Plant growth promoting bacteria induce anti-quorum-sensing substances in chickpea legume seedling bioassay

Abstract

Microorganisms and their hosts communicate through an array of signals. Many physiological processes regulated in quorum sensing (QS) are dependent on auto-inducers, like N-acyl-homoserine lactones (AHLs) as in numerous groups of both gram-positive and gram-negative bacteria. In vitro grown seven-day old chickpea seedlings treated with plant growth promoting bacteria (PGPRs) were used to screen the AHL mimicking and for phytochemical substances like phytohormones and secondary metabolites such as phenolics and flavonoids. Potential anti-quorum sensing (anti-QS) activity surrounding the roots on semi-solid agar lawn of Chromobacterium violaceum (ATCC12742) was observed. Crude protein (4.46–8.30 μg/mL) and methanolic extracts (100 μg/mL) of seedling gave moderate anti-QS activity against CV12742 anti QS bioassay, respectively. Crude protein and methanolic extract of Bacillus amyloliquefaciens (34.00 ± 2.23; 34.00 ± 4.33 mm) and B. subtilis A (27.00 ± 2.10; 3.29 ± 2.16 mm) treated samples showed higher zone of inhibition due to anti-QS activity. Phytohormone analysis using LC–MS for zeatin, auxin and methyl jasmonate (MeJA) indicated that phytohormones were significantly upregulated by 1909.80 ng/g FW, 669.67 ng/g FW and 244.55 ng/g FW, respectively in Pseudomonas brassicacearum treated seedlings compared to control. UHPLC of PGPR treated seedlings showed overly expressed gallic acid, protocatechuic acid, catechin, p-hydroxybenzoic acid, caffeic acid, catechol, vanillin, and ferulic acid in B. amyloliquefaciens treated seedlings compared to others. Enrichment analysis identified significant pathways related to metabolism, biosynthesis of secondary metabolites. The present study indicates that chickpea neutralizes an extensive range of functional responses to AHLs that may play important role in legume host-microbe interactions.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12298-021-01034-x.

Introduction

The communication amongst bacteria with their host through the inter-kingdom signalling is complex and needs extensive research to understand the same. Gene regulation in response to the population density in bacteria is called as quorum sensing (QS) and also involved in microbial cross-talk (Miller and Bassler 2001; Whitehead et al. 2001; Wang et al. 2020). The bacterial signals thus go around monitoring their population and coordinate it to their gene expression vis-à-vis to the occurrence of a defined quorum of cells (Fuqua et al. 1994; Blake et al. 2021). QS may be ascertained by the synthesis of a small diffusible molecules, N-acyl-homoserine lactone (AHLs), being the common QS signal, cause for plant diseases (Monaghan and Zipfel 2012; Singh et al. 2017; Billot et al. 2020). Several AHL degrading enzymes, in various bacterial forms are used as quorum quenchers (QQ) (Chen et al. 2013; Paluch et al. 2020). Thus, AHL signalling therefore induces and affects many aspects of bacterial development, survival, and interaction within eukaryote hosts (Fuqua and Winans 1994; Singh et al. 2017). Higher plants, such as pea, rice, soybean, and tomato produce substances that appear to mimic the activities of AHL and are known to show specific effects on QS regulated behaviours in bacteria (Gao et al. 2003; Asfour 2018). Inhibition of bacterial QS may take place through different mechanisms viz. by inhibition of AHL synthesis, AHL transport and/or secretion, sequestration of AHLs, antagonistic action; and inhibition of targets downstream of AHL receptor binding (Mathesius et al. 2003; Mhlongo et al. 2018).

The plant growth promoting bacteria (PGPRs) can stimulate plant growth directly by an in situ production either by enhancing plant hormones or by enhancing plant nutrient uptake (Pathak et al. 2019). It is reported that plants treated with a consortium of PGPR significantly enhanced the chlorophyll, protein, and sugar contents compared to either irrigated and/or drought conditions (Khan et al. 2020a). PGPRs produce plant signalling molecules such as auxins, cytokinins, gibberellins, abscisic acid (ABA), salicylic acid (SA), ethylene (ET) and jasmonic acid (JA) (Fahad et al. 2015; Khan et al. 2019). Findings have shown that secondary metabolites produced from plants, fungi, eukaryotic and even from animals interfere with bacterial cell-to-cell communication (Srivastava et al. 2020).

Chickpea (Cicer arietinum L.), a member of  Fabaceae family of plants is the third most vital food legumes of the world (FAO 2013). In this study, we have employed the biosensor assay using Chromobacterium violaceum strains CV026, CV31532 and CV12472 to detect the presence of any exogenous AHL/s that may have been leached out by the chickpea roots /seedlings during various PGPR treatments. Chickpea is widely used for the studies in host-microbe interaction including the N-fixing bacterial symbionts, symbiotic mycorrhizae, abiotic stress, phytochemical analysis and various bacterial and fungal pathogens (Cook 1999; Gao et al. 2003; Gopalakrishnan et al. 2018; Bhagyawant et al. 2018a, b, 2019). The objective of this study was to evaluate plant growth promotion and phytochemical analysis of bioactive compounds and their anti-QS activity of PGPRs treated chickpea seedling. This study provides evidence for the developmentally regulated secretion of some of the signal inhibitors both as proteins and/or secondary metabolites. This study seems unique as far as QS studies on PGPRs and more so chickpea seedling as bioassay is concerned.

Material and methods

Plant material

Chickpea (Cicer arietinum L.) seed, variety JG-16 was used on basis of agronomical traits as test plant material. Seeds were obtained from the Rajmata Vijayaraje Scindia Krishi Vishwavidyalaya, Gwalior, India. Healthy chickpea seeds were surface sterilized with 0.01% cetrimide for 5 min and rinsed 6 times with sterile distilled water. Randomly selected 100 g seeds from the bulk of 1000 g seeds were used for treatment with PGPRs.

