Isolation and identification of associated endophytic bacteria from barely seeds harbour non-ribosomal peptides and enhance tolerance to salinity stress

Background Barely Hordeum vulgare L. is considered one of the most important cereal crops with economic and industrial importance in the world, but its productivity is affected by climate change and abiotic stresses. One of the most recent and important microbiological promising aspects is the use of associated microorganisms, especially the endophytic bacteria producers for non-ribosomal peptides which play an important role in promoting plant growth, productivity, and tolerance to biotic and abiotic stresses. This work aims to identify vertically transferred or inherited endophytic bacterial communities in barely seeds, detect the presence of non-ribosomal peptides from these isolated endophytic strains and study their effect on protein patterns as a response to salinity stress. Results From two different tolerant (Giza 126) and sensitive (Giza 123) barely seeds cultivars, six different endophytic bacterial strains were isolated and identified using 16S rRNA. Bacterial strains belonged to Bacillus, Paenibacillus, Staphylococcus and Acinetobacter genera. Three of them have been isolated from both sensitive and tolerant barely cultivar ( Uncultured Staphylococcus , Acinetobacter and Priestia endophytica or Bacillus endophyticus ), while the other three endophytes have been isolated uniquely from the tolerant barely cultivar ( Paenibacillus glucanolyticus , Bacillus cereus and Bacillus sp.). Non-ribosomal peptide synthetases genes NRPs of two lipopeptide families; surfactins and kurstakins have been detected using both bioinformatic analysis and degenerate primers. On the other hand, fragments similar to NRPs genes might be considered new NRPS molecules in Paenibacillus glucanolyticus, Acinetobacter and Priestia endophytica which have been detected using degenerate primers and required whole genome sequencing. The effect of soaking barely seeds exposed to 2.5% NaCl using SDS-PAGE electrophoresis revealed the presence of 24 bands, 10 of them were monomorphic with 41.5%, and 14 were polymorphic with 58.5% polymorphism. Conclusion The overnight soaking and co-cultivation of isolated endophytic strains with barely seeds before planting proved their capability in conferring salt stress tolerance to barely seedlings which appeared in protein patterns. We could consider these barely seeds endophytic among the PGPR strains promising to improve plant growth during abiotic stresses.


Background
Barely Hordeum vulgare L. is considered one of the most important cereal crops in the world, used in bread and beverage industries and provides human health with nutrients that enhance heart health and help prevent cancer [1].One of the most important factors of climate change is salinity which has negative effects on plant growth and crop productivity.Around the world, more than 800 million hectares are affected by salinity [2].Plant-associated bacteria or endophytic bacteria can live inside plant tissues without harming and have an important role in promoting plant growth and tolerance to abiotic and biotic stress [3].One of the most microbiological promising roles of endophytic bacteria is to interact with plants by settling in ecosystem restoration processes.One or more endophytic bacterial species might be harbored by each.The presence and the density of endophytic bacterial species depend on the variables of the bacterial species, host genotypes and environmental conditions [4].Endophytic bacteria can excrete natural products benefits to plants in reducing diseases severity, promoting growth, and inducing plant defense mechanisms [5,6].Among these natural products, lipopeptides are produced by the non-ribosomal peptide synthesis mechanism using a multi-enzymes function synthetases system [7].Surfactins, iturins and fengycins are three different families of non-ribosomal lipopeptides which were discovered between 1949 and 1986 from Bacillus spp.[8].Seed-associated endophytic bacteria role is not well understood yet, but many studies have reported that diverse plant seed microorganisms are critical to seed and plant health [9,10].Recently, the role of seed endophytic community has been studied using multi-omics techniques which improved and significantly increased their understanding role.Seed endophytic isolates have been reported mostly as Proteobacteria, especially γ-Proteobacteria, followed by the Actinobacteria, Firmicutes and Bacteroidetes phyla [11].The seed endophytes genera that are often detected and passed on to the next generation are Bacillus, Pseudomonas, Paenibacillus, Micrococcus, Staphylococcus, Pantoea and Acinetobacter.The presence of these endophytes genera has been reported in rice seeds [12], and Crotalaria pumila seeds during three consecutive years, respectively [13].Plant growth-promoting (PGP) properties have been detected for barely seed endophytes and other plants in vitro and in vivo [12][13][14][15].To our knowledge, there are not enough studies on barely seed endophytes till now.
The interactions between genotypes genes and the environment affected protein and plant response to stress [16,17] which could be understanding using the SDS-PAGE technique tool to explain the genetic diversity and relationships among different crop species/varieties such as wheat and barley genotypes [18,19] and may provide information for the use of genotypes in different breeding experiments used as a rapid, easy, inexpensive evaluation of genetic diversity, because of that it became accepted valuable tool [20,21].
One of the most important techniques employed in biological analysis is the SDS-PAGE technique which can determine shifts in protein bands that could be proteins or enzymes produced due to bio-stress [21][22][23][24][25][26].Electrophoretic pattern changes have been detected in the soluble proteins of different cultivars grown in different environments.Plants, when exposed to certain types of environmental stress conditions, can help organisms to tolerate such stresses by activation of stress genes to produce stress proteins.
This work aims to identify the vertically transferred or inherited endophytic bacterial communities in barely seeds, detect the presence of non-ribosomal peptides from these isolated endophytic strains and study their effect on protein patterns as a response to salt stress.

