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. 2024 Nov 19;24:1095. doi: 10.1186/s12870-024-05826-7

Endophytic Pseudomonas fluorescens relieves intraspecific allelopathy of Atractylodes lancea by reducing ethylene transportation

Ling-Sen Cao 1, Di Wang 1, Chun-Yan Wang 1, Wei Zhang 1, Fei Chen 1, Yaseen Ullah 1, Kai Sun 1,✉, Chuan-Chao Dai 1,✉
PMCID: PMC11575199  PMID: 39558284

Abstract

Background

Endophytes play an important role in promoting plant growth. To date, although many reports provided insight into the function of endophytes in their hosts, few reports focus on their impact on nearby plants. Intraspecific allelopathy in plant community is common and presents a notable challenge to medicinal plant yield and productivity. Atractylodes lancea is a perennial herb that has relatively low yields due to intraspecific allelopathy. The bacterial endophyte Pseudomonas fluorescens ALEB7B has previously been found to increase essential oil content of A. lancea, but the role of ALEB7B in A. lancea allelopathy is still unknown.

Results

Noninoculated A. lancea exhibited growth retardation when it was grown in a community, which was related to ethylene-induced intraspecific allelopathy. Further experiment showed that exposing A. lancea to volatile from noninoculated A. lancea or same concentration of ethylene reduced growth of A. lancea. P. fluorescens-inoculated plants showed reduced ethylene emission and relieved growth retardation on neighboring noninoculated A. lancea. Moreover, P. fluorescens inoculation had little allelopathic effect when receivers were treated with ethylene receptor inhibitor or when emitters were treated with ethylene production inhibitor. Transcriptomic analysis revealed that endophyte ALEB7B altered transcriptional response associated with ethylene response and essential oil production in neighboring A. lancea.

Conclusions

Our results demonstrated that the bacterial endophyte ALEB7B provides fitness benefits for both hosts and neighbors. The allelopathic effect on nearby plants can be alleviated by altering airborne signals, such as ethylene, in endophytic bacteria.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-024-05826-7.

Keywords: Atractylodes lancea, Allelopathy, Bacterial endophyte, Pseudomonas fluorescens, Plant airborne signals, Ethylene

Introduction

Volatile-mediated plant‒plant interactions occur under many conditions [1–3]. These interactions could benefit neighboring plants. For example, tobacco inoculated with mosaic virus emits methyl-salicylate, which upregulates regulating defense-related genes in neighboring plants [4]. However, plant‒plant interactions can also be harmful. Allelopathy is a common phenomenon in terrestrial plant communities and is defined as the effect of chemical agents released by one plant on another [5], which can also occur among the same species [6]. Data analysis from 39 studies of various vegetation types revealed that for 67% of the species pairs, both intraspecific effects and interspecific effects were negative [7]. Many compounds, such as hydroxybenzoic acid, l-fenchone, and 1,8-cineole, have been confirmed as allelopathy agents [8, 9]. For example, the emission of the root volatile β-caryophyllene from Pinus halepensis can suppress the growth of neighboring Lactuca sativa and Linum strictum roots [10]. In addition, the ethylene response is very common under various stress conditions, including cold, heat, drought, growth competition and allelopathy [11–13]. Moreover, ethylene has also been reported to be an inhibitor of root growth on neighboring plants in tobacco [14], indicating that ethylene is not only the responder but also the mediator of allelopathy.

The microbiota plays an important role in plant‒plant interactions [15]. The secondary metabolites produced by endophytes are remarkably similar to volatile organic compounds (VOCs), which are involved in plant‒plant interactions or allopathy [16, 17]. The regulated VOCs include hexenal derivatives, methyl salicylate acid, and terpenoids [1, 4, 18]. For example, Diaporthe isolated from Catharanthus roseus is capable of synthesizing β-caryophyllen [19]. β-Caryophyllene is reported to be the VOC secreted by the roots of Centaurea and is capable of inhibiting the growth of nearby plants [20]. On the other hand, reducing ethylene synthesis or reducing the ethylene response are common in plants with plant growth-promoting microorganisms [15]. It is speculated whether the “beneficial endophyte” benefits only the host but has allelopathic effects on neighbors or whether the endophyte also benefits neighbors in the whole community.

Atractylodes lancea (A. lancea) is both a perennial herb and a medicinal material in China. The medicinal components of A. lancea include but are not limited to β-caryophyllene, hinesol, atractylodin and ß-eudesmol [21, 22]. Dried rhizomes of A. lancea can moderate immunity, have antitumor activity, and alleviate SARS-CoV-2 symptoms [22, 23]. The bacterial endophyte P. fluorescens ALEB7B were previously reported to enhances bioactive terpenoid accumulation in A. lancea [24, 25]. A previous study reported that A. lancea has complicated VOC emissions [26]. Since VOCs are the major agents of communication for plants, it is speculated whether the beneficial coexistence of A. lancea and P. fluorescens could affect VOC emissions and influence the growth of neighbors and what role VOCs play in these tripartite interactions.

