was then added to 2 × YT agar plate to count numbers of colonies (colony forming units, CFU), and each treatment had 5 replicates. The plates were incubated in a growth chamber at 27 °C for 24 h.
Scanning electron microscope images
To verify that bacteria were secreted onto plant wounds during caterpillar feeding, SSB larvae with or without AB treatment were allowed to feed on rice stems for 1 d, and the damaged sections of stems were collected and prepared for scanning electron microscope (SEM) observation. Briefly, the SSB larvae were fixed on the rice stem using the 75% ethanol sterilized plastic tubes and autoclaved cotton according to the methods described by Jiao et al. (2018). After 1 h of feeding, the stems with visible injury caused by both AB-treated larvae and larvae with AB treatment were collected and placed in a centrifuge tube containing formalin fixing solution (Solarbio, Beijing). The samples were stored at 4 °C until being dehydrated and coated with gold. The images were captured using SEM (JSM-6380LV) located at Fujian Academy of Agricultural Sciences (Fuzhou, China).
Effect of antibiotic treatment on larval performance on rice plants
To examine whether AB treatment affect the performance of SSB larvae on rice plants, 2-d-old 3rd-instar larvae with or without AB treatment were fixed on the stems of 40-d-old rice plants using a plastic tube (diameter: 3 cm, length: 6 cm) with both ends plugged with cotton as described above. Thirty larvae with similar size (20 ± 5 mg) from both AB treatment and non-AB treatment were selected and allowed to feed on the stem. After 3 days of feeding, the percentage of successful penetration was determined as the number of larvae successfully penetrating divided by the total number of larvae inoculated (Hou and Han 2010). Then, the larvae were removed, and the mass of larvae that had successfully bored into stems were weighted. The experiments were repeated three times, and each treatment had three replicates.
Effect of OS from AB-treated larvae on defense responses in rice
Oral secretions were collected from both AB-treated larvae and non-AB treated larvae, and diluted 1:9 v/v with sterile MilliQ water. Each rice plant was mechanically wounded using a puncher to punch a 0.5 cm diameter hole in the main stem and 20 μL of diluted OS were applied the wounded sites. Control plants were wounded and treated with 20 μL of sterile water. The leaf tissue around the feeding sites was harvested 24 h later for gene expression analysis. After 48 h, plant tissues around damaged sites were collected for assessing the activities of two key defensive enzyme in rice analysis. The gene transcription in rice plants happens intensively in 24 h after damage occurs (Ye et al. 2012), while induced anti-herbivore enzymes in plants peak at approximately 48 h after the infestation (Constabel et al. 1995; Lin et al. 2022). For insect bioassay, the remaining tissue from each damaged leaf was used to SSB larval growth. All 3rd-instar larvae were allowed to feed on rice stems with different treatment for 3 d, and then the mass gain of larvae on each plant were weighted. Each treatment had more than 30 biological replicates, and the experiment was repeated three times.
Analysis of LOX activities
Lipoxygenase (LOX) catalyzes the initial reaction in JA biosynthesis pathway (Stenzel et al. 2003). In rice plants, LOX could be significantly activated at 48 h after infestated by chewing insect such as rice leaf folder (Cnaphalocrocis medinalis) (Ye et al. 2012). LOX activity was measured as conjugated diene formation with slight modification (Macri et al. 1994). Rice stem samples (0.1 g) were ground in liquid nitrogen and extracted with 1 mL of ice-cold 0.5 M Tris–HCl buffer (pH 7.6) and centrifuged at 12,000 g for 20 min at 4 °C. The supernatant was transferred into a new centrifuge tube and kept at 4 °C until used. The substrate contained 0.5% (v/v) Tween20 and 1.6-mM linoleic acid in 0.1 M PBS (pH 7.6). The reaction was initiated by adding 0.2 mL of supernatant in 4.8 mL of the substrate. An extinction coefficient of 25 mM−1 cm−1 was used to convert absorbance values at 234 nm to nmol of conjugated diene.
Quantification of PI contents
Trypsin protease inhibitor (PI) contents were measured using enzyme linked immunosorbent assay (ELISA) kits (Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China). The stem samples were ground into a fine power in liquid nitrogen using a mortar and pestle, and each sample (0.1 g) was homogenized in 1 mL of 0.01 M Phosphate Buffered Saline (PBS) buffer (pH = 7.4). Samples were centrifuged at 5000 × g for 10 min at 4 °C, and the supernatant was collected into a new 1.5-mL centrifuge tube. The ELISA experiments were performed following the protocols provided with the kits. The optical density values were recorded at 450 nm using a microplate spectrophotometer (BioTek, Vermont, USA). The protein concentrations in plant samples were measured using a bicinchoninic acid (BCA) protein assay kit (Aidlab Biotechnologies Co., Ltd., Beijing, China) according to the manufacturer’s instructions. The amount of protease inhibitor was calculated based on a standard curve, and results were determined as µg protease inhibitor per mg protein.