Plant growth promoting rhizobacteria (PGPR)

Five PGPR strains isolated from potato roots were generously provided by the Mycorrhiza and PGPR laboratory of Prof. K.K.Koul, School of Studies in Botany, Jiwaji University, Gwalior, India. The numbers of bacterial cells for inoculation were approximately adjusted at 10⁸ CFU/seed (Table 1) (Pathak et al. 2019). The PGPRs used are all identified based on 16S rRNA gene sequencing and submitted accession with NCBI, USA.

Table 1.

Bacterial isolates from rhizosphere and non-rhizosphere (bulk) soil on the basis of 16 s rRNA gene sequence (Pathak et al. 2019)

Bacterial origin	Isolates	Bacterial strain	Similarity in percentage (%)	Accession number from NCBI
Rhizosphere	PR1	Bacillus amyloliquefaciens DRKJUPR1	99	KR817726
	PR3	Bacillus subtilis DRKJUPR3	99	KR817727
Non rhizosphere	PB10	Lysinibacillus boronitolerans DRKJUPB10	93	KT031988
	PB11	Pseudomonas brassicacearum DRKJUPB11	96	KR817725
	PB12	Bacillus subtilis DRKJUPB12	99	KR817724

Preparation of bacterial inoculum for chickpea

Five PGPRs viz. Bacillus amyloliquefaciens (KR817726) and Bacillus subtilis A (KR817727) are both rhizospheric soil borne whereas Lysinibacillus boronitolerans (KT031988), Pseudomonas brassicacearum (KR817725) and Bacillus subtilisB (KR817724) are bulk soil borne, were used for the study. Each of these was selected individually for seed treatments. Selected PGPRs were grown as cultures in the nutrient broth (Hi-media, M002, Mumbai, India) kept under shaking incubator at 120 rpm for 48 h at 30 ± 2 °C. Surface-sterilized chickpea seeds (100 g each) were dipped into the respective PGPR broth for an hour under sterile conditions in batches. 100 g seeds soaked in the sterile distilled water served as control (Dasgupta et al. 2015). A batch of 10 seeds from each treatment was spread on sterile petri plates lined with sterile wet filter paper. Both control and treatment petri plates were irrigated with 10 mL solutions containing bacterial suspension. 1 ml of sterile distilled water was added at alternate days to the petri plates for maintaining moisture for germination. These petri plates were incubated at 25 ± 2 °C for seven-days in an incubator under dark conditions.

Sample preparation and quorum sensing bioassay

The micro-organisms used in this study i.e. Chromobacterium violaceum ATCC31532, C. violaceum ATCC12472 and C. violaceum CV026 for AHL activity were obtained from microbial culture repository of Yenepoya research centre, Mangalore, Karnataka, India. Bacterial culture media Luria Bertani (LB) were purchased from M1245 Hi-Media, Mumbai, India. Seven-dayold treated and non-treated, roots only, were ground in the liquid nitrogen and the homogenate from each treatment was stored in a deep freezer at -80 °C. From this, at an appropriate date, respective methanolic (Analytical grade, Hi-Media, Mumbai, India) extraction of 100 mg tissue was done. After centrifugation at 10,000 rpm for 10 min at 4°C the supernatant was preserved at -80 °C for phytochemical analysis and quorum sensing bioassays. Total protein content was analyzed by the method of Bradford (1976).

Detection of PGPR producing N-acyl-homoserine lactones (AHLs)

The AHL biosynthesis activity of the rhizospheric soil and bulk soil bacteria was assayed with cross-streaking with the C. violaceum31532 and C. violaceum CV026, a mini Tn-5 mutant, which responds only to exogenous AHL. B. amyloliquefaciens, B. subtilis A, L. boronitolerans, P. brassicacearum, and B. subtilisB were separately cross streaked with C. violaceum CV026. CV31532 strain was used as a positive control. After incubation for 24 h at 32 °C, strains were observed for whether AHL molecules were produced or not by the PGPR strain treatments. This could be ascertained by the induction of the formation of purple pigmentation in C. violaceum CV026 (McClean et al. 1997; Robson Ee et al. 2013).

Isolation of AHL molecule

The method of Shaw et al. (1997) was adopted for AHL isolation by growing C. violaceum31532 in Luria broth (LB) incubated at 32 °C for 18 h. After centrifugation, the supernatants were pooled and then extracted with acidified ethyl acetate in the ratio 7:3 (ethyl acetate, v/v acetic acid). The partitioned ethyl acetate extracts were finally vacuum dried at 40 °C and reconstituted in acetonitrile. The amount of this partially purified AHL needed for the production of violacein by CV026, was standardized by agar well diffusion plate assay (Fatima et al. 2010).

Detection of anti-quorum sensing activity

Four-day old aseptically grown seedlings were placed on the thin layer of soft agar containing the bacteria. The plates were incubated for 24 h at 32 °C. Inhibition of pigment production near the seedling indicated the quorum sensing inhibition (Teplitski et al. 2000). Agar well diffusion assay with C. violaceum CV12472 and CV026 was performed for the determination of pigment inhibition or enhancement due to seed and seedling extract. LB agar medium plates were spread with 0.1 mL of appropriately diluted (~ 2.5 × 10⁶ CFU/mL) freshly grown cultures and wells of 8 mm diameter. The solvent only was used as a control. Petri plates were incubated at 32 °C for 24 h and the zone of pigment inhibition/ enhancement around the well was determined (Fatima et al. 2010).

