Rhizosphere inoculation of PGPR strain Bacillus cereus BC56 enhances salt tolerance of cucumber

doi:10.21203/rs.3.rs-3590751/v1

Download PDF

Research Article

Rhizosphere inoculation of PGPR strain Bacillus cereus BC56 enhances salt tolerance of cucumber

https://doi.org/10.21203/rs.3.rs-3590751/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 30 Jan, 2024

Read the published version in Plant Growth Regulation →

You are reading this latest preprint version

Salt stress is a major cause of crop failure worldwide. The co-evolution of soil microbes and plants is essential for coping with extreme abiotic environments. In this study, a plant growth-promoting rhizobacteria (PGPR) strain B. cereus BC56 was isolated from the rhizosphere soil of cucumber. BC56 has the ability to produce NH₃ and siderophore, and to solubilize phosphorus to promote cucumber growth. Under 100 mM NaCl treatment, BC56 significantly increased shoot length (1.28-fold), root length (1.16-fold), shoot fresh weight (1.19-fold), root fresh weight (1.54-fold), and chlorophyll fluorescence parameter of ABS/CS_m (1.19-fold), TR₀/CS_m (1.22-fold), ET₀/CS_m (1.52-fold), SPAD (1.12-fold) of cucumber seedlings compared to the cucumber without BC56 inoculation. Physiological and biochemical analysis showed that BC56 could increase the content of total soluble sugars (TSS, 1.36-fold) in cucumber seedlings. BC56 also increased peroxidase (POD, 1.17-fold) and glutathione reductase (GR, 2.59-fold) activity of cucumber seedlings, which has the ability to scavenge reactive oxygen species (ROS) to reduce salt toxicity. However, cucumber seedlings inoculated with BC56 showed a 0.59-fold decrease in abscisic acid (ABA) compared to those uninoculated with BC56. RNA-seq results showed that BC56 induced changes in the expression of a significant number of genes related to photosynthesis, phytohormones, transcriptional regulation, metabolite synthesis and metabolism, and cellular components in cucumber under salt stress, suggesting its role in reducing the deleterious effects of salinity. We concluded that BC56 can alleviate salt stress in cucumber seedlings by affecting photosynthesis, phytohormone levels, osmotic and antioxidant regulation.

PGPR

B. cereus BC56

salt tolerance

cucumber

RNA-seq

Salinization and waterlogging currently affect more than 20% of the world's irrigated land. By 2050, this problem will affect more than 50% of the world's irrigated land if not addressed (Singh, 2021). The high salt content of saline soils limits plant nutrition and water uptake (Gong, 2021). Primary stresses, including osmotic and ionic stresses, are caused by water shortage and nutrient imbalance. These primary stresses lead to oxidative stress and can trigger a number of subsequent stresses (Zhu, 2002). By inhibiting photosynthesis and reducing the amount of available resources, salt stress affects plant growth by causing a variety of physiological and molecular changes, as well as preventing cell division and growth (van Zelm et al., 2020). PGPRs have been reported to have the ability to improve plant salt tolerance in several ways, including induction of the antioxidant system, increase in osmotic regulators, maintenance of phytohormone levels and water balance within the plant, and selective uptake of K⁺ and exclusion of Na⁺ to maintain a high K⁺/Na⁺ ratio (Gao, et al., 2022).

PGPRs are a class of probiotic bacteria that colonize the rhizosphere, promote plant growth and improve plant tolerance to various stresses(Egamberdieva et al., 2019; Gamalero and Glick, 2011; Glick, 2012; Vande Broek et al., 1999). PGPRs exist in many different species, some of which have been commercialised. These species include Pseudomonas, Bacillus, Enterobacter, Klebsiella, Azobacter and Serratia (Glick, 2012). Induced systemic tolerance (IST), a critical component of stress tolerance, is the result of the presence of PGPRs in plants. IST increases the plant's ability to withstand abiotic stresses (J. Yang et al., 2009). There are numerous reports on the use of PGPR to improve salt tolerance in plants. Sharma et al. (2016) isolated salt-tolerant and nitrogen-fixing strains from the rhizosphere of halophytes that can improve groundnut yield under 4–8% NaCl. By controlling pho-tosynthetic molecules, redox potential, stressor metabolites and ion homeostasis, Shahid et al. (2022) found that PGPR increased salt tolerance in radish. According to Gao et al. (2022), PGPR improved the salt-alkali tolerance of soybean soda and promoted plant health in alkaline soils.

Plant salt tolerance mainly involves hormonal regulation, osmotic regulation, antioxidant regulation and ion balance. Among plant hormones, ABA, ET, SA, and jasmonic acid (JA) are considered stress response hormones; others, including auxin, gibberellin (GA), cytokinins (CKs), brassinosteroids (BRs) and strigolactones (SLs), are classified as growth-promoting hormones (Verma et al., 2016). Under salinity stress, osmolytes such as proline, polyols and sugars accumulate. By reducing the osmotic potential in the cytosolic compartment, these osmolytes play a role in controlling osmotic pressure. In addition, they act as signalling molecules to increase ABA levels, influence associated gene expression, and control plant development under salt-stressed conditions (Marusig and Tombesi, 2020). Salt stress generates high levels of ROS in various organelles, leading to cellular damage. Plants have a well-regulated antioxidant mechanism to balance ROS synthesis and scavenging, prevent cellular damage and inhibit ROS overproduction. This mechanism includes active superoxide dismutase (SOD), catalase (CAT) and POD activity. Plants also accumulate glutathione, lipids, and other substances to mitigate oxidative damage by reducing ROS activity (Duan et al., 2012; Gill and Tuteja, 2010; Nadarajah, 2020; Yin et al., 2015). Furthermore, the ability of salt-tolerant plants to maintain cellular ion homeostasis in the face of ion excess is a critical adaptive trait. By decreasing cytoplasmic Na⁺ and increasing cytoplasmic K⁺, an optimal K⁺/Na⁺ ratio can be achieved in the cytoplasm, preventing cell injury and nutrient starvation (Yang and Guo, 2018). However, little work has been reported to elucidate the stress tolerance mechanisms induced by PGPR in cucumber. Therefore, elucidating the complex mechanisms of rhizosphere signalling during plant-PGPR interaction(s) is a subject of intense research.

In this study, a PGPR strain B. cereus BC56 was isolated from the rhizosphere of cucumber. The role of BC56 in improving the salt tolerance of cucumber was also investigated. In addition, the expression of genes regulated by BC56 was investigated by RNA-seq analysis. Morphological, physiological and biochemical, and transcriptomic analyses mutually confirmed the complex mechanisms regulated by BC56. These results will not only be helpful in exploring the mechanisms of PGPR underlying salt tolerance in cucumber, but will also facilitate the development of an effective bioformulation for such problematic soils in the future.