Analysis of gene expression by quantitative real-time PCR
Quantitative real time PCR was used to examine the expression levels of different genes related with defense responses in rice plants. RNA extraction was referred to CWBIO’s Ultrapure RNA kit (ComWin Biotech, Beijing, CW0581M) manufacturers protocol. Briefly, 50 mg of plant tissue was mixed with 1 ml of TRLzon Reagent (ComWin Biotech, Beijing), and allowed to stand at room temperature for 5 min. Chloroform (200 μl) was added, and vigorously shaken for 15 s at room temperature for 2 min, and then centrifuged at 4 °C and 12,000 rpm for 10 min. The pre-extracted RNA was transferred to a new RNase-free tube, an equal volume of 70% ethanol was added, inverted and mixed. RNA washing solution was added to the centrifuge in order, the obtained RNA was stored at − 70 °C. Total RNA (1 µg) was then pipetted for cDNA synthesis using the GoScript TM Reverse Transcription System (Promega Biotech, Beijing). Real-time PCR was performed according to the procedure of Ultra SYBR three-step fluorescence quantitative PCR kit (ComWin Biotech, Beijing). Reaction conditions for thermal cycling were 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 30 s, then 72 °C for 32 s. Fluorescence data were collected during the cycle at 60 °C. The relative transcript levels of the target genes in samples were determined according to the standard curve. A rice actin gene OsActin was used as an internal standard to normalize cDNA concentration. The primers used for qRT-PCR for all tested genes (JA-related genes: OsAOS2 and OsLOX; SA-related genes: OsPAL1 and OsPR-1a) are listed in Additional file 1: Table S1.
Classification of bacteria in larval oral secretion
To isolate bacteria in OS from SSB larvae, the OS was collected from non-AB-treated larvae and cultured on LB agar plates. Single colonies were randomly selected and purified and cultured on BPA medium. A single colony was collected with a 20 µl pipette and transferred to a PCR tube containing 10 µl of sterile water. DNA was obtained by cleavage at 95 °C for 10 min. The next step was PCR amplification. The PCR reaction contained 1 µl of 10 µM 16S rRNA primer (530 F 5′-GTG CCA GCM GCC GCG G-3′ and 1392R 5′-ACG GGC GGT GTG TRC-3′), 12.5 µL of the GoTaq Green Master Mix (Promega), 2 µL of the bacteria DNA, and 8.5 µL of MQ water for a total volume of 25 µL. The PCR conditions as follows: 95 °C for 5 min, followed by 30 cycles of 95 °C for 1 min, 53 °C for 1 min, and 72 °C for 1 min 30 s, and finally 72 °C for 7 min. The PCR production was sent to BioSune bio-company (Fuzhou, China) for sequencing, the 16S rRNA sequences were analyzed using BLAST the nucleotide database of the National Center for Biotechnology Information (NCBI) with sequence similarity greater than 95%.
Effect of oral secreted bacteria on plant induced defenses
The effect of SSB larval OS-associated bacteria on plant defense responses was examined. To determine whether bacterial isolates cultured from SSB larvae oral secretion suppressed plant defenses, rice stems mechanically wounded by a puncher were inoculated with individual bacterial isolate (OD600 = 0.1. c.a. 107 CFU/mL) collected from SSB larval OS and cultured on 2 × YT liquid media. After 48 h, stem tissue around the wounded site was harvested to analyze LOX activity. The impact of individual isolated bacterium on plant defense responses was compared with rice plants treated with wounding and application of liquid media. Leaf samples were frozen in liquid nitrogen and stored at − 80 °C until analysis.
Generation of axenic and gnotobiotic SSB larvae
Axenic and gnotobiotic SSB larvae were produced by following the methods described by Mason et al (2019b) with a small modification. All the processes of axenic rearing were conducted in a laminar flow hood. The SSB eggs were sterilized as described above, and the larvae were maintained on autoclaved diet. To generate gnotobiotic larvae from these axenic larvae, bacteria were administered to axenic larvae through inoculation of artificial diet. Liquid cultures of both Enterobacter and Acinetobacter were freshly grown overnight on 2YT media (16 g/L tryptone; 10 g/L yeast extract; 5 g/L NaCl). Cells were collected and re-suspended in 10 mM of sterilized MgCl2 solution. Each group of insects (5 larvae) was provided with approximate 107 cells, while control insects received an identical volume of MgCl2 solution. Insects were allowed to feed on diet 1-cm diameter diet cube inoculated with individual bacterial isolate UV sterilized diet cups for 2 days before using in the subsequent experiments.
Detection of bacteria from gnotobiotic larvae deposited onto plants
To determine whether gnotobiotic larvae secreted the same bacteria onto damaged plants, surface-sterilized gnotobiotic larvae were placed on the rice stems confined by sterilized tubes, and undamaged plants were treated with an empty tube. The rice stems were wiped with 70% ethanol before the infestation of SSB larvae. After 24 h of feeding, the tubes were removed, and the boring sites on the stems were detached by sterilized scissors. The collected tissues were suspended in 2.5 mL tube with 1 mL liquid 2 × YT media and cultured overnight in a rotary shaker at 200 rpm at 27 °C. Ten microliters of the culture were heated at 95 °C for 10 min. DNA released from the bacterial cells was amplified with specific primers showed in Additional file 1: Table S1. Gel electrophoresis was conducted to examine the PCR products, which were then sequenced and verified.
Effect of OS from gnotobiotic larvae on defense responses in rice
Twenty microliters of OS collected from larvae inoculated with Enterobacter were applied to the wounded sites of rice stems using a puncher. Plant tissue around the damaged area was harvested for measuring the expression levels of plant defense signaling pathway-related genes (24 h later) and LOX activity (48 h later) according the detailed methods described above. For insect bioassay, 3rd instar larvae were allowed to feed on plants pre-treated with OS collected from axenic or gnotobiotic larvae. After 3 days of feeding, the mass gain of larvae on each plant was weighted. Each treatment had more than 30 replicates, and the experiment was repeated three times.
The normal distribution and homogeneity of all data sets were verified before analysis. Minitab 16 (Minitab Inc., State College, USA) was used for data analysis. Data were analyzed by one-way analysis of variance (ANOVA) followed by Fisher’s test or unpaired student’s t-test (significance level, p < 0.05). The sample size and number of replicates for all the assays and analysis are indicated in the legends of the corresponding figures. All figures were generated using GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, United States).