Sample preparation and phytohormone analysis using LC–MS

The endogenous levels of plant hormones were estimated by liquid chromatography-mass spectroscopy (LC–MS) using a protocol modified from (Pan et al. 2010). Root tissue (200 mg) from each sample was homologized using a mortar and pestle (IKA, Staufen, Germany) suspended in 500 μL of extraction solvent (2-propanol/Milli-Q water/concentrated HCl; 2:1:0.002 v/v/v) and 1 mL of chilled dichloromethane was added, and mixing was done for 30 min. Dichloromethane purchased from Avantor Performance, Panoli, Gujarat, India. The organic and aqueous phase was separated by centrifuging at 10,000 g for 5 min at 4°C. The aqueous phase (900 μL) was transferred to a fresh Eppendorf tube, and dried completely in vacuum using a Speed Vac (Thermo Scientific) for 45 min. Dried samples were dissolved in chilled methanol 70 μL and subsequently transferred to an injection vial. The separations of phytohormones were done on a Hyperreal GOLD C₁₈ column (2.1 × 75 mm, 2.7 µm) (Thermo Scientific). Analysis was done using Exactive^™ Plus Orbitrap mass spectrometer (Thermo Fisher, 188 Scientific, USA) coupled with Ultra performance Liquid Chromatography (UPLC) (Waters, Milford, MA, USA) (Tyagi et al. 2015). The samples were then reconstituted in 70µL of LC–MS grade methanol and Milli-Q water solvent in the ratio of 1:1 (v/v). These were properly mixed using vortex spin and centrifuged for 5 min at 4°C and then transferred to auto-sampler vials to be kept in the sampler tray maintained at 4 °C for analysis. Unlabeled phytohormones were purchased from Sigma-Aldrich Co. (Steinheim, Germany).The detection of abscisic acid (ABA), gibberellic acid (GA), jasmonic acid (JA), and salicylic acid (SA) was performed in all ion fragmentation (AIF) mode (range of m/z 50–450) with positive heated electrospray ionization (ESI) in negative ion mode. The zeatin, auxin (IAA), indole butyric acid (IBA), methyl jasmonate (MeJA), and epibrassinosteroid were analyzed using Turbo ion spray source in positive ion mode. The levels of endogenous hormones were quantified by using standard curves for the following growth regulators-ABA, zeatin, IAA, MeJA, and SA (Kumari et al. 2017).

Phenols and flavonoids analysis using UHPLC

The standard chemicals like phenolic acids (rutin hydrate, gallic acid, p-hydroxybenzoic acid, syringic acid, vanillic acid, ellagic acid, quercetin dihydrate, protocatechuic acid, ferulic acid, p-coumaric acid, catechol, caffeic acid, catechin, Naringin, saponin, Quercetin, vanillic acid) were procured from Sigma Aldrich Co. (St. Louis, MO, USA). All other solvents used were of HPLC grade. Standards were dissolved in HPLC grade methanol. 20 μL of test sample representing 10 ng/μL from a working stock of 1 mg/mL was injected into the system. Fresh tissues (1g) of respective treatments were frozen with liquid nitrogen pulverized with the help of pestle and mortar (IKA, Staufen, Germany) extracted with 80% methanol in a 1:1 (w/v) ratio and vortexed for 1 h. Cell debris was pelleted by centrifugation at 10,000 g (Sorvall lynx 6000, Thermo Scientific, MA, USA) for 10 min and supernatants were evaporated to 100 μL. After filtration through 0.22 μm filters, samples were subjected to ACCELA UHPLC (Thermo Fisher Scientific, Bremen, Germany) with four solvent delivery systems, a quaternary pump including a photo diode array detector. The separation was achieved over a reverse-phase Luna C18 column (5-micron particle size, i.d. 4.6 × 250 mm) (Phenomenex, USA). UHPLC chromatographic detection was done using a photo diode array UV detector at three different wavelengths of 272, 280, and 310 nm. The absorption maxima of analyzed compounds were observed. Protocols for the standards and the samples were kept similar (Seal 2016).

Protein–protein interaction networks (PPI) and pathway enrichment analysis of differentially expressed genes

On the bases of biochemical and morphological parameters, B. amyloliquefaciens treated root chickpea var. JG-16 induced was selected for 2D Gel electrophoresis and identified by MS–MS analysis (unpublished data). Phenylalanine ammonia-lyase 1 (Cicer arietinum) was computed and identified by the protein–protein interaction from latest version of STRING (11.0) database (https://string-db.org/) (Cao et al. 2014; Szklarczyk et al. 2019). Differentially expressed protein coding genes were used as input for pathway enrichment analysis using GeneSCF tool (Subhash and Kanduri 2016), wherein, 24,851 gene set as background to perform enrichment analysis using KEGG ‘cam’ (Cicer arietinum) as reference database in GeneSCF. Significant terms are filtered by considering p value < 0.05.

Statistical analysis

The comparisons of the plant growth-regulating effects of PGPR bacteria for both treated and controls were conducted in an absolute randomized design. Each treatment was replicated 3–5 times, and the whole experiment was independently repeated three times. The treatment effects were derived by calculating the mean ± standard deviation using Microsoft Excel, 2010.

Results

Quorum sensing bioassay

Detection of N-acyl-homoserine lactones (AHLs) producing PGPR

Five PGPRs viz. B. amyloliquefaciens, B. subtilis A, L. boronitolerans, P. brassicacearum and B. subtilisB have been subjected to detection for any AHLs mediated QS activity against CV026. As B. amyloliquefaciens, B. subtilis A, L. boronitolerans and B. subtilis B are Gram-positive forms and do not produce the AHLs molecules, since gram-positive bacteria are known to lack AHLs. However, they use different cell-to-cell signalling molecules and pathways (Robson Ee et al. 2013). In the present study, during interactions of PGPRs, it was observed that the AHL molecule movement from PGPRs against CV026 was absent (Fig. 1).

Fig. 1.

Chromobacterium violaceum CV026 used as biosensor strain for detecting exogenous activity with PGPRs. i CV026 produces purple pigment in response to CV31532 which is naturally producing violacein pigment, ii CV026 strain with B. amyloliquefaciens (PR1), iii CV026 strain with B. subtilis A (PR3), iv CV026 strain with L. boronitolerans (PB10), v CV026 with P. brassicacearum (PB11), vi CV026 strain with B. subtilis B (PB12). No pigment induction was observed with CV026 by any of the PGPRs when compared to controls

Isolation of AHL molecule

Assay with CV026 required standard amount of natural AHL extracted from CV31532 strain. CV31532 showed the production of violacein pigment under an influence of naturally extracted AHLs (Fig. 2). The results of the biological assays were examined with QS indicator strains C. violaceum CV31532 which is a naturally AHLs producing bacteria and used as a positive control vis-à-vis a mutant CV026 which is a type of non AHLs producing bacteria. This strain has therefore, made it easy to detect any such activity. CV026 strains produce a response specifically to C4 and C6 AHLs. Our observations show that there is an AHL mediated induction of purple pigment violacein (Fig. 2). The Bacillus group of PGPRs are gram-positive hence are unable to produce AHL molecules and as such no violacein through AHL is mediated (Fig. 1).