2.1. Plant materials and growth conditions

Cucumber seeds (Jinchun No. 4) were obtained from the Cucumber Research Institute, Tianjin Academy of Agricultural Sciences, China. Cucumber seeds were soaked in 75% (v/v) ethanol for 40 sec and washed 3 times with sterile water. The seeds were then sterilized with 2% (v/v) NaClO for 8 min and washed 3 times with sterile water. The sterilized cucumber seeds were transferred to Hoagland’s medium (pH 6.0) containing 0.5% (w/v) agar, and plants were grown under a 16 h light/8 h dark cycle at 28℃. After the first true leaf emerged, seedlings were transferred to 1/4 strength Hoagland’s solution and the solution was replaced every 4 days until the second true leaf emerged.

2.2. Isolation of PGPR strains

Bacteria were isolated from 10 g of rhizosphere soil collected from cucumber roots after shaking loose soil from the roots. Morphologically discrete bacterial colonies were subcultured on Luria-Bertani (LB) solid medium at 28°C for 12 h. Bacterial isolates were supplemented with 60% (v/v) sterilized glycerol and stored at -80°C for future use.

The isolated bacteria were inoculated into liquid LB medium at 28°C and 140 rpm in an incubator shaker for 12 h, the centrifuged bacteria were diluted with ddH₂O to a final concentration of 10⁶ CFU/mL for use.

Cucumber seeds that had been sterilized were put in Petri dishes with 1 mL of a bacterial suspension. After frequently checking on the germination of the seeds for 4–7 days in a 28°C light incubator (16 h of light and 8 h of darkness), the growth-promoting strains were chosen for the subsequent studies. Sterilized cucumber seeds were placed in Petri dishes containing 1 mL of a bacterial suspension. After frequent checking of the germination of the seeds for 4–7 days in a 28°C light incubator (16 h light and 8 h dark), the growth-promoting strains were selected for the following studies.

2.3. Systematic identification of PGPR strains

The EasyPure Bacteria Genomic DNA Kit (TRANS, China) was used to extract the whole genome of bacteria. Bacterial genomic DNA was used as a template for 16S rDNA amplification. In addition, the universal primer pairs 27F/1492R and KOD-Plus-Neo (TOYOBO, Japan) were used for amplification. Tsingke Biotechnology Co., Ltd. (Hangzhou, China) sequenced the amplified DNA products. Using BLAST analysis, the PCR sequences were used as queries to search for closely related sequences in the NCBI database. MEGA 7 was used to generate the most probable phylogenetic tree (Kumar et al., 2016). The 16S rRNA gene sequences were deposited in the GenBank nucleotide database.

2.4. Assay of growth promoting ability of PGPR strains

The strain was inoculated on solid LB medium at 28°C for 12 h, and then subcultured on fresh medium. The NH₃ production capacity was measured as described by Cappucino and Sherman (1992). The methods for measuring siderophore, phosphorus solubility and IAA production were taken from Pérez-Miranda et al. (2007), Son et al. (2006) and Glickmann and Dessaux, (1995), respectively.

2.5. Growth conditions of PGPR strains and treatment on cucumber

BC56 was inoculated into liquid LB medium at 28℃ and 170 rpm in an incubator shaker for 12 h. The centrifuged bacteria were diluted to 10⁸ CFU/mL for use.

Cucumbers were grown in 1/4 Hoagland until the two-leaf stage. Four treatments were then prepared as follows: (1) CK: seedlings without any treatment; (2) BC56: seedlings with BC56 inoculation to a final concentration of 10⁶ CFU/mL; (3) Salt: seedlings with 100 mM NaCl treatment and without BC56 inoculation; (4) BC56 + salt: seedlings with 100 mM NaCl treatment and BC56 inoculation to a final concentration of 10⁶ CFU/mL.

2.6. Morpho-physiological analysis of cucumber

Length and fresh weight of roots and shoots were measured at 0, 6, 10, and 14 days after inoculation (DAI). To measure dry biomass, samples were harvested at 14 DAI, and data were recorded after drying at 115℃ for 7 days.

Photosynthetic efficiency was measured using Pocket PEA (Hansatech, UK) at 0, 6, 10, and 14 DAI. The SPAD values of cucumber leaves were measured at 14 DAI using SPAD-502 (Konica Minolta, Japan).

2.7. Determination of TSS and proline

Two days after treatment, 0.5 g of fresh leaf sample was homogenized in 80% (v/v) ethanol or sulfosalicylic acid, which is used to measure TSS and proline content respectively. Measurements of TSS and proline were performed as described by Irigoyen et al. (1992) and Bates et al., (1973), respectively.

2.8. Quantification of ABA

Leaf samples were taken at 10 DAI. ABA was quantified by HPLC using Shimadzu LC-20AT (Shimadzu, Japan). The method for measuring ABA content was adapted from Xiong et al. (2014).

2.9. Measurements of Na⁺ and K⁺

Cucumber seedlings were treated with or without BC56 for 10 days under salt stress, 0.5 g shoot and root tissues were harvested and digested according to the method described by Wolf (1982). Na⁺ and K⁺ were determined by atomic absorption spectrophotometry (Optima 2100DV, Perkin Elmer, USA).

2.10. Determination of antioxidant enzymatic activity

Cucumber seedlings were harvested at 4 DAI and enzymatic antioxidants were extracted by grinding 0.2 g of fresh leaves. SOD and GR activities were measured as described by Giannopolitis and Ries, (1977) and Smith et al. (1988), respectively. CAT and POD activities were determined according to the previously reported method (Chance and Maehly, 1955).

2.11. RNA extraction, cDNA library construction, and RNA-seq

Total RNA was extracted from the second true leaf of cucumber seedlings with four treatments at 7 DAI. Liquid nitrogen was used to rapidly freeze the plant tissue and TRIzol (Thermo Fisher Scientific, USA) was used to extract the RNA. The NanoDrop ND 2000 (NanoDrop, USA) and Bio-analyzer 2100 (Agilent, USA) were used to measure the amount and purity of total RNA. LC Bio Technology CO. (Hangzhou, China) used the Illumina NovaseqTM 6000 platform (Illumina, USA) for double-ended sequencing. Gene functions were annotated on the NCBI website (https://www.ncbi.nlm.nih.gov/genome/?term=cucumis+sativus), and for GO enrichment analysis, all DEGs were mapped to GO terms in the Gene Ontology database (http://www.geneontology.org/). For KEGG analysis, all DEGs were mapped to KEGG pathways in the Kyoto Encyclopedia of Genes and Genomes (https://www.kegg.jp/kegg/).