Materials and methods

Microorganisms and growth media

P. fluorescens (ALEB7B) was previously isolated from the leaves of A. lancea and preserved at the China Center for Type Culture Collection (CCTCC AB 2013331) [25]. ALEB7B was maintained in Luria-Bertani (LB) medium supplemented with 2% agar. To make bacterial suspensions, ALEB7B were grown in LB media at 28 °C with agitation (180 rpm) for 24 h, washed 3 times with sterilized distilled water (SDW) and resuspended in SDW. The bacterial suspension was diluted to 106 CFU/ml (OD600 = 1.0) for inoculation.

Plant growth conditions and microbial inoculation

In this study, A. lancea was collected from Maoshan, Jiangsu Province, China [27]. Qi-ying Yang (College of Life Sciences, Nanjing Normal University, China) made the initial, formal identification. The voucher specimens were planted in the botanical garden of Nanjing Normal University (China). In this study, A. lancea was reproduced via aseptic tissue culture as previously described [28]. Before the experiments were performed, the plants were grown in 1/2 MS media supplemented with 0.3 mg·ml− 1 1-naphthylacetic acid and 1.5% sucrose. The plants were grown under controlled growth conditions (14-h light:10-h dark photoperiod with 200 µmol m− 2 s− 1 photosynthetically active radiation, 18–25 °C) for 7–8 weeks. The plants were subsequently transferred to appropriate vessels for the following experiments.

Briefly, ALEB7B-treated A. lancea were treated with 200 µL of bacterial suspension, while control A. lancea were treated with 200 µL of sterilized double-distilled H2O.

Pot experimental design

After 60 days of growth in 1/2 MS media, A. lancea plants were carefully washed with SDW to remove the remaining culture media. The A. lancea plants were subsequently transferred to plastic cups (10 cm in diameter and 20 cm in height) filled with a mixture of 1/2 potting soil and 1/2 nutrient soil (Xing-xing-xiang-nong Co., Gan Zhou, Jiangxi Province, China). The transferred plants were incubated under controlled growth conditions (12-h light:12-h dark photoperiod with 200 µmol m− 2 s− 1 photosynthetically active radiation, 18–25 °C) for 7 days.

To evaluate the effects of A. lancea (with or without P. fluorescens inoculation) on neighboring A. lancea plants, 10 or 5 plant bodies of A. lancea were placed in a transparent plastic box (with vents). Each plant body of A. lancea was placed 10 cm apart from neighboring A. lancea plants, forming a 5 × 2 array (Fig. 1A). Five plant bodies of A. lancea plants with lower intensity were treated with SDW (LP). Ten plant bodies of A. lancea were treated with SDW (CK). Five plant bodies of A. lancea were P. fluorescens-inoculated (PI), whereas the other 5 neighboring plants were treated with SDW (NP). The experiments were performed 3 times.

Fig. 1.

Fig. 1

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Effects of bacterial inoculation on A. lancea in pot experiments. (A) Experiment set A. lancea in the pot experiment. All A. lancea were planted in plastic cups filled with a mixture of 1/2 roseite and 1/2 nutrient soil and divided into 4 groups. Each of the 5 or 10 A. lancea plants were placed in a transparent plastic box. CK was treated with sterilized distilled water (SDW); A. lancea plants with lower intensity (LP) of were treated with SDW; P. fluorescens-inoculated A. lancea (PI) was inoculated with Pseudomonas P. fluorescens; and the NPs were neighbors of the PI, which were also treated with SDW. (B) shoot length, (C) root length, (D) fresh weight, and (E) dry weight. (Different letters indicate a significant difference according to one-way analysis of variance (ANOVA) with Duncan’s multiple comparison test (p < 0.05), n ≥ 5)

Gas exchange assay and plant sampling

An instrument was designed to evaluate the effects of phyllospheric changes on A. lancea (Fig. 2A). Each pair of bottles, including an emitter bottle (left side) and an acceptor bottle (right side), was connected by a latex tube. Purified air (filtered through a 4 A molecular sieve, silica (80–100 mesh), and activated charcoal (80–100 mesh)) served as the gas source. Every two days, 150 ml of phyllospheric gas was removed from the system. The treatment was maintained for 7 or 14 days before sampling.

Fig. 2.

Fig. 2

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Effects of exposing A. lancea to P. fluorescens-inoculated or SDW-treated A. lancea. (A) Diagram of a gas transfer system. A gas exchange system consists of three filter bottles (successively containing a molecular sieve, active carbon and silica gel), an emitter bottle, a receiver bottle and a glass column. After the gas was extracted, the filter bottles and glass columns were removed, and the open tubes were sealed to avoid contamination. (B) Experimental design of the gas transfer experiment. E represents the plants from the emitter bottle of each treatment, whereas R represents the plants from the receiver bottle of each treatment. The plants in the negative control groups (EC and RC) were treated with SDW. The emitter plants in the positive group (EPI) were treated with SDW, while the receiver plants (RPI) were inoculated with P. fluorescens suspension. The emitter plants in the treatment group were inoculated with P. fluorescens suspension, while the receiver plants were treated with SDW. (C-F) Effects of bacterial inoculation and gas exchange on the growth of A. lancea over time. (C) Shoot length, (D) root length, (E) fresh weight, and (F) dry weight. The data are presented as the means ± SEs (n = 4). Statistical differences between nontreatment (RC) and gas-treated samples inoculated with A. lancea were analyzed via Student’s t test. (“*” and “**” represent significant differences between RC and RB; “*” represents p < 0.05; “**” represents p < 0.01)