Fig. 2.

Induction of violacein formation by Chromobacterium violaceum CV026 in the presence of natural C6 AHL extracted from CV31532

Detection of anti-quorum sensing activity

QS modulatory activity of chickpea seedling using C. violaceum strains as bioassay showed inhibition of pigment production in CV12472 while CV026 does not show any activity with soaked seed, root extract, and/or seedling extract. Assay with PGPR treated four-day old seedling showed the inhibition of violacein pigment surrounding the root exudates on C. violaceum strain on soft LB agar plate after 24 h incubation. It may, therefore, be that the natural compounds exuded from the root part of seedlings passes anti-QS activity or produces a bio-control activity with an indirect presence of PGPRs. The AHLs signalling inhibition activity by all the five PGPRs used here was significantly high (Fig. 3). Observations with crude protein extract of whole chickpea seedling showed moderate anti-QS activity with CV12742 assay at protein concentration 4.46–8.30 μg/mL (Fig. 4A and 5A). Besides, crude protein in 100 µL volume the methanolic extracts of PGPRs treated root from seedlings were also tested with 100 μg/mL volumes, and these extracts were effective as anti QS against the CV12472 assay (Figs. 4C, D and 5C, D; Supplementary Table 1–4).

Fig. 3.

Summarized activity of violacein inhibition of Chromobacterium violaceum CV12472 by seven-dayold germinated chickpea seedling treated with PGPRs and non-treated chickpea seedling along with soaked seed on Luria agar plate i Soaked seed, ii aseptically grown control seedling (non-treated) iii B. amyloliquefaciens (PR1), iv B. subtilis A (PR3) treated seedling, v L. boronitolerans (PB10), vi P. brassicacearum (PB11), vii B. subtilis B (PB12) treated seedling. PGPR treated seedling showed a clear zone of pigment inhibition. Scale used 100 mm

Fig. 4.

CV12472-agar plate bioassay resulting in localized violacein pigment inhibition (mm) against crude chickpea protein from whole seedling (A) and root (B) and methanolic extracts of seedling (C) and root (D) treated with B. amyloliquefaciens (PR1), B. subtilisA (PR3), L. boronitolerans (PB10), P. brassicacearum (PB11) and B. subtilis B (PB12). B. amyloliquefaciens (PR1) B. subtilis A (PR3) and B. subtilisB (PB12), show the largest zone of inhibition

Fig. 5.

CV12472-agar plate bioassay resulted in localized pigment inhibition against crude chickpea protein from whole seedling (A) and root (B) and methanolic extracts of seedling (C) and root (D) treated with ii B. amyloliquefaciens, (PR1) iii B. subtilis A (PR3), iv L. boronitolerans (PB10), v P. brassicacearum (PB11) and vi B. subtilis B (PB12). i of a–d indicates soaked seed (a), Control (non-treated) seed (b) and Tris–HCl solution (for crude protein) or methanol (for methanolic extract) (c)

Phytohormone analysis using LC–MS

Growth regulator level in chickpea root exudates of seven-day old chickpea seedlings treated with already mentioned five PGPRs viz. B. amyloliquefaciens, B. subtilis A, L. boronitolerans, P. brassicacearum, and B. subtilis B along with their controls were analysed. Test and control extracts were quantified by using HighPerformance Liquid Chromatography (HPLC/ESI–MS). Out of eight examined regulators, a total of five phytohormones viz. zeatin, IAA, MeJA, ABA and GA were detected in the targeted chickpea root samples (Figs. 6 and 7). Amongst five phytohormones, zeatin, MeJA, ABA and IAA showed higher levels in PGPR treated seven days old root exudates. Zeatin level is found highest among the other regulators and gibberellic acid 3 (GA₃₎ the least. Comparatively, the IAA level in soaked seed was absent and too low in the control root exudates (Fig. 6 and Supplementary Table 5). ABA hormone level was highest in P. brassicacearum i.e., 1642 ng/g fresh weight though significantly higher than control in B. amyloliquefaciens and L. boronitolerans, it was par below control in others. Our results suggest that JA signalling mediates plant-bacteria interactions in the root-soil vicinity once a pathogen or an herbivorous insect attack. This study shows that the concentration of MeJA (244.53 ng/g FW) was maximally present in the treatment by P. brassicacearum. This infers therefore, that in the chickpea seedling, roots are the largest provider of growth regulators, and fresh and dry matter contents are also higher in these samples (Fig. 6 and Supplementary Table. 5).

Fig. 6.

Quantification of growth regulators from PGPRs viz. B. amyloliquefaciens (PR1), Bacillus subtilis A (PR3), L. boronitolerans (PB10), P. brassicacearum (PB11) and B. subtilisB (PB12) treated with chickpea seedling root FW (fresh weight) respectively and and control root along with the simply soaked seed. A Zeatin, B IAA, C MeJA, D ABA and E GA3

Fig. 7.