2.12. Quantitative real-time PCR analysis

Using a CFX96 Touch Real-time PCR Detection System (Bio-Rad, USA) and TB Green® Premix Ex TaqTM (Tli RNaseH Plus) (Takara, Japan), qRT-PCR was carried out on the extracted RNA using a PrimeScriptTM RT reagent Kit (Takara, Japan). Primer Premier 5.0 (PREMIER Biosoft, Canada) was used to create the primers, which are given in Supplementary Table S4 according to the gene ID. The 2^−ΔΔCT approach was used to assess the relative expression level data (Livak and Schmittgen, 2001). Actin transcript levels served as an internal benchmark.

2.13. Statistical analysis

All experiments were performed with at least three biological replicates. Student’s t-tests were performed using SPSS Statistics 27.0.1 (IBM, USA) to detect differences.

3.1. PGPR isolation and characterization

After screening PGPRs from cucumber rhizosphere, BC56 was selected as a PGPR strain based on its ability to promote cucumber seedlings growth (Fig. S1). Specifically, BC56 was identified for its growth-promoting ability, NH₃ production, siderophore production and phosphate solubility (Table S1). Analysis of the 16S rDNA sequence showed that BC56 has 100% sequence homology with B. cereus (accession NR074540.1) and the sequence of BC56 was submitted to the GenBank database under accession number OQ271230 (Fig. S1).

3.2. Effect of different NaCl concentration on cucumber growth

To determine the appropriate salt tolerance of cucumber seedlings, two-leaf stage cucumber seedlings were grown in 1/4 Hoagland's solution supplemented with different NaCl concentrations. As the NaCl concentration increased, the growth of the cucumber seedlings decreased progressively. At 125 mM NaCl, the growth of cucumber seedlings almost stopped (Fig. S2). Therefore, we use 100 mM NaCl to treat cucumber for future use.

3.3. Effects of B. cereus BC56 on cucumber growth under salt stress

BC56 had no significant effect on shoot length and fresh weight of cucumber seedlings not exposed to salt (Fig. 1a and 1c). The root length of BC56-uninoculated seedlings was approximately 29.9 cm, whereas the root length of BC56-inoculated seedlings was approximately 32.8 cm at 14 DAI (Fig. 1b). These results showed that BC56 could promote the growth of roots rather than shoots of cucumber seedlings.

Under 100 mM NaCl treatment, the shoot length of BC56-inoculated seedlings was 1.37-fold and 1.28-fold longer than that of BC56-uninoculated seedlings at 10 and 14 DAI, respectively (Fig. 1d). The root length of BC56-inoculated seedlings was 1.81-, 1.16- and 1.16-fold longer than that of BC56-uninoculated seedlings at 6, 10, and 14 DAI, respectively (Fig. 1e). BC56-inoculated seedlings were 1.19- and 1.57-fold heavier than BC56-uninoculated seedlings in shoot fresh weight and root fresh weight at 14 DAI, respectively (Fig. 1f). These results indicated that BC56 could counteract the decrease in biomass of cucumber under salt stress.

3.4. Effects of B. cereus BC56 on biochemical index of cucumber under salt stress

To investigate the effect of BC56 on cucumber photosynthesis under salt stress, several chlorophyll fluorescence parameters were determined. The results showed that the values of ‘ABS/CS_m’, ‘TR₀/CS_m’, and ‘ET₀/CS_m’ of BC56-inoculated seedlings were 1.19-, 1.22- and 1.52-fold higher than those of BC56-uninoculated seedlings, respectively (Fig. 2a), indicating that BC56 improved the absorption and transmission capacity of light energy in cucumber.

The SPAD values of both BC56-uninoculated and BC56-inoculated seedlings showed a decreasing trend under salt stress, and that of BC56-inoculated seedlings decreased more slowly than that of BC56-uninoculated seedlings. The leaf SPAD value of BC56-inoculated seedlings (27.69) was significantly higher than that of BC56-uninoculated seedlings (24.65) at 14 DAI (Fig. 2b). It can be speculated that BC56 was able to improve the photosynthesis of cucumber leaves under salt stress.

To investigate whether BC56 affects osmotic regulation, ion balance and phytohormone regulation in cucumber under salt stress, TSS, Pro, Na⁺, K⁺, and ABA levels were measured. Under salt stress, the TSS content in BC56-inoculated seedlings was 1.36-fold as much as that in BC56-uninoculated seedlings while there was no significant difference in proline content between the two groups (Fig. 2c and 2d). The ABA content in BC56-inoculated seedlings was 0.59-fold that in BC56-uninoculated seedlings (Fig. 2e). Although there was no significant difference in Na⁺ and K⁺ content between BC56-uninoculated and BC56-inoculated seedlings, the K⁺/Na⁺ ratio in BC56-inoculated seedlings was significantly lower than that in BC56-uninoculated seedlings in roots (Fig. 2f, 2g and 2h). These results inferred that the salt stress of cucumber alleviated by BC56 might be involved in sugar and endogenous hormone metabolism as well as K⁺ and Na⁺ homeostasis.

3.5. Effects of B. cereus BC56 on activity of antioxidant enzymes

To investigate the effect of BC56 on antioxidant regulation in cucumber under salt stress, the activity of some antioxidant enzymes was determined. Under salt stress, the activity of POD and GR was 1.17-fold and 2.59-fold higher, respectively, in BC56-inoculated seedlings than in BC56-uninoculated seedlings (Fig. 3a and 3b). No significant difference in CAT and SOD activity was observed between BC56-uninoculated seedlings and BC56-inoculated seedlings (Fig. 3c and 3d). Thus, BC56 increased the activity of antioxidant enzymes under salt stress.

3.6. Transcriptomic analysis of cucumber in response to BC56 inoculation and salt stress

After eliminating low-quality reads and performing quality checks, 489 million high-quality, clean reads were obtained. Principal Component Analysis (PCA) showed that the samples were clustered into three major groups. In addition, CK and BC56 + Salt samples were closely clustered, and the gene expression level in three samples was similar to each other (Fig. 4c and S3), indicating that the sample reproducibility is qualified.