Group division was set as follows: A. lancea treated with SDW in an accepter bottle next to 4 emitter bottles with A. lancea treated with SDW were collected as the negative control (RC). A. lancea in the accepter bottle inoculated with P. fluorescens next to the bottle with emitter A. lancea treated with SDW were collected as the positive control (RPI). A. lancea treated with SDW in an accepter bottle next to an emitter bottle with A. lancea inoculated with P. fluorescens were collected as the treatment group (RB). Each treatment had 4 replications. The plants were harvested after 7 or 14 days of inoculation. The plants were subsequently washed with SDW. The cleaned plant samples were measured and dried at 37 °C for essential oil extraction or stored at -80 °C for other experiments.

Plant essential oil extraction and quantification

Essential oil extraction was performed as previously described [29]. Briefly, the samples acquired were dried at 37 °C for 24 h and then ground into powder. Fifty milligrams of powder was extracted with 0.5 ml of cyclohexane at 4 °C for 24 h and vortexed for 1 min. After centrifugation at 6000 × g for 1 min, the supernatant was filtered with a 0.22 μm Teflon filter and transferred to a 2-ml sample vial. A 7890 A gas chromatograph coupled with a 30 m × 0.25 mm × 0.25 μm film thickness HP-5 fused silica column (Agilent, Santa Clara, California, United States) and a flame ionization detector were used for analysis. A 1 µL sample was injected in split-less mode and isolated with the following oven temperature program: 5 min hold at 40 °C, 10 °C min-1 ramp to 270 °C, and 10 min hold at 270 °C. All the compounds were verified with chemical compound standards (analytically pure).

Plant volatile collection and analysis

The collection of volatile compounds from the plants was performed as described in a previous study [30]. Volatiles from A. lancea plants treated with SDW and A. lancea plants inoculated with P. fluorescens suspension were collected. The plants were treated for 7 days before the experiment. The bottles containing the plants were sealed. Purified air (filtered through a 4 A molecular sieve, silica (80–100 mesh), and activated charcoal (80–100 mesh)) was used as the gas source and was maintained at a flow rate of 1 ml·s− 1. After extraction for 2 h to remove the remaining VOCs, the air outlets were connected to a glass tube containing 50 mg 80/100 mesh Porapak-Q (Waters, Milford, MA, USA). After two days of collection, the VOCs were eluted twice with 500 µL of dichloromethane (chromatographically pure, Merck, Darmstadt, Germany). After the two eluates were combined, 300 ng of nonyl acetate (Sigma–Aldrich) was added as an internal standard. Sixteen peaks were identified as compounds (Table S2).

For qualitative analysis, a mixture of VOCs from SDW-treated A. lancea and ALEB7B suspension-treated A. lancea was injected into a GCMS (GCMS-QP2020, Shimadzu Corp., Kyoto, Japan) coupled with an Rxi-5ms column. The components were identified by comparing the retention index and mass spectra in the NIST library (NIST 08). Impurities from solvent or capillary chromatographic columns, including divinylbenzene, 4-vinyl pyridine, n-vinyl 2-pyrrolidone, and ethylene glycol dimethacrylate, were removed from the list of compounds. Six of the available compounds were verified with chemical compound standards. 3-Carene, D-limonene and benzothiazole were purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). β-Caryophyllene was purchased from Tokyo Chemical Industry (Tokyo, Japan). Valencene was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Lavender lactone was synthesized by Summer Pharmacy Co. (Shanghai, China) and confirmed by 1H-NMR.

For the qualitive analysis, the method was the same as that for the essential oil quantification described above. Each treatment had 3 replications.

Gas sampling and ethylene quantification

Gas samples were collected as follows: Two days before gas sampling, bottles containing plants were sealed with a silica cap. Two days later, side-open syringes coupled with a three-way valve were used to transport 20 ml of phyllospheric gas to 20 ml gas injection vials. Each treatment had 3 replications. The quantification was performed on an Agilent 7890 A gas chromatograph (GC) coupled with a flame ionization detector fitted with an HP-PLOT Q column (30 m × 0.32 mm × 0.20 μm). The ethylene peak was verified with standard ethylene gas.

RNA isolation and qPCR analysis

For relative expression analysis, two leaves (30–60 mg) from each sample were harvested. The leaf tissues were frozen in liquid nitrogen and ground into powder. Isolation of total RNA was performed following the instructions for the RNA Isolator Total RNA Extraction Reagent (Vazyme Biotech Co., Ltd., Jiangsu, China). The RNA samples were stored at -80 °C until use. First-strand cDNA was synthesized via a HiScript III 1st Strand cDNA Synthesis Kit (Vazyme Biotech Co., Ltd., Jiangsu, China) according to the manufacturer’s instructions. The RNA was quantified via a Nanodrop spectrophotometer (NanoDrop 2000 C; Thermo Scientific).

Real-time PCR analyses were performed on a 7500 Real Time PCR System using AceQ qPCR SYBR Green Master Mix (Vazyme Biotech Co., Ltd., Jiangsu, China). The PCR conditions were as follows: denaturation at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s and extension at 72 °C for 30 s. Primers were designed via Primer-BLAST from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov) and verified via SDS‒PAGE. The gene elongation Factor 1 alpha (EF1α) gene was used as the reference gene to normalize gene expression via the 2-ΔΔCt method [31]. All primers used are shown in Table S1.