Phytohormone standard chromatograms for both positive and negative mode. A For positive mode the peak details are 1. Zeatin 2. Indole-3acetic acid 3. Indole butyric acid 4. Methyl Jasmonate, B Negative mode 1. Salicylic acid 2. Gibberellic acid 3. Abscisic acid 4. Jasmonic acid with their peak retention time as their standards

Phenols and flavonoid analysis using UHPLC

The UHPLC analyses of methanolic extracts of PGPRs treated and non-treated root exudates revealed the presence of all twelve standards in varied amounts. A typical UHPLC Chromatogram of all the standard mixtures recorded at 272 nm is presented in Fig. 8H. As shown in the chromatograms, all investigated compounds had responses at 272 nm, where they were successfully identified. The constituents under investigation were also identified by the recorded absorption spectra, which were comparable for B. amyloliquefaciens, B. subtilis A, L. boronitolerans, P. brassicacearum, and B. subtilis B treated root exudates, non-treated root exudates and simply soaked seeds. Seeds soaked with distilled water (Fig. 8A) were having gallic acid, p-hydroxybenzoic acid, catechol, rutin, quercetin, and control non-treated seeds (Fig. 8B) were having gallic acid, protocatechuic acid, p-hydroxybenzoic acid, catechol, ferulic acid, and quercetin as per the comparative standards used. There were other compounds too which could not be identified or characterized because of their absence or presence in non-detectable amount. As for the PGPR treated root extract, B. amyloliquefaciens (Fig. 8C) treated root extract was having very promising results in the form of number of comparative standards identified as well as their amount when compared to the intensity of the peak in µAU with other treated treatments. In B. amyloliquefaciens, total 10 out of 12 standards were present followed by P. brassicacearum having 8 (Fig. 8F), B. subtilis A (Fig. 8D) and L. boronitolerans having 7 (Fig. 8E) and B. subtilis B had 5 standard comparative compounds (Fig. 8G), respectively. Also, among all the PGPR treated seed UHPLC profile in comparison to other PGPR treatments, treatment with B. amyloliquefaciens composed with high quantity of gallic acid, protocatechuic acid, catechin, p-hydroxybenzoic acid, caffeic acid, catechol, vanillin, and ferulic acid expressed by the seedling root. Quercetin expression steadily decreased in B. amyloliquefaciens, L. boronitolerans as compared to control non-treated seeds. Rutin was present in L boronitolerans and B. amyloliquefaciens while naringenin was present in P. brassicacearum only. Interestingly, out of all PGPR treated seeds compared to control, B. amyloliquefaciens showed comparative much better results in the present study (Fig. 8).

Fig. 8.

UHPLC chromatogram of mixed phenolic acid standards and root extracts at 280 nm. A Soaked seed in Distilled water, B Control non-treated, C B. amyloliquefaciens (PR1) treated, D B. subtilis A (PR3) treated, E L. boronitolerans (PB10) treated, F P. brassicacearum (PB11) treated, G B. subtilis B (PB12) treated chickpea seven-day old root extract (H). Standard used for analysis are 1. Gallic acid—4.67 min, 2. Protocatechuic acid—6.01 min, 3. Catechin—6.66 min, 4. p-Hydroxybenzoic acid—7.72 min, 5. Caffeic acid—7.93 min, 6. Catechol—8.18 min, 7. Rutin Hydrate—8.50 min, 8. p-Coumaric acid—10.42 min, 9. Vanillin—10.62 min, 10. Ferulic acid—11.22 min, 11. Quercetin—17.85 min and 12. Naringenin chalcone—19.02 min

Protein–protein interaction (PPI) and pathway enrichment analysis of differentially expressed genes

Protein–protein interaction (PPI) is known to successfully predict these as key regulatory/functional entities, which are involved in the plant–microbe interaction. STRING v 11.0 database was searched for the retrieval of interacting gene/proteins network based on either experimental or predicted interactions. Total 10 interactions appeared with Phenylalanine ammonia-lyase 1 (Cicer arietinum) LOC101496077 NBCI gene with exhibiting intercoms (Fig. 9). In the PPI network analysis of the result of chickpea- B. amyloliquefaciens DRKJUPR1 interactions in silico, it was observed that total of eleven proteins were detected using the STRING. It was found that in the chickpea and Bacillus spp. interaction metabolic pathways, the biosynthesis of secondary metabolites and biosynthesis of amino acids were enriched (Fig. 9 and Table 2). Of these enrichments, the most representative pathways were primarily metabolic pathways followed by secondary metabolite pathways and amino acids. The differentially expressed 149 proteins (unpublished data) out of that the Phenylalanine ammonia-lyase 1 (Cicer arietinum) showed to be associated with 15 metabolic pathways. KEGG pathway enrichment for differentially expressed genes. It was found that 14 KEGG pathways are enriched with terms related to photosynthesis, carbon fixation, carbon, fructose, and mannose metabolism (Figs. 10 and 11).

Fig. 9.

Protein–protein interaction network of protein phenylalanine ammonia-lyase 1 (Cicer arietinum L.) NCBI accession no (XP_004493977.1; Gene ID LOC101496077 with predicted functional proteins. Total 10 proteins were identified (Blue line represents known interaction from curated databases while experimentally determined known interaction is shown by pink line. Dark green line depicts predicted interactions (gene neighborhood), black line represents co-expression and light green line shows text-mining). The most representative metabolic pathways were as follows: Metabolic pathways (blue), Biosynthesis of secondary metabolites (red) and Biosynthesis of amino acids (yellow) (PPI enrichment p value: 9.47e−05) (color figure online)

Table 2.

Network interactions of phenylalanine ammonia-lyase 1 (Cicer arietinum L.) LOC101496077 NBCI gene and ID: XP_004493977.1 with predicted functional proteins

NCBI accession number	Predicted functional partners	Score
XP_004514691.1	Histidinol-phosphate aminotransferase, chloroplastic-like isoform X1 (414 aa)	0.936
XP_004510731.1	tyrosine/DOPA decarboxylase 1-like (516 aa)	0.932
XP_004493836.1	Tyrosine decarboxylase 1-like (489 aa)	0.932
XP_004511252.1	Probable aminotransferase TAT2-like (416 aa)	0.929
XP_004497538.1	4-coumarate—CoA ligase-like 7-like (558 aa)	0.886
CYP73A19	Trans-cinnamate 4-monooxygenase; Controls carbon flux to pigments essential for pollination or UV protection, to numerous phytoalexins synthesized by plants when challenged by pathogens, and to lignins; Belongs to the cytochrome P450 family (506 aa)	0.854
XP_004516098.1	4-coumarate—CoA ligase-like 1-like (555 aa)	0.851
XP_004500376.1	Trans-cinnamate 4-monooxygenase-like; Belongs to the cytochrome P450 family (505 aa)	0.842
XP_004511424.1	4-coumarate—CoA ligase 2; Upstream in-frame stop codon (590 aa)	0.841
XP_004487067.1	4-coumarate—CoA ligase-like 7-like (539 aa)	0.840
XP_004514691.1	Histidinol-phosphate aminotransferase, chloroplastic-like isoform X1 (414 aa)	0.936

Fig. 10.