Gene expression was normalized using the fragment per kilobase of transcript per million mapped reads (FPKM) method. A total of 2,547 significant differentially expressed genes (DEGs) with log₂-fold change ranging from ≥ + 1.0 to ≤ − 1.0 and P < 0.05 were identified from the comparative analysis of the four datasets (Salt vs CK; BC56 + Salt vs CK; BC56 + Salt vs Salt; BC56 vs CK). There were 845 DEGs (593 up-regulated and 863 down-regulated) in Salt vs CK, 1048 DEGs (494 up-regulated and 554 down-regulated) in BC56 + Salt vs CK, 845 DEGs (593 up-regulated and 252 down-regulated) in BC56 + Salt vs Salt, 1456 DEGs (350 up-regulated and 403 down-regulated) in BC56 vs CK (Fig. 4a). The Venn diagram showed that 953 genes were unique DEGs between Salt vs CK and BC56 + Salt vs CK transcriptional responses, suggesting that BC56 activated these regulatory pathways under salt stress. While a significant overlap of 337 DEGs was observed between BC56 + Salt vs CK and BC56 vs CK transcriptional response, suggesting activation of a common regulatory pathway by BC56 in the presence or absence of salt stress (Fig. 4b).

Expression levels of 9 randomly selected DEGs from each sample were detected by qPCR to further verify the validity of the DEGs characterized by transcriptomic analysis. The correlation between the log₂ fold change in qPCR and RNA-seq showed a correlation value of 0.9305 for a total of 36 data points, confirming the validity of the transcriptomic data (Fig. 4d).

3.7. GO and KEGG analysis of the identified DEGs in cucumber

The GO annotation contains three main classification methods: biological process, molecular function, and cellular component. BC56 + Salt vs Salt GO enrichment analysis shows that some DEGs were enriched in response to water deprivation, salt stress, oxidative stress, and some terms related to plant hormones (auxin, abscisic acid, ethylene, salicylic acid) appeared in the GO annotation, suggesting that BC56 may mainly affect the plant hormone regulatory network of cucumber seedlings under salt stress, and other regulatory pathways including antioxidant regulation, water stress, salt stress, etc. In addition, a large number of DEGs were enriched in the chloroplasts GO term, indicating that BC56 also mainly affected the photosynthesis-related pathways (Fig. 5a).

KEGG pathway analysis of DEGs in different comparison groups revealed that in the BC56 + salt vs salt comparison group, DEGs were mainly enriched in eight pathways, namely ‘peroxisome’, ‘plant hormone signal transduction’, ‘MAPK signaling pathway–plant’, ‘metabolic pathway’, ‘biosynthesis of secondary metabolites’, ‘phenylpropanoid biosynthesis’, ‘starch and sucrose metabolism’, ‘flavonoid biosynthesis’ and ‘photosynthesis-antenna proteins’ (Fig. 5b and S4). Most of the regulatory genes were up-regulated in these pathways which were activated by BC56 under salt stress (Table S2).

3.8 B. cereus BC56 partially restored the expression of the genes altered by salt stress in cucumber

The FPKM between these four samples was clustered in the heat map (Fig. S3). Notably, one gene cluster was up-regulated by salt stress, and the expression level of this cluster in BC56 + Salt was partially restored to that of CK and the BC56-inoculated samples (Table S3). According to GO analysis, these genes were related to response to IAA, JA, ABA, ET, GA, transmembrane transport, transcriptional regulation, metabolite synthesis and metabolism, and cellular components. These results indicated that BC56 mainly activated the expression of genes involved in maintaining intracellular communication, cell structure, and cellular metabolism to promote cucumber growth, and thus alleviated the effects of salt stress.

3.9. Functional analysis of DEGs

In order to uncover the mechanism underlying BC56-mediated response to salt stress, some genes related to photosynthesis, phytohormones, peroxidase, sugar metabolism and signaling were selected to analyze their expression levels by qRT-PCR. Three genes LOC101203342 (chlorophyll a-b binding protein of LHCII type 1-like), LOC101218675 (chlorophyll a-b binding protein P4, chloroplastic-like) and LOC101211276 (chlorophyll a-b binding protein of LHCII type 1-like) are related to chlorophyll a-b binding protein, which are involved in the absorption and transfer of light energy. The expression levels of these three genes under BC56 + Salt treatment were 3.3-, 3.43- and 3.21-fold higher than those in salt-treated seedlings, respectively (Fig. 6a). This result indicated that BC56 enhances photosynthesis by promoting the absorption and transfer of light energy in cucumber under salt stress, which is consistent with the improvement of the cucumber chlorophyll content and photosynthetic capacity by BC56 (Fig. 2a and 2b).

The genes LOC101212508 (abscisic acid 8'-hydroxylase 1), LOC101212264 (protein REVEILLE 1-like isoform X1), LOC101204103 (ACC oxidase 2) and LOC101222233 (allene oxide cyclase 3, chloroplastic) are associated with ABA degradation, auxin synthesis, ethylene synthesis and jasmonic acid synthesis, respectively. The expression levels of the four genes under BC56 + Salt treatment were 1.8-, 2.18-, 1.75- and 7.7-fold higher than those in salt-treated seedlings respectively (Fig. 6b), suggesting that phytohormone plays a role in the BC56-mediated response to salt stress in cucumber.

The expression levels of LOC101217567 and LOC101218982 under BC56 + Salt treatment were 3.06- and 3.42-fold higher than those in salt-treated seedlings, respectively (Fig. 6c). The functional annotations of the two genes are ‘peroxidase N1-like’ and ‘peroxidase 45-like’ respectively, suggesting that the alleviation of salt stress by BC56 in cucumber was associated with POD activity.

BC56 also affected the expression of genes responsible for sugar metabolism and signaling in cucumber under salt stress. LOC101218588 (alkaline/neutral invertase A, mitochondrial) and LOC101210545 (inactive beta-amylase 4, chloroplastic) are related to sucrose and starch metabolism. LOC101216977 (2-oxoisovalerate dehydrogenase subunit alpha 1, mitochondrial) is involved in the response to sucrose. The expression levels of these three genes under BC56 + Salt treatment were 4.02-, 11.24- and 1.95-fold higher than those in salt-treated seedlings, respectively (Fig. 6d).

4.1. B. cereus BC56 enhances the growth of cucumber under salt stress

Several PGPR strains, such as B. amyloliquefaciens FZB42, B. amyloliquefaciens SQR9, have been shown to improve salinity tolerance in Arabidopsis and cucumber, with the ability to produce IAA, siderophores, and solubilize phosphorus (X. H. Chen et al., 2007; Li et al., 2014; Y. Liu et al., 2016; Mpanga et al., 2020; Talboys et al., 2014). In this study, we isolated a PGPR strain B. cereus BC56 that could improve the salinity tolerance of cucumber with the ability to produce NH₃ and siderophore as well as solubilize phosphorus (Fig. 1 and S1, Table S1). However, BC56 had no effect on promoting growth of cucumber without salt stress under hydroponic conditions (Fig. 1a, 1b and 1c), suggesting that BC56 promotes cucumber growth by stimulating limited element uptake in soil without salt stress.