RNA sequencing experiment set

A. lancea plants adjacent plants to nontreated A. lancea, as well as P. fluorescens-treated A. lancea and A. lancea plants adjacent to P. fluorescens-treated A. lancea, were selected for RNA-seq analysis.

RNA extraction and sequencing were performed following the manufacturer’s protocol (S1). All the clean reads were assembled viausing Trinity software. For functional annotation, the longest transcript of each gene was defined as the ‘unigene’. Nucleotide sequences of all unigenes were searched against the current versions of Nr, COG, and KEGG viausing BLASTX with a cutoff E -value of 10− 5. Differentially expressed genes (DEGs) between two different samples were calculated according to the transcripts per million reads (TPM) method. DEGs with a |log2FC|>1 and a Q value < = 0.05 were considered to be significantly differentially expressed. The Illumina sequencing data of A. lancea plantlets were submitted to the NCBI Sequence Read Archive (SRA) under the accession number PRJNA900630 (available at https://dataview.ncbi.nlm.nih.gov/object/PRJNA900630?reviewer=jgjj6f2k2c2bmmannvnbseqf67). The transcriptome data of A. lancea are shown in Table S3.

Quantitative assay of l-aminocyclopropane-l-carboxylic acid (ACC) deaminase activity

The activity of ACC deaminase (ACCD) was analyzed according to a previous description [32]. P. fluorescens ALEB7B was pretreated in minimal medium supplemented with ACC as the sole nitrogen source for 24 h. After centrifugation, the bacterial cells were washed with SDW 3 times and then rehydrated. One hundred microliters of toluenized cells were transferred to a microcentrifuge tube, and 10 µg of ACC was added as the material. After 1 h of incubation at 37 °C, the content of α-ketobutyrate was measured as the amount of ACC consumed by ACC deaminase. The experiment was performed in triplicate. Another 100 µL of toluenized cells was diluted and used to determine the protein concentration via a BSA protein assay kit (Solarbio Life Science, Beijing, China).

Statistical analysis

The statistical analyses were performed via SPSS 22.0 (SPSS Inc., Chicago, IL, USA). Comparisons between two treatments were made via Student’s t test. When comparisons were conducted among three or more treatments, one-way analysis of variance (ANOVA) was performed, followed by Duncan’s multiple comparison test (P < 0.05) to test whether the differences among the values obtained in these groups were significant. All experiments were performed with at least three individual replicates. The data are expressed as the mean ± standard error of the mean (SEM).

Results

P. fluorescens ALEB7B inoculation alleviates intraspecific allelopathy in neighboring plants of A. lancea hosts

To investigate the allelopathic effects of A. lancea on nearby A. lancea and the potential alleviating effects of P. fluorescens on the allelopathy of A. lancea, 5 or 10 A. lancea plants were kept in diaphanous boxes 10 cm from nearby A. lancea (Fig. 1A).

Compared with A. lancea plants treated with SDW(CK), A. lancea plants with lower intensity (LP) presented increased root length (p = 0.011349) at 14 days after inoculation (dai) and increased fresh weight (p = 0.000730) and dry weight at 21 dai (p = 0.04930) (Fig. 1D, E). Similarly, compared to CK, after 21 days of inoculation, neighboring A. lancea of P. fluorescens-inoculated A. lancea (NP) increased root length by 33% (p = 0.044764) and weight gain by 60% (p = 0.046974) (Fig. 1C, D, E). Additionally, the LP and NP treatment resulted in an increased total essential content compared to CK (p = 0.0142695 and 0.061838896) (Fig. S1A).

To evaluate the agents transferred to nearby A. lancea and their effects on nearby A. lancea, a specially designed gas exchange instrument was used to reproduce the phenomenon and collect the VOCs (Fig. 2A). P. fluorescens ALEB7B reportedly contributes to the growth and high-efficiency essential oil accumulation of A. lancea. Direct inoculation with an ALEB7B suspension (RPI) was performed as a positive control. In the negative control set, the emitters and acceptors (RC) were treated with SDW. In the experimental set, the emitters were inoculated with a bacterial suspension, whereas the receivers (RB) were treated with SDW (Fig. 2B). The plant samples were collected at 7 and 14 dai. At 14 dai, inoculation with P. fluorescens increased the growth and essential oil accumulation of nearby A. lancea. At 14 dai, the shoot length, root length, fresh weight and dry weight of the A. lancea plants in the RB group significantly increased by 22% (p = 0.000095), 46% (p = 0.027140), 23% (p = 0.008014) and 29% (p = 0.022873), respectively (Fig. 2C-F). Moreover, compared with RC, inoculation with ALEB7B increased the accumulation of β-caryophyllene, atractydin, hinesol, zingiberene, and total essential oil (p = 0.018188521) of neighbors (Fig. S2). To exclude the possibility that the change in the receivers was due to the airborne transmission of bacterial cells, a bottle of LB medium (2% agar) was set below ALEB7B-treated plants (EB). At 14 dai, no bacterial colonies were observed in the LB medium.