Bar plot shows significantly enriched pathways with p < 0.05 from our MS data. The color gradient represents its significance level of enrichment [Score =  − log10 (p value)] and the number on top of each bar represents number of differentially regulated genes from MS data enriched in individual pathway

Fig. 11.

KEGG pathway of cam01110: Biosynthesis of secondary metabolites (Cicer arietinum L.). Pathway entered from cam_M00012 Glyoxylate cycle

Discussion

QS signalling molecules in C. violaceum are produced by specific QS systems, such as LuxI/LuxR (Ge et al. 2020). An earlier report says that Pseudomonas is a gram-negative soil bacterium however, a species P. brassicacearum produces signalling molecules that are different from AHL. The synthesis of Pseudomonas quinolone signal (PQS) transcription is said to be mediated by the P. aeruginosa. Two pair system of LuxI/LuxR regulates QS, homologous to Lasl/LuxR and RhlL/RhlR which are present in P. aeruginosa (Nazzaro et al. 2013). Several strains of Pseudomonas have been studied for their ability to colonize plant-related niches, such as the rhizosphere (e.g. P. aeruginosa, P. fluorescens, and P. putida), where they can act as plant growth-promoting rhizobacteria by antagonizing plant-deleterious microorganisms and through the production of traits that directly influence plant disease resistance and growth (Venturi and Keel 2016; Ortiz-Castro and López-Bucio 2019).

The effect of Auto inducer (AI) on the root development of chickpea opens new possibilities to identify plant growth-promoting bacterial strain on AI or AHL mimic compound production. Further, Bacillus species is reported to induce plant growth promotion through their ability to solubilize nutrients and produce phytohormones, secondary metabolites, and volatile organic compounds (VOCs) (Aloo et al. 2019). After chickpea seed treatment with different PGPRs viz. B. amyloliquefaciens, B. subtilis A, L. boronitolerans, P. brassicacearum and B. subtilis B, almost all PGPRs showed significant increase in the germination percentage and vigor index of chickpea seedling. Irrespective of the treatment, increase in the seed germination was about 11%, whereas the vigor index increased by 29-48%. Maximum fresh weight increment, 77%, was due to B. subtilis A and 73% due to B. amyloliquefaciens treatments. However, dry matter content showed almost two-fold increases in B. amyloliquefaciens and B. subtilis A compared to control. Additionally, other treatments showed high and significant dry matter content increment ranging between 52 and 84% with PGPR treatments. More than 60% increase is shown in shoot length by the PGPRs. Changes in total phenolic content (TPC), total flavonoid contents (TFC), total protein and antioxidant activity in seven-day old chickpea seedling is also observed (Manuscript submitted).

It is reported that several Bacillus species inhibit the quorum sensing (QS) activity through QS-interrupting strategies i.e. quorum quenching (QQ), enzymes viz. lactonases, acylases and oxidorreductases which degrade the QS signalling of other pathogenic bacteria (Dong et al. 2001). Lactones hydrolytic enzymes from Bacillus spp. have been identified in various QQ bacterial models (Rosier et al. 2021). In the present study, we have identified that PGPRs used for chickpea seed priming consequently, modulate the proteome and metabolome of the root exudates. Notably, the crude protein extract probably possesses QQ enzymes and/ or degrading proteins and peptides. On the other hand, methanolic extracts containing phytochemicals i.e. phenols and flavonoids modulate the QS activity of C. violaceum. Besides this anti-microbial activity of the PGPR treated root samples and control have already been observed with CV12472 (supplementary table1; 2). QQ represents interesting biocontrol strategy against phytopathogens, strategies involve PGPRs treated and metabolites thereof that interfere directly and indirectly break the fatty acid chain to the AHL ring. Vega et al. (2020) have highlighted the PGPR i.e. Staphylococcus equorum strain EN21 treated tomato plants under sterile condition used to attenuate phytopathogens virulence through QQ approach (Baltenneck et al. 2021).

In previous studies, it has been reported that the Bacillus cereus group of bacteria has the capacity of enzymatic inactivation of AHLs (Saeki et al. 2020). Further, genetic analyses revealed that AHL inactivation enzymes were almost in homology to that of AHL-lactonase gene (aiiAi) from Bacillus species strain 240B1 and were known to act by hydrolysing the lactone bond in the AHL (Dong et al. 2001; Kusada et al. 2019). In short, the AHLs dependent quorum quenching (QQ) can broadly have two classes viz. AHL lactonase and AHL acylases. Enzymes, which degrade AHLs have interestingly been worked out in AHLs producing bacteria and therefore,b could seemingly play a role in quorum sensing regulation (Huang et al. 2003).

The CV026 reporter strain is a mutant of wild type C. violaceum ATCC31532, which cannot synthesize N-hexanoyl-L homoserine lactone (C6-HSL). Herein, an assay with CV026 strain required a standardized amount of natural AHL extracted from CV31532 strain. In this study the chickpea treated with PGPRs inhibited the AHL mediated quorum sensing activity. Growing seedling of pea showed inhibition of pigment production in CV12472 and CV026 strains while seedlings of the other six legumes could not influence pigment production (Fatima et al. 2010). There are earlier reports to demonstrate that chickpea does not show any anti-QS activity (Fatima et al. 2010; Kalia 2013). However, post PGPRs treatment, chickpea seedlings did show the observable anti-QS as well as bacterial inhibition activity after 24 h. The progressively greater inhibition of AHL-induced violacein was observed in the PGPRs viz. B. amyloliquefaciens, B. subtilis A, L. boronitolerans, P. brassicacearum and B. subtilisB treatments when compared to control. This, therefore, infers that PGPRs can induce this through anti-QS molecules. Secreted or exudated metabolites are either species or genus specific and can be differentially modified depending upon their source of secretion. Given the strong complexity and specificity, root extracts and exudates have the capacity to develop a complex system of information for inter and also about the communication events amongst the rhizosphere organisms (Sasse et al. 2018; Mhlongo et al. 2018).