4.2. B. cereus BC56 modulates physiological and biochemical properties of cucumber under salt stress

Studies have shown that PGPRs can enhance salt tolerance in plants by regulating photosynthesis and significantly increasing chlorophyll and carotenoid content or photosynthetic efficiency (Barnawal et al., 2017; Shi et al., 2022). BC56 increased chlorophyll content, light absorption/capture and electron transfer capacity (Fig. 2a and 2b), and also activated the photosynthesis-related genes were activated in cucumber under salt stress (Fig. 6a and S4, Table S3), suggesting that BC56 conferred salt tolerance to cucumber by maintaining photosynthesis.

The PGPR-plant interaction attenuated the detrimental effects of high salt concentrations on plant cells by reducing the Na⁺ content of plants with a concomitant increase in the K⁺/Na⁺ ratio, helping cells to maintain their structure and survival (El-Esawi et al., 2018; Garg and Manchanda, 2009; Shabaan et al., 2022). Although the decreased K⁺/Na⁺ ratio was detected in cucumber roots under salt stress (Fig. 2f), BC56 had no significant effect on Na⁺, K⁺ levels in cucumber, and K⁺/Na⁺ ratio in cucumber shoots, (Fig. 2g and 2h), suggesting that BC56 had no beneficial effect on the K⁺/Na⁺ ratio of cucumber under salt stress and alleviated salt stress on cucumber irrelative of the ion balance pathway.

Ali et al. (2022) and Nawaz et al. (2020) found that PGPR can increase the content of soluble sugars and proline under salt stress. In our experiment, BC56 increased TSS content and had no effect on proline content (Fig. 3c and 3d). Some DEGs were enriched in 'starch and sucrose metabolism' pathway, and the expression level of this pathway related genes LOC101218588 and LOC101210545 were increased after BC56 inoculation (Fig. 6d), suggesting that BC56 can maintain the cellular water potential by increasing the TSS content (Fig. 5b).

PGPR can enhance the activity of antioxidant enzymes (Patani et al., 2023; Yasmeen et al., 2020). BC56 increased the activities of POD and GR in the leaves of cucumber seedlings under salt stress (Fig. 4a and 4b). DEGs were enriched in 'response to oxidative stress' and 'peroxisome', and the expression of POD-related genes LOC101217567 and LOC101218982 were upregulated by BC56 under salt stress (Fig. 6c). These results indicated that BC56 indirectly promoted the activities of antioxidant enzymes (POD and GR) through increasing ROS-scavenging gene expressions to reduce the damage caused by ROS in cucumber seedlings under salt stress.

4.3. B. cereus BC56 mediated plant hormone regulation under salt stress

Plants have two pathways for coping with stress under abiotic stress: one is ABA-dependent and the other is ABA-independent (Yamaguchi-Shinozaki and Shinozaki, 2006; Yoshida et al., 2014). In addition, ABA levels in the plant were increased by inoculation of some PGPRs (Jahromi et al., 2008). We found that the expression level of the ABA degradation gene LOC101212508 was increased and the ABA content was decreased by BC56 (Figs. 4c, 6b) under salt stress. This result was consistent with B. amyloliquefaciens SQR9 (Chen et al., 2016). Thus, BC56 may enhance salt tolerance of cucumber in ABA-independent ways.

Other plant hormones have also been implicated in salt stress, with RNA-seq showing that plant hormone signalling is enriched by PGPRs (Liu et al., 2022; Wang et al., 2022). Rhizobia, Arbuscular, and Ecto mycorrhizal fungi can form lateral roots by regulating auxin homeostasis (Buendia et al., 2018). Serratia nematodiophili promotes plant growth at low temperatures by producing GA and ABA to reduce SA and JA levels (Asaf et al., 2017). In this study, BC56 affected genes related to response to several plant hormones (Fig. 5a and 5b, Table S2). The expression of auxin-, ET- and JA-related genes LOC101212264, LOC101204103 and LOC101222233 were up-regulated by BC56 under salt stress (Fig. 6b), suggesting that BC56 may improve salt tolerance in cucumber by regulating these phytohormone levels.

4.4. B. cereus BC56 enhances photosynthesis, metabolite biosynthesis, and metabolism to protect cucumber from salt stress

Chauhan et al. (2019) found that B. amyloliquefaciens-SN13 enriched rice genes in 'biosynthesis of secondary metabolite', 'carbohydrate, lipid, amino acid and nucleic acid metabolism', 'energy metabolism' under salt stress, these genes were also enriched by BC56 (Fig. 5). In addition, flavonoids are stress-inducible metabolites that are important for plant-microbe interactions (He et al., 2022). Flavonoid biosynthesis was represented among other secondary metabolite biosynthesis in response to B. amyloliquefaciens-SN13 inoculation (Chauhan et al., 2019). The situation was the same after rhizosphere inoculation with BC56 under salt stress (Fig. 5b). It can be concluded that BC56 could improve the ability of cucumber to release energy and biosynthesize secondary metabolites, including flavonoids, and these secondary metabolites could directly or indirectly improve salt tolerance in cucumber.

In the present study, a molecular mechanism of B. cereus BC56-mediated 'plant-microbe-stress' interaction was established. Under salt stress, BC56 could significantly promote the growth of cucumber seedlings by improving the salt tolerance of cucumber. According to physiological and biochemical indexes and transcriptome analysis of cucumber, it was found that under salt stress, BC56 can maintain chlorophyll content of cucumber seedlings and improve photosynthetic capacity under salt stress; affect plant hormone regulation pathway, reduce ABA content to alleviate cell senescence, active auxin-related to promote plant growth, and active JA/ET-related signalling pathways to directly respond to salt stress. BC56 can also increase the TSS content and maintain the water potential of the cell; enhance the activity of POD and GR to remove excess ROS; activate the expression of genes including membrane transport, metabolite synthesis and metabolism, transcriptional regulation, indirectly improving salt tolerance. These mechanisms lead to the restoration of physiological functions and improved salt tolerance of cucumber (Fig. 7). This study provides implications for the use of PGPR strains such as BC56 in assisting plants to tolerate salt stress in agricultural production, in addition to their traditional application for growth promotion and biocontrol.

Transcriptome date availability statement

Online repositories contain the datasets used in this investigation. The following link will take you to the repository's name(s) and accession number(s): https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE227178.