P. fluorescens ALEB7B-alleviated intraspecific allelopathy in neighboring plants is related to reduced ethylene emission

To determine whether P. fluorescens could increase essential oil accumulation by altering the emission of volatile organic compounds (VOCs), VOCs from A. lancea treated with SDW (EC) or ALEB7B suspension (EB) were collected. Three VOCs, 3-carene (p = 0.022904), D-limonene (p = 0.015033) and benzothiazole (p = 0.022904), changed significantly at 3 dai (Fig. S3A-F). The effects of these VOCs on A. lancea as single compounds or mixtures were tested. These compounds increased the contents of caryophyllene oxide, hinesol, β-eudesmol, and atractylon but decreased the biomass of A. lancea (Figs. S3, 4).

Since ethylene is in gas form which can be volatized to nearby plants, it is interesting whether the concentration of ethylene in the air is down-regulated by ALEB7B, leading to the alleviation of allelopathy. To test these hypotheses, phyllospheric air and evaluated ethylene emissions were collected. A. lancea spontaneously releases ethylene to the phyllospheric air (Fig. 3A). When inoculated with P. fluorescens, the rate of ethylene emission decreased by 65% at 7 dai (Fig. 3A) (p = 0.033608).Similarly, at 14 dai, inoculation with ALEB7B also reduced the rate of ethylene emission by 77% (Fig. 3A, B) (p = 0.020475).

Fig. 3.

Fig. 3

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Ethylene emission of A. lancea. (A) Ethylene production of A. lancea treated with SDW or bacterial suspension and filtered air at 0, 7, and 14 dai. (B) Chromatogram of the ethylene measurement. The three curves represent gas from A. lancea treated with SDW (EC), A. lancea inoculated with P. fluorescens (EB) and an ethylene-diluted standard at 7 dai, bottom to top

To evaluate the growth inhibition effect of biologically relevant concentrations of ethylene, a comparison was made among receivers exposed to fresh air (RN), volatiles from nontreated A. lancea plants (RC), relevant concentrations of ethylene from nontreated or P. fluorescens-treated plant volatiles (RMP and RMB), emitters treated with the ethylene synthesis inhibitor AOA (RAOA), and the ethylene response inhibitor 1-MCP-treated receivers exposed to volatiles from nontreated plants (RMCP) (Fig. 4A). Compared with untreated A. lancea (RN), A. lancea exposed to the relevant concentration of ethylene from nontreated plant volatiles (RMP) had a shorter root length (p = 0.020475) and lower dry weight (p = 0.020475) (Fig. 4C, E).Furthermore, A. lancea exposed to the relevant concentration of ethylene from nontreated plant volatiles (RMP) did not significantly differ from A. lancea exposed to nontreated plant volatiles (RC). However, A. lancea exposed to the relevant concentration of ethylene from P. fluorescens-inoculated plant volatile (RMB) presented greater shoot length (p = 0.014553), longer root length (p = 0.024484) and better dry weight (p = 0.031825) than RMP did (Fig. 4B-E). Compared with A. lancea exposed to fresh air (RN), the shoot length, root length, weight and dry weight of the plants in the RMB treatment hardly differed (Fig. 3B-E). Additionally, the neighbors of emitters treated with the ethylene synthesis inhibitors AOA (RAOA) and 1-MCP, an ethylene response pathway inhibitor-treated receiver (RMCP), presented increased growth (in shoot length, root length, weight and dry weight, p < 0.05) compared with that of RC (Fig. 4E).

Fig. 4.

Fig. 4

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Effect of ethylene on the growth of A. lancea. (A) Experimental design. RMP: A. lancea treated with ethylene at the same concentration as untreated A. lancea (0.3 PPM 12 h− 1); RMB: A. lancea treated with ethylene at the same concentration as ALEB7B-inoculated A. lancea (0.1 PPM 12 h− 1). (B-E) Fresh weight, dry weight, shoot length, root length and total essential oil content of A. lancea at 14 dai. Different letters indicate a significant difference according to one-way analysis of variance (ANOVA) with Duncan’s multiple comparison test (p < 0.05) (n = 4)

Gas chromatography analysis of the essential oil content revealed similar results. Compared with RN, treatment with nontreated plant volatiles or the relevant concentration of ethylene from SDW-treated plant volatiles (RMP) significantly decreased the β-caryophyllene content (p = 0.041968). In contrast, compared with RMP, RMB resulted in better β-caryophyllene production (p = 0.012992) (Fig. S5).

P. fluorescens ALEB7B inoculation reduces ethylene emission via the inhibition of plant ethylene biosynthesis and the expression of bacterial ACC deaminase

To determine why ethylene production decreases, the expression of genes involved in ethylene production, including l-aminocyclopropane-l-carboxylic acid synthetase ((AlACCS)) and l-aminocyclopropane-l-carboxylic acid oxidase (AlACCO) were tested. The expression level of AlACCS was reduced in A. lancea treated with P. fluorescens (EB) at both 7 dai (p < 0.000001) and 14 dai (p < 0.000001) (Fig. 5B), whereas the expression level of AlACCO decreased only at 14 dai and did not differ at 7 dai (Fig. 5C). In addition, ALEB7B carries a gene that produces the same protein as the 1-aminocyclopropane-1-carboxylate deaminase from P. fluorescens strain DR133. Moreover, ALEB7B exhibited ACC deaminase (ACCD) activity of 0.3819 ± 0.007 μm α-ketobutyrate (mg− 1 protein h− 1) after pretreatment with minimum medium with ACC as the sole nitrogen source. The colonization of ALEB7B in A. lancea and the expression of ACCD were subsequently detected in EB instead of EC at 7 dai and 14 dai (Fig. 5D, E).