The methanolic and protein extract of PGPR treated chickpea seedling and root exudates showed an effective anti-QS activity which inhibited the growth of AHL indicator C. violaceum 12472. It is therefore, pertinently reported in this study, that PGPR treated chickpea root exudates and extracts produced some phytochemicals viz. phenols, flavonoids, polyphenols and proteins and protein-like compounds, being synthesized as a result of mutual response.

The phenolic extracts of the edible plant Rubus rosifolius inhibit pigment production, cluster movement, and biofilm formation in C. violaceum12472 (Oliveira et al. 2016; Yang et al. 2020). Similarly, UHPLC analysis of PGPR treated root expressed quantitatively higher amounts of gallic acid, protocatechuic acid, catechin, p-hydroxybenzoic acid, caffeic acid, catechol, vanillin, and ferulic acid. The proteomic studies reveal that AHL based QS regulation in Azospirillum regulates the functions during rhizosphere competence and adaptation in plant roots (Boyer et al. 2008). It is reported that co-cultivation of PGPR strains B. subtilis GB03, B. amyloliquefaciens IN937 with Arabidopsis thaliana under in vitro conditions and subjected to bacterial volatiles diffusion increased the A. thaliana growth. The two strains jointly produce volatile organic compounds (VOCs) like 3-hydroxy-2-butanone (acetoin) and 2, 3 butanediol (Mhlongo et al. 2018). Zhang et al. (2014) reported that citric acid identified from cucumber root exudates and extracts did attract B. amyloliquefaciens SQR9 and induced the formation of biofilm. In the case of banana root, fumaric acid exudates attracted, and stimulated B. subtilis N11 for biofilm formation (Qu et al. 2020).

Being a symbiotic relation the rhizobacteria either secrete or exudate molecules which are favourable to the plant. Different types of low carbon number molecules exhibited by the root exudates play a key role to activate or suppress the transcription factor for biosynthesis of phytohormone (Khan et al. 2020b). For example, tryptophan the indole-3-acetic acid precursor, are reported to be concentrated in the root tip region (Mhlongo et al. 2018). Flavonoid can act as modulators of auxin transport, and induce nodule development in numerous legumes (Ferguson and Mathesius 2014). Likewise, B. amyloliquefaciens treated chickpea root expressed putative indole-3-acetic acid-amido synthetase GH3.9 and Auxin-responsive protein IAA12-like isoform X2 in the present study. UHPLC analysis confirmed the presence of flavonoids, therefore probably a cross talk exists and so induce the plant growth enhancement. Auxin responses help detect nodule primordia through the expression of auxin-responsive reports (GH2 or DR5) during the indeterminate nodule development. Auxin probably plays various corresponding roles in infection, vascular tissue development and differentiation and cell cycle control. Dominantly auxin importantly plays a specific role in the overlap to auxin and PGPR induced response in roots (Mathesius 2020). ABA-responsive proteins were identified; ABA being crucial in plant growth and lateral root development and has also been involved in plant defense (Kwon et al. 2016). Auxin-responsive and flavonoid synthesis proteins were induced. The secretion of plant metabolites that mimic QS compounds were found, and these may have the potential to disrupt QS signalling by associated bacteria (Hartmann et al. 2014). Changes are known to result in PGPR interference with the main hormonal pathways which regulate plant root development; auxin, ethylene, and cytokinin being the main and to a lesser extent are GA, and ABA (Vacheron et al. 2013).

The novelty of this study seems to reflect that these QS and anti-quorum sensing inhibitors (QSI) and/or secondary metabolite synthesis being under auxin control IAA, being the main auxin produced by higher plants (Matilla and Krell 2018). In our data 100 mg fresh root from PGPR treated samples show a very good amount of IAA hormone upregulation compared to controls non-treated and soaked chickpea seed. In support of this notion, several studies have shown that microbially-produced IAA is involved in the suppression of plant defence responses, development of plant diseases, and plant growth promotion (Kunkel and Harper 2018). In mung bean plants, 3-oxoC10-HL induced adventitious root formation and mimicked auxin action through modifying the downstream second messenger's hydrogen peroxide, nitric oxide, or cGMP, suggesting the role of these messengers as mediators of root branching (Ortiz-Castro and López-Bucio 2019).

Production of zeatin (cytokinins) has been documented in several PGPRs viz., Bradyrhizobium japonicum, Paenibacillus polymyxa, and Pseudomonas fluorescens (Vacheron et al. 2013). Cytokinins are well-known to control root meristem differentiation, plant cell division, and induce proliferation of root hairs, but do inhibit lateral root formation and primary root elongation (Ghosh et al. 2018). The biosynthesis of phytohormone seems to be bacterial strain specific, and there are many such reports available to substantiate (Cassán et al. 2009; Ghosh et al. 2018). JA synthesis is activated following an interaction with symbiotic microbes that induce systemic resistance (ISR). Activation of the JA signalling pathway via application of MeJA is reported to alter the composition of carbon-containing compounds released by root. These compounds are implicated to decide the microbial community and its structure in the rhizosphere (Carvalhais et al. 2013). In this study too the quantitative and qualitative differential in hormone levels seems to be related to various PGPR types. In the LC–MS analysis a total of five phytohormones viz. Zeatin, IAA, MeJA, GA3 and ABA were detected in seven-dayold chickpea PGPR treated root segments; Zeatin and IAA levels being found to be the highest.