Funding

This research was funded by the National Natural Science Foundation of China (No. 31902106), the Key Research and Development Program of Zhejiang Province (2021C02063-6), the National Natural Science Foundation of China (No. 31571636), the Natural Science Foundation of Zhejiang Province (LY20C130006), Hangzhou Scientific and Technological Major Project (202203A01), State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products (2010DS700124-ZZ1805).

Competing interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Author contributions

Conceptualization of research (H-R.S.,G-W.W.,H-S,W and H,W), design of the experiment (H-R.S. and G-W.W), execution of experiments and data collection (H-R.S), analysis of data and interpretation (H-R.S.,G-W.W. and R-Z.H), and preparation of the manuscript (H-R.S.,G-W.W.,H.W.,R-Z.H. and X.G.). All authors contributed to the manuscript revision, and read, and approved the submitted version.

Acknowledgments

We acknowledge Heqing Liu from the Zhejiang Academy of Agricultural Sciences for providing several instruments for this study.

Ali, B., Wang, X., Saleem, M. H., Sumaira, Hafeez, A., Afridi, M. S., Khan, S., Zaib-Un-Nisa, Ullah, I., Amaral Júnior, A. T. do, Alatawi, A., and Ali, S. (2022). PGPR-mediated salt tolerance in maize by modulating plant physiology, antioxidant defense, compatible solutes accumulation and bio-surfactant producing genes. Plants, 11(3), 345. https://doi.org/10.3390/plants11030345
Asaf, S., Khan, M. A., Khan, A. L., Waqas, M., Shahzad, R., Kim, A.-Y., Kang, S.-M., and Lee, I.-J. (2017). Bacterial endophytes from arid land plants regulate endogenous hormone content and promote growth in crop plants: an example of Sphingomonas sp. and Serratia marcescens. Journal of Plant Interactions, 12(1), 31–38. https://doi.org/10.1080/17429145.2016.1274060
Barnawal, D., Bharti, N., Pandey, S. S., Pandey, A., Chanotiya, C. S., and Kalra, A. (2017). Plant growth-promoting rhizobacteria enhance wheat salt and drought stress tolerance by altering endogenous phytohormone levels and TaCTR1 / TaDREB2 expression. Physiologia Plantarum, 161(4), 502–514. https://doi.org/10.1111/ppl.12614
Bates, L. S., Waldren, R. P., and Teare, I. D. (1973). Rapid determination of free proline for water-stress studies. Plant and Soil, 39(1), 205–207. https://doi.org/10.1007/BF00018060
Buendia, L., Girardin, A., Wang, T., Cottret, L., and Lefebvre, B. (2018). LysM receptor-like kinase and lysM receptor-like protein families: an update on phylogeny and functional characterization. Frontiers in Plant Science, 9. https://doi.org/10.3389/fpls.2018.01531
Cappucino JC, Sherman N. In: Microbiology: a laboratory manual. 3rd ed. New York: Benjamin/Cumming Pub. Co.; 1992.
Chance, B., and Maehly, A. C. (1955). [136] Assay of catalases and peroxidases. In Methods in Enzymology (pp. 764–775). https://doi.org/10.1016/S0076-6879(55)02300-8
Chauhan, P. S., Lata, C., Tiwari, S., Chauhan, A. S., Mishra, S. K., Agrawal, L., Chakrabarty, D., and Nautiyal, C. S. (2019). Transcriptional alterations reveal Bacillus amyloliquefaciens-rice cooperation under salt stress. Scientific Reports, 9(1), 1–13. https://doi.org/10.1038/s41598-019-48309-8
Chen, L., Liu, Y., Wu, G., Veronican Njeri, K., Shen, Q., Zhang, N., and Zhang, R. (2016). Induced maize salt tolerance by rhizosphere inoculation of Bacillus amyloliquefaciens SQR9. Physiologia Plantarum, 158(1), 34–44. https://doi.org/10.1111/ppl.12441
Chen, X. H., Koumoutsi, A., Scholz, R., Eisenreich, A., Schneider, K., Heinemeyer, I., Morgenstern, B., Voss, B., Hess, W. R., Reva, O., Junge, H., Voigt, B., Jungblut, P. R., Vater, J., Süssmuth, R., Liesegang, H., Strittmatter, A., Gottschalk, G., and Borriss, R. (2007). Comparative analysis of the complete genome sequence of the plant growth–promoting bacterium Bacillus amyloliquefaciens FZB42. Nature Biotechnology, 25(9), 1007–1014. https://doi.org/10.1038/nbt1325
Duan, J., Zhang, M., Zhang, H., Xiong, H., Liu, P., Ali, J., Li, J., and Li, Z. (2012). OsMIOX, a myo-inositol oxygenase gene, improves drought tolerance through scavenging of reactive oxygen species in rice (Oryza sativa L.). Plant Science, 196, 143–151. https://doi.org/10.1016/j.plantsci.2012.08.003
Egamberdieva, D., Wirth, S., Bellingrath-Kimura, S. D., Mishra, J., and Arora, N. K. (2019). Salt-tolerant plant growth promoting rhizobacteria for enhancing crop productivity of saline soils. Frontiers in Microbiology, 10. https://doi.org/10.3389/fmicb.2019.02791
El-Esawi, M., Alaraidh, I., Alsahli, A., Alzahrani, S., Ali, H., Alayafi, A., and Ahmad, M. (2018). Serratia liquefaciens KM4 improves salt stress tolerance in maize by regulating redox potential, ion homeostasis, leaf gas exchange and stress-related gene expression. International Journal of Molecular Sciences, 19(11), 3310. https://doi.org/10.3390/ijms19113310
Gamalero, E., and Glick, B. R. (2011). Bacteria in agrobiology: plant nutrient management. Bacteria in Agrobiology: Plant Nutrient Management, 17–46. https://doi.org/10.1007/978-3-642-21061-7
Gao, Y., Zou, H., Wang, B., and Yuan, F. (2022). Progress and applications of plant growth‐promoting bacteria in salt tolerance of crops. International Journal of Molecular Sciences, 23(13). https://doi.org/10.3390/ijms23137036
Garg, N., and Manchanda, G. (2009). Role of Arbuscular mycorrhizae in the alleviation of ionic, osmotic and oxidative stresses induced by salinity in Cajanus cajan (L.) Millsp. (pigeonpea). Journal of Agronomy and Crop Science, 195(2), 110–123. https://doi.org/10.1111/j.1439-037X.2008.00349.x
Giannopolitis, C. N., and Ries, S. K. (1977). Superoxide dismutases. Plant Physiology, 59(2), 309–314. https://doi.org/10.1104/pp.59.2.309
Gill, S. S., and Tuteja, N. (2010). Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiology and Biochemistry, 48(12), 909–930. https://doi.org/10.1016/j.plaphy.2010.08.016
Glick, B. R. (2012). Plant growth-promoting bacteria: mechanisms and applications. Scientifica, 2012(1), 1–15. https://doi.org/10.6064/2012/963401
Glickmann, E., and Dessaux, Y. (1995). A critical examination of the specificity of the Salkowski reagent for indolic compounds produced by phytopathogenic bacteria. Applied and Environmental Microbiology, 61(2), 793–796. https://doi.org/10.1128/aem.61.2.793-796.1995
Gong, Z. (2021). Plant abiotic stress: new insights into the factors that activate and modulate plant responses. Journal of Integrative Plant Biology, 63(3), 429–430. https://doi.org/10.1111/jipb.13079
He, D., Singh, S. K., Peng, L., Kaushal, R., Vílchez, J. I., Shao, C., Wu, X., Zheng, S., Morcillo, R. J. L., Paré, P. W., and Zhang, H. (2022). Flavonoid-attracted Aeromonas sp. from the Arabidopsis root microbiome enhances plant dehydration resistance. The ISME Journal, 16(11), 2622–2632. https://doi.org/10.1038/s41396-022-01288-7
Irigoyen, J. J., Einerich, D. W., and Sánchez‐Díaz, M. (1992). Water stress induced changes in concentrations of proline and total soluble sugars in nodulated alfalfa ( Medicago sativa ) plants. Physiologia Plantarum, 84(1), 55–60. https://doi.org/10.1111/j.1399-3054.1992.tb08764.x
Jahromi, F., Aroca, R., Porcel, R., and Ruiz-Lozano, J. M. (2008). Influence of salinity on the in vitro development of glomus intraradices and on the in vivo physiological and molecular responses of mycorrhizal lettuce plants. Microbial Ecology, 55(1), 45–53. https://doi.org/10.1007/s00248-007-9249-7
Kumar, S., Stecher, G., and Tamura, K. (2016). MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33(7), 1870–1874. https://doi.org/10.1093/molbev/msw054