Plant samples
Sensitive (Giza 123) and tolerant (Giza 126) barely seeds were obtained from Field Crops Research Institute, Agricultural Research Center (ARC), Giza, Egypt.

Bacterial strains isolation and medium
About 20 seeds of each cultivar sensitive (Giza 123) and tolerant (Giza 126) were surface sterilized according to [27].The attached dust was removed by washing with tap water, then with ethanol 70% for 1 min and 5 min with 5% sodium hypochlorite.After that, the seeds were rinsed with sterilized distilled water three times, and then, seeds were sterilized one more time with ethanol 70% for 10 s and rinsed with sterilized distilled water three times.After that, soaking seeds in 25 ml sterilized distilled water overnight was recommended to become soft and easy to mash.This mixture was incubated on two different cultures media YPDA (20 g peptone, 10 g yeast extract, 20 g dextrose and 15 g agar in 1litertotal volume, pH 6.5) and 1/10 869 [0.1 g glucose, 0.5 g Nacl, 1 g tryptone, 0.5 g yeast extract and 15 g agar in 1-L total volume, pH 6.5 [28].Three replicates for each cultivar were prepared in addition to one negative control (without surface sterilization) and positive control (100 µl of the last rinsed water) at 28 C until the appearance of colonies.Each resulting colony was cultured on LB solid plate separately at 30 C overnight till growth.Physiological identification for strains followed by DNA extraction and 16srRNA isolation has been performed.

Identification of barely seeds fungi
A sterilized needle was used to obtain fungal tissues from a petri dish.Fungal tissues were placed on a clean microscopic slide.Fungal spores were photographed with a fixed camera attached to a compound microscope at 200 × magnification (Olympus microscope camera, Japan).Fungal key identification texts such as [29] were consulted for aiding in accurate identification.

Identification of endophytic bacteria isolated from barely
The grown colonies were total genomic DNA extracted by a DNA extraction kit (Qiagen, USA).Bacterial 16srDNA universal primers were used as described by using primers 27F (AGA GTT TGATCMTGG CTC AG) and 1525R (AAG GAG GTG WTC -CARCC) [30] in a Biometra thermocycler.The PCR amplification was programmed for 30 cycles as follows: an initial denaturation for 5 min at 95•C, a denaturation step for 1 min at 95•C, an elongation step for 1 min at 55•C and finally, an extension step for 1.30 min at 72•C.The final extension was at 72•C for 10 min.The PCR product was purified by a PCR purification kit (Qiagen, USA) and sent to Macrogen Inc. (Seoul, South Korea) for sequencing.The closest match sequences were identified using BLAST search of the NCBI GenBank website.