Major groups of compounds that are responsible for antimicrobial activity from plants include phenolics, phenolic acids, saponins, flavonoids, quinones, tannins, terpenoids, coumarins, and alkaloids (Asfour 2018). Variations in the structure and chemical composition of these compounds result in differences in their QS inhibitory action (Asfour 2018). It is reported that flavonoids are essential signalling molecules and control organogenesis of legume nodules and actinorhiza (Abdel-Lateif et al. 2012). Similarly, in the present study compounds like gallic acid, protocatechuic acid, catechin, p-hydroxybenzoic acid, caffeic acid, catechol, vanillin, and ferulic acid were expressed quantitatively higher by B. amyloliquefaciens treatment than the other PGPR treatments. Flavonoids di-phenyl benzopyrone derivatives from the root exudates are known to induce bacterial nod genes. QS Inhibition has also been reported in various fabaceae members like Glycyrrhiza glabra, Pterocarpa moniliformis, and Bauhinia acuruana. Vandeputte et al. (2011) stated that the phenolic naringenin action most probably manifests by a combination of (1) reduction of the production of both AHL molecules and (2) the capacity of the LuxR-type transcription factors to perceive their cognate molecules and thus results into a consequent reduction of the expression of QS-related genes (Asfour 2018; Gorlenko et al. 2020).

In this study the HPLC data shows the Rutin presence as identified in L. boronitolerans treatments. Rutin, vitexin and orientin reportably are very active in E.coli and C. violaceum in the QS test system (Husain et al. 2019). Quercetin (80 μg mL) showed a significant reduction in QS-dependent phenotypes such as violacein production, biofilm formation, exopolysaccharide (EPS) production, motility, and alginate production in a concentration-dependent manner. It can act as a competitive inhibitor for the signalling compound toward lasR receptor pathway (Gupta et al. 2015; Asfour 2018). These results suggest that the hormone pathways involved in plant defensive responses usually operate through complex networks of regulatory interactions. Studies have shown that bacteria have the potential to recognize plant-derived compounds (flavonoids) that help in rhizobia-legume interactions through many molecular signalling pathways (Mandal et al. 2010). Flavonoids can be both antagonistic and agonistic for rhizobia species (Mohd-Radzman et al. 2013). A role has been suggested for the phenylpropanoid pathway in plant resistance to pathogen attack where the production of monolignols strengthens the lignification of cell walls and provides mechanical resistance against pathogens (Jaeck et al. 1992; Yadav et al. 2020).

In addition to providing a physical barrier, flavonoids are also metabolic products from the phenylpropanoid pathway with important role in resistance. Phenylalanine ammonia-lyase (PAL) is the precursor enzyme of the phenylpropanoid pathway (Barros and Dixon 2020). The first step in this pattern leads to salicylic acid (SA) or by the determination of phenylalanine to cinnamic acid formation; the later being catalysed by PAL (Yuan et al. 2020). In the present study proteomic analysis, 2D, and MS-MS analysis of the treated and control chickpea root, PGPRs showed auxin presence and PAL, the key enzyme in phenylpropanoid metabolism (manuscript submitted elsewhere).

Proteins participate in complex networks of biochemical interactions both at functional and structural levels. These are therefore involved at DNA, RNA, other proteins, lipids, and other small metabolite levels too (Gautam et al. 2019; Ruas and Guerra-Sá 2020). 14 KEGG pathways were significantly enriched with terms related to metabolic pathways, secondary metabolic and amino acid pathways and as such metabolites as affected by B. amyloliquefaciens PGPR treatment of chickpea. Importantly, these metabolic pathways and those already mapped pathways are interlinked and thus cross talk in the KEGG online database which are assigned for multiple pathway metabolic reactions, including both necessary catabolic and anabolic ones. The necessary ones include those needed for carrying out processes for maintenance including glycolysis, pentose pathways, electron transport chain and other primary pathogen (Ruas and Guerra-Sá 2020). One of these, the phenylpropanoid pathway; being an important defense-related pathway, contained at least 96 DEGs in chickpea. These included the gene encoding phenylalanine ammonia-lyase (PAL)-a key regulator and the branch point enzyme between primary and secondary metabolism and other genes such as for 4-coumerate: CoA ligase 5-cinnamoyl-CoA reductase, caffeoylshikimate esterase, caffeoyl-CoA 3-O-methyltransferase, 4-hydroxyphenylpyruvate dioxygenase (Zha et al. 2019).

PGPRs thus may be modulating the transcriptome of the root, since these are now known to have biocontrol activities. They change the level of anti-QS or QS signals. They may also be improving the generation of new compounds therefore, producing direct or indirect responses in the plants. These compounds can also protect the plant from pathogenic microbes in the rhizosphere. The zone of inhibition was visible around the root exudates of the seedling and thus can be stated that PGPR treated seedling present an anti-QS activity. This study thus seems to present a maiden report in that the chickpea shows anti-QS activity with C.violaceum by showing violacein pigment inhibition in the chickpea root assay.

Conclusion

World over plant pathogenic microbes effect and cause diseases in wide range of agriculture crops. Antibiotics and chemicals have been used for many years to overcome the plant microbial infection. Consistent use of chemical pesticides and antibiotics effectively reduced the crop yield and induce improved resistance to these treatments. Serious environmental pollution has the subsequent results. Alternative strategies are therefore required to combat crop diseases and therefore induce improved plant growth. Mycorrhiza and PGPRs can be one of these alternatives. The extensively studied genera of PGPR include Bacillus and Pseudomonas (dos Santos et al. 2020). In this study it is being asserted that degrading enzymes probably lactonases, acylases and various phytochemicals especially phenol and flavonoids lead to major QS perception/ disruption. The enhancement of plant growth and nutrient uptake induced by beneficial rhizobacteria has been associated with the biosynthesis of phytohormones like auxins, gibberellins, jasmonates, zeatin and abscisic acid. KEGG enrichment analysis identified significant metabolic pathways, biosynthesis of secondary metabolites and biosynthesis of amino acids in PGPR treated seedlings.The results here may strongly suggest Bacillus spp., isolated from potato root rhizosphere as potential organism among the PGPRs to promote plant growth, development and yield. Both B. amyloliquefaciens and B. Subtilis A strains are competent to induce the plant–microbe interaction and these PGPRs are also competent to modulate the root exudates of chickpea to increase the plant defence mechanisms (Deryabin et al. 2019). Studies on quorum sensing systems demonstrate that PGPR bacteria too seem to have developed several forms of intra and inter species communication and additionally also anti-QS as an attractive alternative for anti-infective therapy.

Supplementary Information

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Declarations

Conflict of interest

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