Characterization of plant growth-promoting rhizobacteria (PGPR) in Persian walnut associated with drought stress tolerance

There is a lack of information on the rhizosphere of nut-bearing trees where microbial populations can benefit roots and tree growth. The current research aimed at discovering plant growth-promoting rhizobacteria (PGPR) in the rhizosphere of soil samples from around the root zone of six walnut trees, each of which was considered as a genotype, i.e. ‘TT1’, ‘TT2’, ‘SS2’, ‘ZM1’, ‘Chandler’ and ‘Haward’. The trees grew in different arid and semiarid regions of Iran and Turkey. The strains were isolated and identified based on different morphological and biochemical markers. Drought-stress tolerance was assessed in the case of each isolate through their transfer to culture medium, containing polyethylene glycol (PEG6000) at 0 and 373.80 g L−1. Resilient strains were analyzed for measuring their ability to produce siderophore, hydrogen cyanide (HCN), Indole-3-acetic acid (IAA) and Gibberellic acid (GA3). In sum, 211 isolates were identified, of which a large number belonged to the Bacillus genus and, specifically, 78% of the strains were able to grow under drought stress conditions. The genus Arthrobacter was only detected in the rhizosphere of ‘ZM1’, ‘Haward’ and ‘TT1’ genotypes. In 4% of the strains, IAA production exceeded 53 mg L−1, while a high level of phosphorus solubility was verified in 6% of the strains. No strain was found to have the capability of producing HCN. The strains were screened for drought-tolerance, which resulted in the discovery of two promising strains, i.e. ZM39 and Cha43. Based on molecular identification through amplification and sequencing of the 16S rDNA gene, these two strains seemed to belong to Bacillus velezensis and Bacillus amyloliquefaciens, respectively. The discovery of new PGPR strains could probably assist walnut trees in improving their mechanisms of adaptation to drought stress.

Drought stress tolerance assessment for the isolates. Single colonies were cultured and transplanted into 500 ml containers containing NB. They were aerobically grown on a rotary vibrator at 150 rpm for 48 h at 27 °C. This was followed by adding distilled water along with a suspension of bacteria, reaching a final concentration of 10 9 ml mL −1 . Various strains were transferred to a culture medium containing PEG 6000 at 0 and 373.80 g L −1 which was equivalent to osmotic stress at 0 and − 1.5 MPa. The growth rate of isolates in a pilot study was previously recorded at different PEG 6000 concentrations. Screening tests on these strains were calculated based on the growth rate of bacteria by measuring the optical density of their growth medium at 600 nm, using spectrophotometric analysis. Cultures that were able to grow in the presence of PEG 6000 were analyzed further for biochemical traits through a factorial (PEG 6000 at 0 and 373.80 g L −1 as factors) based on completely random design.
fied type of the Sperber (Sp) medium 15 , and was supplemented with inositol hexaphosphate. Instead of tricalcium phosphate; however, inositol hexaphosphate was used. The test was performed on both solid and liquid media. The Sp medium, contained 2.5 g L −1 calcium phytate, was distributed in petri dishes under sterile conditions. Each petri dish was divided into six equal parts and the different fresh isolates were cultured by the dipping method in a separate petri dish, with three replicates, and stored in an incubator at 28 °C. The colony diameter grew, leaving different diameters of the transparent halos from the dissolution of the phosphate around each colony. The diameters were measured at intervals of 1, 2, 4, 8 and 10 days, and then the diameter of the halo was measured against the colony diameter in each isolate. Isolates which had halo-to-colony diameter ratios of more than 1.5 mm were evaluated by culture methods on liquid medium. At this stage, the modified Sp medium of 2.5 g L −1 calcium phytate was used. Phosphorus was measured in inoculated and control (un-inoculated) media. The preparation of the standard curve was similar to the mineral method. Amylase activity was performed using a modified version of Nack-Moon et al. 16 method. After incubation at 30 °C for 5 days, the production of amylase, which was detected using soluble starch (1%), was screened by adding iodine to the culture. Liquid starch medium was used as control. Discoloration confirmed the presence of amylase production of bacterial isolates that decompose starch.
Siderophore and hydrogen cyanide production. The CAS method 17 was used for quantitative measurement of siderophore produced by the strains. The reaction mixture included a cell-free extract of supernatant (0.1 ml) which was mixed with 0.5 ml of the CAS assay solution along with 10 μl of a shuttle solution (0.2 M 5-sulfosalicylic acid). The reaction mixture was stored at room temperature for 10 min, and then the absorbance was measured at 630 nm using a UV-VIS spectrophotometer (SL164, Systronics). For the control treatment, all of the compounds were used, except the extract which was obtained from the cell. Each siderophore unit was calculated using the following formula: where Ar is the absorbance at 630 nm of reference (CAS assay solution + uninoculated media), and As is the absorbance at 630 nm of the sample (CAS assay solution + supernatant).
Production of HCN was done using a nutrient agar medium containing 0.44% glycine 18 . The surface of the agar was streaked with one-day culture and was coated with Whatman No. 1 filter paper. It was immersed in 2% sodium carbonate solution and 0.5% picric acid. This was followed by storage at 30 °C for 72 h. The changes observed in the color of the filter paper ranged from yellow to orange, red and brown. These colors indicate respectively the low, medium and high levels of HCN production by the strains. A quantitative analysis of HCN was done by analyzing strips of filter paper, which were changed by picric acid and sodium bicarbonate. After inoculating the tubes and keeping them at 30 °C for seven days, the strips were stored in double-distilled water and the changes in color were observed at 625 nm.
Estimations of Indole-3-acetic acid and gibberellins. Primarily, bacterial isolates were grown in NB for 72 h at 37 °C in a shaker. IAA was eluted by methanol according to a method described by Żur et al. 19 . The supernatant comprised formic acid (0.1%) aqueous solution (solvent A) and acetonitrile: methanol (1:1) mixture (solvent B). The MRM (Multiple Reaction Monitoring) mode was used for monitoring each analyzed compound in which the most abundant ion product functioned as the quantifier. Also, another abundant ion product was used for identifying the phytohormones. For the extraction of gibberellic acid (GA 3 ), extracts were prepared with a mixture of iso-propanol/H 2 O/concentrated HCl (2:1:0.002, v:v:v). Then, they were centrifuged and purified within a series of steps. A re-dissolved operation was performed with a final concentration in 100 L methanol. Half of the volume was subjected to an ESI-triple quadrupole mass spectrometer device (HPLC-ESI-MS/ MS, Applied Biosystems, USA) which was equipped with a reverse-phase C18 Gemini column (150 9 2.00 mm, 5-lm particle size, Phenomenex, USA) 20 .