Detection of NRPS encoding genes for identified endophytic strains by bioinformatics tools
Sequenced genomes of related identified endophytic strains were obtained from the National Center for Biotechnology Information (USA) NCBI and analyzed with the Anti-Smash website to detect the secondary products and the gene clusters.PKS-NRPS website (http:// nrps.igs.umary land.edu/ nrps) was used to identify the NRPS genes, annotation, description of modules and domains, specificity prediction of adenylation (A) domains, potential primary structure of the peptide.The last step was to compare the predicted peptides structures obtained by PKS/NRPS website with the Norine database (http:// bioin fo.lifl.fr/ norine) which includes a database of 1185 non-ribosomal peptides to know whether the predicted peptide is an existing molecule or a new one.
A final extension step was lanced at 72 °C for 5 min.
The amplified fragments were analyzed by agarose gel electrophoresis (1.2%), and then gels were visualized by the Gel Doc system (Bio-Rad).Fragments were purified from gels by a PCR purification kit (Qiagen, USA) and sent to Macrogen Inc.(Seoul, South Korea) for sequencing.

Phylogenetic tree
The sequences were aligned to the 16S rDNA database on GenBank using BlastN, and the closest sequences were used to construct the phylogenetic tree which was performed by Mega (Version 6.0) with a neighbor-joining method [33].

Bacteria preparation for salinity experiment
The isolated endophytic bacteria strains from barely seeds (B1, B2, B3, B4, B5 and B6) as shown in Table 3 were grown in LB liquid media overnight culture at 30 °C.

SDS-PAGE protein banding patterns
Samples preparations and extraction of total proteins were performed according to [34] and analyzed according to the method of [35].Separating gel of 15% were prepared, whereas protein fractionations were performed exclusively on vertical slab gel (19.8 cm × 26.8 cm × 0.2 cm) using the vertical electrophoresis apparatus manufactured by Cleaver, UK.The gel was photographed and scanned by Gel Doe Bio-Rad System (Gel Pro analyzer V.3) followed by protein bands analysis, by the Total Lab program.

Isolation and identification of endophytes from sensitive and tolerant barely seeds cultivars
Fifty colonies were grown on both YPDA and 1/10 diluted 869 media which were screened physiologically under microscope after staining with gram.After the screening, six different bacterial endophytes were selected for identification, three of them were isolated from both sensitive (Giza 123) and tolerant barely cultivar (Giza 126) (Uncultured Staphylococcus, Acinetobacter and Priestia endophytica or Bacillus endophyticus), while the other three endophytes have been isolated uniquely from the tolerant barely cultivar (Paenibacillus glucanolyticus, Bacillus cereus and Bacillus sp.).The identification using 16 s rRNA revealed six different strains Acinetobacter johnsonii, Bacillus cereus, Bacillus sp., Paenibacillus glucanolyticus, priestia endophytica and Staphylococcus pasteuri.
Two experiments 1 (without plant extract) and 2 (with plant extract) using YPDA and 1/10 diluted 869 media have proved its significance; using 1/10 diluted 869 (rich) media have delivered the highest diversity of endophytes, as well as the highest numbers of cultivable endophytes, while the addition of plant extract to the medium composition significantly increased the numbers of isolated bacteria but had small effect on the cultivable endophytes diversity (Table 2).

Identification of barely seeds fungi
The identification of barely cultivated fungi based on colony color and conidial shape revealed the presence of three different fungal species: Alternaria alternate, Aspergillus niger and Rhizopus stolonifera.