Fig. 5.

Fig. 5

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Effects of P. fluorescens inoculation on ethylene production gene expression. (A) Experimental set. (B,C) Relative gene expression of AlACCS and AlACCO in A. lancea treated with SDW or bacteria at 7 dai and 14 dai. (D) Bacterial concentration of P. fluorescens in P. fluorescens-inoculated A. lancea. (E) Expression of ACCD in untreated A. lancea (EC) and P. fluorescens-inoculated A. lancea (EB). (“*” represents p < 0.05, n = 4)

To assess ethylene production at the receiver site, the expression levels of AlACCS and AlACCO in A. lancea subjected to different treatments were measured, including fresh air (RN), volatiles from nontreated A. lancea (RC) and volatiles from bacterially treated A. lancea (RB), were measured. Compared with those under RN, none of these treatments resulted in a significant change in the expression of ethylene-producing genes (Fig. 6A), but the transcription of the ethylene-responsive factors AlERF1A and AlERF109 in RC was significantly greater than that in RN (p = 0.002013, p = 0.000134 respectively) and RB (p = 0.009448, p = 0.000190 respectively) (Fig. 6C).

Fig. 6.

Fig. 6

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Effects of P. fluorescens on ethylene production and ethylene-related gene expression in their receivers. (A) AlACCS and AlACCO of A. lancea at 14 dai. RN: A. lancea treated with filtered air; RC: A. lancea treated with volatiles from untreated A. lancea; RB: A. lancea treated with volatiles from P. fluorescens-inoculated A. lancea. (B) Diagram of the ethylene pathway. (C) Ethylene relative gene expression of different receiver groups. Different letters indicate a significant difference according to one-way analysis of variance (ANOVA) with Duncan’s multiple comparison test (p < 0.05) (n = 4)

For the pot experiments, ten randomly selected ethylene-responsive factor genes were selected to represent the ethylene response level. First, the 10 ERFs of A. lancea were tested with 10 ppm ethylene. The expression of AlERF109, AlERF1B, AlERF3, and AlERF071 increased in response to ethylene at 2 days (Fig. S6A). The expression of AlERF4 decreased in response to ethylene. At 7 dai, the PI treatment downregulated AlERF3, whereas the NP treatment downregulated AlERF3 and AlERF109 and upregulated AlERF4. At 14 dai, the PI treatment downregulated AlERF109 and AlERF071. NP treatment downregulated AlERF109. Both treatments upregulated AlERF4. At 21 dai, both the PI treatment and NP treatment downregulated AlERF3 and AlERF071 (Fig. S6B-F). These results showed that the ethylene response level could be the reason for the reduced growth and essential oil accumulation in the CK. On the other hand, at 7 dai, the ethylene production gene did not significantly differ between CK and NP or between CK and PI (Fig. S7), but there was a significant increase in ACCD in the PI treatment (Fig. S7G), which is consistent with the results obtained from the bottle experiment above.

Decreasing ethylene is fundamental to enhance the growth and essential oil accumulation of nearby A. lancea

Interestingly, AlACCS expression in A. lancea treated with volatiles from P. fluorescens-treated plants (RB) did not significantly differ from that in A. lancea treated with fresh air (RN) or volatiles from P. fluorescens-treated plants (RB) (Fig. 6A). However, compared with those treated with volatiles from SDW-treated plants (RC), fresh air-treated plants (RN) and P. fluorescens-treated plants (RB) presented significantly decreased transcription of AlERF1A and AlERF109, which act downstream of ethylene, as observed via RT‒qPCR analysis (Fig. 6C). Conversely, exposure to volatiles from P. fluorescens-inoculated plants (RB) significantly increased the transcription level of AlERF4 and decreased the transcription levels of AlERF1A and AlERF109 (Fig. 6C). ERF4 negatively responds to ethylene, suppressing ethylene signal transduction [33]. The differential expression of ERFs rescued P. fluorescens.

To determine the role of ethylene transmission in growth promotion and essential oil accumulation effects, an experiments to evaluate the importance of P. fluorescens growth promotion and essential oil accumulation effects was conducted. Whether this effect persisted when the ethylene receptor in receiver plants was blocked with 1-MCP (RB−MCP) or when ethylene emission in emitter plants was blocked with AOA (RB−AOA) was measured (Fig. 7A). Our results showed that P. fluorescens could effectively rescue intraspecies allelopathy in nontreated plants. However, when P. fluorescens was applied at the emitter site, there was no significant difference in root length, dry weight or fresh weight. Furthermore, gas chromatography analysis was performed on both the AOA and 1-MCP treatments (Fig. 7B-E). The essential oil content was similar (Fig. S8).

Fig. 7.