Li, B., Li, Q., Xu, Z., Zhang, N., Shen, Q., and Zhang, R. (2014). Responses of beneficial Bacillus amyloliquefaciens SQR9 to different soilborne fungal pathogens through the alteration of antifungal compounds production. Frontiers in Microbiology, 5. https://doi.org/10.3389/fmicb.2014.00636
Liu, Q., Li, P., Cheng, S., Zhao, Z., Liu, Y., Wei, Y., Lu, Q., Han, J., Cai, X., Zhou, Z., Umer, M. J., Peng, R., Zhang, B., and Liu, F. (2022). Protoplast dissociation and transcriptome analysis provides insights to salt stress response in cotton. International Journal of Molecular Sciences, 23(5), 2845. https://doi.org/10.3390/ijms23052845
Liu, Y., Chen, L., Zhang, N., Li, Z., Zhang, G., Xu, Y., Shen, Q., and Zhang, R. (2016). Plant-microbe communication enhances auxin biosynthesis by a root-associated bacterium, Bacillus amyloliquefaciens SQR9. Molecular Plant-Microbe Interactions®, 29(4), 324–330. https://doi.org/10.1094/MPMI-10-15-0239-R
Livak, K. J., and Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods, 25(4), 402–408. https://doi.org/10.1006/meth.2001.1262
Marusig, D., and Tombesi, S. (2020). Abscisic acid mediates drought and salt stress responses in vitis vinifera—a review. International Journal of Molecular Sciences, 21(22), 8648. https://doi.org/10.3390/ijms21228648
Mpanga, I. K., Ludewig, U., Dapaah, H. K., and Neumann, G. (2020). Acquisition of rock phosphate by combined application of ammonium fertilizers and Bacillus amyloliquefaciens FZB42 in maize as affected by soil pH. Journal of Applied Microbiology, 129(4), 947–957. https://doi.org/10.1111/jam.14654
Nadarajah, K. K. (2020). ROS homeostasis in abiotic stress tolerance in plants. International Journal of Molecular Sciences, 21(15), 5208. https://doi.org/10.3390/ijms21155208
Nawaz, A., Shahbaz, M., Asadullah, M., Imran, A., Marghoob, M. U., Imtiaz, M., and Mubeen, F. (2020). Potential of salt tolerant PGPR in growth and yield augmentation of wheat (Triticum aestivum L.) under saline conditions. Frontiers in Microbiology, 11(October), 1–12. https://doi.org/10.3389/fmicb.2020.02019
Patani, A., Prajapati, D., Ali, D., Kalasariya, H., Yadav, V. K., Tank, J., Bagatharia, S., Joshi, M., and Patel, A. (2023). Evaluation of the growth-inducing efficacy of various Bacillus species on the salt-stressed tomato (Lycopersicon esculentum Mill.). Frontiers in Plant Science, 14(March), 1–17. https://doi.org/10.3389/fpls.2023.1168155
Pérez-Miranda, S., Cabirol, N., George-Téllez, R., Zamudio-Rivera, L. S., and Fernández, F. J. (2007). O-CAS, a fast and universal method for siderophore detection. Journal of Microbiological Methods, 70(1), 127–131. https://doi.org/10.1016/j.mimet.2007.03.023
Shabaan, M., Asghar, H. N., Zahir, Z. A., Zhang, X., Sardar, M. F., and Li, H. (2022). Salt-tolerant PGPR confer salt tolerance to maize through enhanced soil biological health, enzymatic activities, nutrient uptake and antioxidant defense. Frontiers in Microbiology, 13. https://doi.org/10.3389/fmicb.2022.901865
Shahid, M., Al-Khattaf, F. S., Danish, M., Zeyad, M. T., Atef Hatamleh, A., Mohamed, A., and Ali, S. (2022). PGPR Kosakonia radicincitans KR-17 increases the salt tolerance of radish by regulating ion-homeostasis, photosynthetic molecules, redox potential, and stressor metabolites. Frontiers in Plant Science, 13. https://doi.org/10.3389/fpls.2022.919696
Sharma, S., Kulkarni, J., and Jha, B. (2016). Halotolerant rhizobacteria promote growth and enhance salinity tolerance in peanut. Frontiers in Microbiology, 7. https://doi.org/10.3389/fmicb.2016.01600
Shi, L.-N., Lu, L.-X., Ye, J.-R., and Shi, H.-M. (2022). The endophytic strain ZS-3 enhances salt tolerance in Arabidopsis thaliana by regulating photosynthesis, osmotic stress, and Ion homeostasis and inducing systemic tolerance. Frontiers in Plant Science, 13. https://doi.org/10.3389/fpls.2022.820837
Singh, A. (2021). Soil salinization management for sustainable development: A review. Journal of Environmental Management, 277(May 2020), 111383. https://doi.org/10.1016/j.jenvman.2020.111383
Smith, I. K., Vierheller, T. L., and Thorne, C. A. (1988). Assay of glutathione reductase in crude tissue homogenates using 5,5′-dithiobis(2-nitrobenzoic acid). Analytical Biochemistry, 175(2), 408–413. https://doi.org/10.1016/0003-2697(88)90564-7
Son, H. J., Park, G. T., Cha, M. S., and Heo, M. S. (2006). Solubilization of insoluble inorganic phosphates by a novel salt- and pH-tolerant Pantoea agglomerans R-42 isolated from soybean rhizosphere. Bioresource Technology, 97(2), 204–210. https://doi.org/10.1016/j.biortech.2005.02.021
Talboys, P. J., Owen, D. W., Healey, J. R., Withers, P. J., and Jones, D. L. (2014). Auxin secretion by Bacillus amyloliquefaciens FZB42 both stimulates root exudation and limits phosphorus uptake in Triticum aestivium. BMC Plant Biology, 14(1), 51. https://doi.org/10.1186/1471-2229-14-51
van Zelm, E., Zhang, Y., and Testerink, C. (2020). Salt tolerance mechanisms of plants. Annual Review of Plant Biology, 71(1), 403–433. https://doi.org/10.1146/annurev-arplant-050718-100005
Vande Broek, A., Lambrecht, M., Eggermont, K., and Vanderleyden, J. (1999). Auxins upregulate expression of the indole-3-pyruvate decarboxylase gene in Azospirillum brasilense. Journal of Bacteriology, 181(4), 1338–1342. https://doi.org/10.1128/jb.181.4.1338-1342.1999
Verma, V., Ravindran, P., and Kumar, P. P. (2016). Plant hormone-mediated regulation of stress responses. BMC Plant Biology, 16(1), 86. https://doi.org/10.1186/s12870-016-0771-y
Wang, W., He, Y., Wu, Z., Li, T., Xu, X., and Liu, X. (2022). De novo transcriptome sequencing of Capsicum frutescens. L and comprehensive analysis of salt stress alleviating mechanism by Bacillus atrophaeus WU-9. Physiologia Plantarum, 174(4). https://doi.org/10.1111/ppl.13728
Yamaguchi-Shinozaki, K., and Shinozaki, K. (2006). Transcriptional regulatory networks in cellular responses and tolerance to dehydration and sold stresses. Annual Review of Plant Biology, 57(1), 781–803. https://doi.org/10.1146/annurev.arplant.57.032905.105444
Yang, J., Kloepper, J. W., and Ryu, C. (2009). Rhizosphere bacteria help plants tolerate abiotic stress. Trends in Plant Science, 14(1), 1–4. https://doi.org/10.1016/j.tplants.2008.10.004
Yang, Y., and Guo, Y. (2018). Elucidating the molecular mechanisms mediating plant salt-stress responses. New Phytologist, 217(2), 523–539. https://doi.org/10.1111/nph.14920
Yasmeen, T., Ahmad, A., Arif, M. S., Mubin, M., Rehman, K., Shahzad, S. M., Iqbal, S., Rizwan, M., Ali, S., Alyemeni, M. N., and Wijaya, L. (2020). Biofilm forming rhizobacteria enhance growth and salt tolerance in sunflower plants by stimulating antioxidant enzymes activity. Plant Physiology and Biochemistry, 156, 242–256. https://doi.org/10.1016/j.plaphy.2020.09.016
Yin, X. M., Huang, L. F., Zhang, X., Wang, M. L., Xu, G. Y., and Xia, X. J. (2015). OsCML4 improves drought tolerance through scavenging of reactive oxygen species in rice. Journal of Plant Biology, 58(1), 68–73. https://doi.org/10.1007/s12374-014-0349-x
Yoshida, T., Mogami, J., and Yamaguchi-Shinozaki, K. (2014). ABA-dependent and ABA-independent signaling in response to osmotic stress in plants. Current Opinion in Plant Biology, 21, 133–139. https://doi.org/10.1016/j.pbi.2014.07.009
Zhu, J.-K. (2002). Salt and drought stress signal transduction in plants. Annual Review of Plant Biology, 53(1), 247–273. https://doi.org/10.1146/annurev.arplant.53.091401.143329