Detection of secondary metabolite genes involved in some isolated endophytic strains from barely seeds by Anti-smash
The identified barely endophytic bacterial strains-related genomes were analyzed by Anti-smash which allows the   of bacillibactin synthetase gene, six genes cluster of unknown NRPS product and three genes cluster responsible for the biosynthesis of kurstakin lipopeptide (Fig. 2 and Table 4).Anti-smash analysis of Paenibacillus glucanolyticus genome sequence (accession no: CP028366.1)revealed 14 regions of identified secondary metabolites, four of which are NRPS and PKS products.Bacillibactin, PKS-NRPS hybrid unknown product, unknown PKS product and one synthetase gene encoded for tryptophan (Fig. 3 and Table 4).Bacillus cereus complete genome sequence analysis by Anti-smash revealed the presence of 9 regions of identified secondary metabolites, three of which are NRPS products.In bacillibactin, two regions were identified as fengycin with a similarity of 40%, which was well analyzed by PKS-NRPS and identified as kurstakin (Fig. 4 and Table 4).
Bacillus cereus and Priestia endophytica are found to harbor kurstakin synthetases genes encoded for kurstakin lipopeptide which consists of three gene cluster heptapeptide as shown in Fig. 4.
Three gene clusters were identified from Priestia endophytica.One NRP (Fig. 2A) consists of one gene encoded for bacillibactin.The gene cluster revealed in Fig. 2B consists of five genes and is a Type I PKs-NRPs hybrid BGC (unknown NRPs) with a size of approximately 30 kb.The PKs module consists of a ketosynthase (KS) domain, an acyltransferase (AT) domain, an acyl carrier protein (ACP) domain and a thioesterase (TE) domain which incorporates the polyketide moiety of malonyl-CoA, while the NRPs modules incorporate six amino acid residues.Based on anti-SMASH analysis, this unknown molecule shows a low similarity of 28% to the paenilamicin BGC, synthesized by pam BGC from Paenibacillus larvae DSM25430 which shows antibacterial and antifungal activity.This pam cluster has a size of ∼60 kb and consists of five NRPs genes, two Type I PKs genes and two Type I PKs-NRPs hybrid genes.Additionally, B. endophyticus FH5 has been found to harbor Type I PKs-NRPs hybrid BGC but consists of only three NRPS genes and one Type I PKS gene and differs from the pam cluster of Paenibacillus larvae DSM25430.Other NRPs of 25 kb total size (Fig. 2C) consist of three genes encoding for 24 domains: seven condensation (C) domains, seven adenylation (A) domains, seven thiolation (T) domains, two epimerization (E) domains and one thioesterase (TE) domain.This BGC catalyzes the primary formation of a lipopeptide product highly similar to kurstakin.

NRPs synthetase genes detection in endophytic isolates strains by degenerate primers
Kurstakin degenerate primers amplified fragments of 1162 bp with uncultured Staphylococcus, Paenibacillus glucanolyticus and Priestia endophytica (Fig. 5).These results agree with bioinformatic detection of kurstakin synthetase genes in Paenibacillus glucanolyticus and Priestia endophytica, while it seems different with Fig. 2 NRPS identified synthetases genes by PKS-NRPS analysis system for Priestia endophytica isolated endophyte from barely seeds.A Bacillibactin synthetase gene; B Unknown NRPS-PKS synthetases genes; C Kurstakin synthetases genes bioinformatic detection of kurstakin synthetase genes in Bacillus cereus which may refer to that this strain belongs to another Bacillus species or by the absence of kurstakin synthetase genes.Plipastatin degenerate primers amplified no specific fragments for plipastatin synthetase genes which indicates the absence of this lipopeptide in all isolated strains from barely (Fig. 5).
Surfactin degenerate primers led to the amplification of surfactin synthetase partial gene fragment with the expected size of 431 bp in both strains Bacillus cereus and Bacillus sp., while it led to the detection of  fragments similar to a NRPs gene in Paenibacillus glucanolyticus, Acinetobacter and Priestia endophytica (Fig. 6).These strains are highly recommended for whole genome sequencing because they may harbor new NRPS molecules.Bioinformatic analysis results of related complete genomes of endophytic isolates from barely seeds match with degenerate primers results in all isolated strains except in Bacillus cereus and Uncultured Staphylococcus which appear to need more analysis by sequencing their complete genomes to verify their NRPs presence.Uncultured Staphylococcus strain has no related complete genome sequence on NCBI to detect the NRPs synthetases genes by bioinformatics, while the use of degenerate primers led to the detection of kurstakin synthetases genes in the strain.Bioinformatic analysis of Bacillus cereus-related complete genome sequence on NCBI revealed the presence of bacillibactin synthetase gene and kurstakin synthetase genes cluster, while the use of degenerate primers has proven the absence of kurstakin and the presence of surfactin synthetase genes cluster (Table 5).