Fig. 7

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Effects of ethylene inhibitors on receptor plants when emitters are treated with SDW or P. fluorescens. (A) Experimental set. (B-E) Growth situation of A. lancea. Error bars with different letters indicate a significant difference according to one-way analysis of variance (ANOVA) with Duncan’s multiple comparison test (p < 0.05) (n = 4)

Transcriptome analysis confirmed that ethylene is involved in growth promotion and essential oil accumulation

To confirm the growth promotion and essential oil accumulation effects of P. fluorescens inoculated with A. lancea on neighboring A. lancea, a transcriptome analysis was conducted to compare CK, PI and NP on Day 14. In total, 127,720 genes were identified, of which 61,571 (48.21%) were annotated. Compared with the CK treatment, the PI treatment upregulated 1082 genes and downregulated 141 genes, whereas the NP treatment upregulated 2188 genes and downregulated 1407 genes (Fig. 8A). PI and NP shared 655 (27.37%) differentially expressed genes (DEGs) (Fig. 8C). KEGG enrichment analysis revealed that both the PI treatment and NP treatment altered the pathways involved in DNA replication, terpenoid backbone synthesis and plant hormone transduction (Fig. 8B, D).

Fig. 8.

Fig. 8

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Functional distribution of differentially expressed genes (DEGs) between P. fluorescens-inoculated A. lancea (PI), neighboring A. lancea of the PI (NP) and untreated A. lancea (CK). (A) DEGs between the PI, NP and CK groups. (B) KEGG pathway enrichment of the top 10 DEGs in the PI vs. CK comparison. Red indicates genes whose expression was upregulated, whereas blue indicates genes whose expression was downregulated. (C) Venn diagram of DEGs between PI vs. CK and between NP vs. CK. (D) KEGG pathway enrichment of the top 10 DEGs between the NP group and CK group. (E,F) Expression of DEGs associated with terpenoid synthesis, plant growth and the ethylene response. The color changing from blue to red indicates that log2 (FPKM + 1) gradually changes from small to large

Both PI treatment and NP treatment upregulated genes associated with the MVA pathway, including hydroxymethylglutaryl-CoA synthase (AlHMGS), 3-hydroxy-3-methylglutaryl-coenzyme A reductase (AlHMGR2), and geranyl-diphosphate synthase (AlGGPS1) (Fig. 8E). Interestingly, NP treatment downregulated 1-deoxy-D-xylulose 5-phosphate reductoisomerase (AlDXR), a gene associated with the MEP pathway (Fig. 8E). This downregulation could be used as a strategy to acquire more material for the MVA pathway.

Both treatments also downregulated the ethylene signaling pathway, as evidenced by the significant downregulation of ethylene-responsive transcription Factor 109 (AlERF109), AlERF017, AlERF3, and AlERF5 (Fig. 8F). Moreover, in the NP, AlERF1 and AlERF2 were also downregulated. All these factors are known as positive responders to ethylene [34, 35].

Discussion

Intraspecific allelopathy is a major issue in medicinal plant culture. On the other hand, endophytes are capable of regulating secondary metabolism and VOCs. As a usage of endophytic against allelopathy via metabolism regulation of host, our results proved that endophytic P. fluorescens ALEB7B could promote the growth of noninoculated neighbors of the host by reducing host ethylene emission.

Intraspecific allelopathy is a part of plant‒plant communication and a common phenomenon in the plant community, especially for these medicinal plant species. Most previous studies focused on terpenoids, flavonoids or aromatic compounds, such as baicalin released from Scutellaria baicalensis, as allelopathy media [36]. ALEB7B incubation the VOCs change cause by ALEB7B did not bring benefit to neighbor plants nor last more than 5 days (fig. S3, S4). These results indicating that the change of typical VOCs is not the reason of relieving of allelopathy. Like previous studies reporting that ethylene can be emitted as a VOC and an inhibitor of root growth in neighboring plants [14], our experiments revealed that ethylene, as an allelopathy medium, plays an important role in the allelopathic effect of A. lancea on intraspecific neighbors (Fig. 3). In our experiment, an ethylene emitter at a rate of 2.5 nL g− 1 f.wt h− 1 is sufficient to cause allelopathy effect upon an none-inoculated A. lancea (Fig. 3). Our results are consistent with those of previous studies in which ethylene was shown to have allelopathic effects on Nicotiana plants, attenuating and inhibiting root growth [14]. It is reasonable that ethylene also plays an important role in allelopathy transduction. Interestingly, different species presented similar ethylene emission rates. A previous study reported that peanut roots emit ethylene at a rate of 5 nL g− 1 f.wt h− 1, although in their research, ethylene is a direct regulator of rhizosphere microbes [37]. IIn potato, this rate is approximately 0.2–2 nL g− 1 f.wt h− 1 [38]. The similar ethylene emission rates suggest that an intraspecific allelopathic effect mediated by ethylene could exist among many species.