SupplementaryMaterial.docx

Download PDF

Journal Publication

published 30 Jan, 2024

Read the published version in Plant Growth Regulation →

Editorial decision: Minor revisions
28 Nov, 2023
Reviewers agreed at journal
10 Nov, 2023
Reviewers invited by journal
10 Nov, 2023
Editor assigned by journal
10 Nov, 2023
First submitted to journal
08 Nov, 2023

You are reading this latest preprint version

Rhizosphere inoculation of PGPR strain Bacillus cereus BC56 enhances salt tolerance of cucumber

Status:

Journal Publication

Version 1

Abstract

Figures

1. Introduction

2. Materials and methods

2.1. Plant materials and growth conditions

2.2. Isolation of PGPR strains

2.3. Systematic identification of PGPR strains

2.4. Assay of growth promoting ability of PGPR strains

2.5. Growth conditions of PGPR strains and treatment on cucumber

2.6. Morpho-physiological analysis of cucumber

2.7. Determination of TSS and proline

2.8. Quantification of ABA

2.9. Measurements of Na+ and K+

2.10. Determination of antioxidant enzymatic activity

2.11. RNA extraction, cDNA library construction, and RNA-seq

2.12. Quantitative real-time PCR analysis

2.13. Statistical analysis

3. Results

3.1. PGPR isolation and characterization

3.2. Effect of different NaCl concentration on cucumber growth

3.3. Effects of B. cereus BC56 on cucumber growth under salt stress

3.4. Effects of B. cereus BC56 on biochemical index of cucumber under salt stress

3.5. Effects of B. cereus BC56 on activity of antioxidant enzymes

3.6. Transcriptomic analysis of cucumber in response to BC56 inoculation and salt stress

3.7. GO and KEGG analysis of the identified DEGs in cucumber

3.9. Functional analysis of DEGs

4. Discussion

4.1. B. cereus BC56 enhances the growth of cucumber under salt stress

4.2. B. cereus BC56 modulates physiological and biochemical properties of cucumber under salt stress

4.3. B. cereus BC56 mediated plant hormone regulation under salt stress

4.4. B. cereus BC56 enhances photosynthesis, metabolite biosynthesis, and metabolism to protect cucumber from salt stress

5. Conclusions

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1

2.9. Measurements of Na⁺ and K⁺