Soluble Proteins electrophoresis
Protein profiling assessment by SDS-PAGE for barley samples of different treatments showed different banding patterns.Many alterations in protein patterns have been observed based on the relative mobility of gel proteins in barley leaves treated with different endophytic bacteria isolated from barley seeds B1, B2, B3, B4, B5, B6,

Discussion
In this work, we study the role of barely seeds endophytic bacteria vertically transferred or inherited in tolerance to salinity stress and focus on the role of a group of natural products secreted by these bacteria called non-ribosomal peptides NRPs which consider alternatives to traditional chemical substances.The use of 1/10 diluted 869 (rich) media has delivered the highest diversity of endophytes, as well as the highest numbers of cultivable endophytes, while the addition of plant extract to the medium composition significantly increased the numbers of isolated bacteria but had a small effect on the cultivable endophytes diversity [36].These endophytes have been found to belong to three main families: Bacillaceae, Paenibacillaceae and Staphylococcaceae.These results agreed with [36] who reported that highland barley seeds harbored the genus Bacillus.The use of Anti-Smash in detecting secondary metabolites from bacterial complete genomes facilitates and economizes the time of researchers [37].Here, we analyzed three complete genome sequences of Paenibacillus glucanolyticus, Bacillus cereus and Priestia endophytica endophytes using Anti-Smash which revealed the presence of gene clusters of bacillibactin and kurstakin in addition to the use of PKS-NRPS website which detected the presence of gene clusters of bacillibactin and surfactin in Bacillus spp.endophytic strain.These results are agreed with [36,38] who identified and characterized Bacillus endophytes species which are widely used as producers of growth-promoting substances, bioactive compounds and metabolites with antimicrobial effects.
To our knowledge, endophytes in barely seeds and consequently the detection of NRPs genes have not been studied to date, but kurstakin synthesis has been reported in several studies from Bacillus thuringiensis and Bacillus cereus group.Also, Paenibacillus spp.included in the group of plant growth-promoting rhizobacteria and biofertilizers [8] which produced a wide spectrum of NRPs as: polymyxins, fusaricidins, tridecaptins and other secondary metabolites weapons against Grampositive, Gram-negative and fungal competitors [39,40].Detecting NRPS genes by using degenerate primers was reported before by [32,33] who confirmed the detection of kurstakin synthetase genes in Bacillus thuringiensis using kurstakin AKs-F/TKs-R primers pair and detection of surfactin synthetase genes in Bacillus subtilis using surfactin degenerate As1-F/Ts2-R primers pair, respectively.We here detected the presence of fragments similar to an NRPs gene in Paenibacillus glucanolyticus, Acinetobacter and Priestia endophytica using surfactin degenerate As1-F/Ts2-R primers pair, which considered in agreement with [33] who detected fragments similar to an NRPs gene in B. thuringiensis berliner 1915 using the same primers.
Salinity treatment of barely seeds previously soaked in different endophytic strain suspensions has revealed many alterations in protein profile using SDS-PAGE.The results were in agreement with those of [21] who used SDS-PAGE to study the genetic diversity between wheat genotypes and scored 31 total bands among the genotypes and maximum polymorphism of 95.8%.Also, SDS-PAGE was used to identify twelve durum wheat Genotypes and detected a total number of 22 bands, 18 of them were polymorphic bands with 81.8% polymorphism, including two unique bands [41].A total number of 36 bands have been detected by [42] arranged between 124 to 12 kDa, 8 bands of which were polymorphic under salt stress in barley.In addition, upon transition from control to stress environment, [43] detected that band numbers increased in both cultivars and compared to the susceptible cultivar, resistant cultivar showed more number of bands.The use of all the six bacterial strains might play an important role in plant growth promotion by  plant pathogens growth inhibition, and/or by producing several secondary metabolites that can help in improving crop yield.These results agreed with those of [44] who used it as an eco-friendly alternative tool in the agricultural sector for the detection of plant defense to assist the host in sustaining different abiotic and biotic stresses.We observed that the best bacterial treatment under salinity stress 2.5% NaCl was the co-cultivation between all barely isolated endophytic strains B 1.2.3.4.5.6 which revealed a synergistic effect by the interaction between the endophytic bacteria.It is known that the plant-associated beneficial rhizosphere and/or endosphere bacteria are considered biocontrol agents that protect their host plants against pathogens directly or by promoting the plant's induced systemic resistance (ISR) [3,45] which depend on increasing the chemical or physical barrier of the host plant.Indeed, several elicitors involved in ISR have been identified in Bacillus subtilis GB03 [18,29] including antibiotics [8], organic volatile compounds and biosurfactants [lipopeptides families such as; surfactin] in Paenibacillus polymyxa C13 [46].Our results revealed a significant effect of the use of Bacillus genus members (strains B2, B4, B5 and B6) in addition to Staphylococcus (B1 strain) and Acinetobacter (B3 strain) members in conferring salt stress tolerance to barely seedlings.These results agree with [47] which reported that Bacillus amyloliquefaciens NBRI-SN13 (SN13) plant growth-promoting rhizobacteria (PGPR), confer salt stress tolerance  to rice seedlings and are known to modulate endogenous level of phytohormones in plants as a member of Bacillus genus [47,48] and a Bacillus producing lipopeptide families (surfactin, fengycin or plipastatin and iturin).
According to these results, we could consider barely seeds endophytic strains among the PGPR which are promising to improve plant growth during abiotic stress.