Endophytic microorganisms are commonly found in most plants, and represent a promising opportunity to relieve allelopathy. The symbiosis between plants and microorganisms could help not only in nutrient absorption or resistance to pathogens [39, 40] but also in the direct manipulation of hormone compounds such as gibberellin or IAA [41, 42], which is common. In addition to IAA, P. fluorescens can degrade the precursor of ethylene, ACC [43]. Thus, microorganisms such as ALEB7B provide a raising opportunity to relieve allelopathy. Although many studies have provided great insight into the interaction between endophytes and their hosts, for most endophytes, their role in the plant community is still unknown. Previous studies have focused on the degradation of allelopathic compounds, or the absorption of allelopathic compounds, by biological or nanomaterials [44, 45]. It is reasonable to hypothesize that endophytes would show an increased allelopathy against noninoculated neighbors for two reasons. One thing is because of the growth-promoting promotion effect of endophytes. The other is because during symbiosis, there is an increased likelihood that the host will produce more secondary metabolites, such as terpenes or flavonoids, which exhibit allelopathic effects against neighbors [46, 47]. In our research, ALEB7B showed strong activity and reduced the amount of ethylene emitted from A. lancea to a rate of 0.5 nL g− 1 f.wt h− 1 (Fig. 3A), whereas gene expression related to ethylene synthesis did not show siginificance1 (Fig. 6). These results suggest that the change in the ethylene response is due to changes in the ethylene emission change of emitters. These results are also an extension of a previous study on how bacteria with ACC deaminase affect their hosts. Endophytes, including endophytic bacterium, have advantages for their hosts, such as growth promotion, pathogen resistance and abiotic stress resistance [48]. However, our findings indicate that endophytic P. fluorescens ALEB7B not only benefits not only its host A. lancea plants but also its neighbors.

Recruiting endophytic microorganisms like P. fluorescens ALEB7B could be a natural way to relieve intraspecific allelopathy. The absence of venting in simple green systems can result in a reduced yield of crops due to ethylene accumulation. As a solution, P. fluorescens is not only an endophytic bacterium but also a soil bacterium that can be recruited by emitting VOCs such as a-pinene [38]. The existence of P. fluorescens in the field may explain why the allelopathy of ethylene has been disregarded during the growth phase in former research. In this study, the results from both bottle and pot experiments demonstrated that A. lancea inoculated with P. fluorescens strain ALEB7B exhibited enhanced growth and increased essential oil in host and nearby A. lancea (Figs. 1B-E and 2C-F). The allelopathy-selected microbiomes relieve the allelopathic effect can be also seen in other species [49].

In conclusion, our results reveal a novel allelopathic mechanism mediated by a classic hormone, ethylene, produced by neighboring plants. This chemical agent decreases the root length and dry weight of nearby A. lancea. Fortunately, we discovered that this harmful effect can be suppressed by the endophytic bacterium P. fluorescens. Consequently, improved growth of nearby plants can be achieved (Fig. 9). Our research revealed that P. fluorescens ALEB7B down-regulated the emission of ethylene, which act as a VOC that inhibits the growth of neighbors in A. lancea. These findings indicate that the endophytic P. fluorescens ALEB7B benefit not only the host, but also the neighbor plants. This study provides a new perspective on how endophytic bacteria achieve their growth promotion effect and highlights the ability of P. fluorescens to counteract the negative effects of ethylene as an airborne phytohormone signal.

Fig. 9.

Fig. 9

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Schematic summarizing how P. fluorescens relieves intraspecific allelopathy in A. lancea

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (6.6MB, xls)
Supplementary Material 2 (6.9MB, docx)

Acknowledgements

We thank Dr. Yong Jia for advices to our experiment design.The manuscript has a former version as a preprint at Authorea (Doi: 10.22541/au.165886750.01373206/v1). Link: https://www.authorea.com/users/497625/articles/578608-endophytic-pseudomonas-fluorescens-relieves-intraspecies-allelopathy-in-atractylodes-lancea-by-reducing-ethylene-emissions. The license “Non-exclusive, no reuse license” was chosen and the license term is as follows: The copyright holder for this preprint is the author/funder. All rights reserved. No further reuse allowed without permission.

Abbreviations

SDW

Sterilized Distilled Water

VOC

Volatile Organic Compound

Author contributions

LSC, KS and CCD formulated the idea and designed the experiments. LSC performed the experiments, analyzed the data and edited the manuscript; DW and CYW provided the plant material; FC directed the data analysis of the transcriptome; WZ corrected the experimental design. YU revised the manuscript. All authors read and approved the final manuscript.

Funding

This work was financially supported by Key Project at Central Government Level: The ability establishment of sustainable use for valuable Chinese medicine resources (2060302), and a project funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.

Data availability

The Illumina sequencing data of A. lancea plantlets were submitted to the NCBI Sequence Read Archive (SRA) under the accession number PRJNA900630 (available at https://dataview.ncbi.nlm.nih.gov/object/PRJNA900630?reviewer=jgjj6f2k2c2bmmannvnbseqf67).

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Kai Sun, Email: sunkainnu@sina.cn.

Chuan-Chao Dai, Email: daichuanchao@njnu.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (6.6MB, xls)
Supplementary Material 2 (6.9MB, docx)

Data Availability Statement

The Illumina sequencing data of A. lancea plantlets were submitted to the NCBI Sequence Read Archive (SRA) under the accession number PRJNA900630 (available at https://dataview.ncbi.nlm.nih.gov/object/PRJNA900630?reviewer=jgjj6f2k2c2bmmannvnbseqf67).

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