Conclusion
The use of endophytic bacteria is considered an alternative method to improve plant stress tolerance.According to this work, the overnight soaking and co-cultivation of these endophytic strains harbor NRPs synthetase clusters of two different lipopeptide families, surfactin and kurstakin with barely seeds before planting proved their capability in conferring salt tolerance to barely seedlings which appeared in protein patterns.We could consider these barely seeds endophytic among the PGPR strains which are promising in improving plant growth during abiotic stresses.

Fig. 1
Fig. 1 Phylogenetic tree for the seven isolated endophytic strains from barely seeds and the related strains from GenBank

Fig. 3
Fig. 3 NRPS identified synthetases genes by Anti-smash and PKS-NRPS analysis system for Paenibacillus glucanolyticus isolated endophyte from barely seeds.A Bacillibactin synthetases gene; B Unknown NRPS synthetase gene

Fig. 7
Fig. 7 Total and polymorphic with unique bands of SDS polyacrylamide gel electrophoresis of barley treated with endophytic bacteria under 2.5% NaCl

Fig. 8
Fig. 8 SDS-PAGE of barely soluble protein under the effect of endophytic bacteria and salt stress

Table 1
list of primers used for NRPS synthetases and 16S rRNA gene amplification Barley (Hordeum Vulgare) verity Giza 138 salinity sensitive was used to select the best bacteria-treated seeds under salinity stress.The barely seeds were soaked in overnight culture bacteria, 10 seeds with three replicates for each treatment B1, B2, B3, B4, B5, B6, B1.3, B 2.4.5.6,B1.2.3.6 and B1.2.3.4.5.6 for 24 h.The barley-treated grains with bacteria were germinated in pots.The growing seedling plants were treated with 2.5% NaCl after two weeks of germination for 48 h the control and treatments were collected for protein extraction.

Table 2
Diversity of microbial isolates from barely tolerant and sensitive seeds under different media conditions canolyticus and Bacillus cereus) were analyzed by Antismash due to the availability of their complete genome sequences on GenBank (NCBI), while the other three isolates were analyzed depending on their encoded protein data on GenBank followed by annotation and NRPS synthetases domains and modules detection using PKS-NRPS website.Two endophytic strains: Staphylococcus and Acinetobacter, have shown the absence of NRPS genes, while two strains: Bacillus sp.(In bacteria) and Priestia endophytica, have shown the presence of NRPS genes.Bacillus sp.(In bacteria) have shown the presence of bacillibactin synthetase gene, and two partial genes involved in surfactin biosynthesis, while Priestia endophytica encoded protein analysis revealed the presence

Table 3
GenBank accession numbers of the endophytic isolated strains from barely seeds with closest relative sequences similarity

Table 5
Non-ribosomal peptides synthetase genes involved in known lipopeptides synthesis detected by degenerate primers

Table 6
Densitometric analysis represents leaves water soluble protein electrophoretic patterns of barely treated with endophytic bacteria under 2.5% NaCl.(+) means presence and (−) means absence of bands

Table 7
The total and polymorphic with unique bands of SDS polyacrylamide gel electrophoresis of barley treated with endophytic bacteria under 2.5% NaCl B = bacteria 123456 = no. of bacteria Bold values indicate better results than other treatments