Molecular identification of the bacterial strains.
Two strains were then selected for further molecular characterization and identification. For this purpose, amplification occurred through PCR reaction, followed by subsequent sequencing of the 16S rDNA gene. The cultures were grown in LB for DNA extraction at 26 °C on a shaker (250 rpm). DNA was extracted using the DNeasy Tissue Kit (Qiagen). DNA concentration and quality were determined using a spectrophotometer and by gel electrophoresis on agarose (1%). The thermal cycles of the PCR operated according to a protocol used by Rees and Li, 2004. The amplified products were sent to Macrogen (a South Korean company) for sequencing the samples in both directions. The NCBI database was used as a platform for the comparison of sequence data by the BLAST program. The sequences of representative strains were submitted to the GenBank database and accession numbers were obtained.

Results and discussion
The physical and chemical characteristics of soil samples from the rhizosphere of each genotype differed from another (Table 1). In total, 205 non-identical bacteria were found in the rhizosphere surrounding the walnut roots (  21 . In confirming a large range of diversity among the bacterial strains, our results were in agreement with previous findings reported by Vega et al. 22 in a research that led to the isolation of large numbers of bacterial strains from the rhizosphere of coffee plants. In the current study, the strains were primarily screened for their ability to tolerate drought stress, while considering important physiological and biochemical traits relevant to plant growth promoters. In the absence of drought stress, phosphate-solubilizing activity corresponded with high amounts of dissolved phosphorus, which were caused by strains ZM4 (256 mg L −1 ), ZM18 (239 mg L −1 ), ZM44 (237 mg L −1 ), Cha13 (236 mg L −1 ) and Cha27 (235 mg L −1 ) (Table 3). Meanwhile, drought stress led to high amounts of dissolved phosphorous in strains ZM39 (248 mg L −1 ), ZM18 (254 mg L −1 ), Cha15 (241 mg L −1 ) and Cha43 (269 mg L −1 ). High amounts of siderophore production were recorded in strains Cha38 (24.5%), Haw25 (23.7%), ZM4 (22.7%) and Haw20 (21.4%) under normal conditions, whereas drought stress induced high levels of siderophore production by Cha43 (28.6%), ZM39 (28.4%), Haw25 (25.8%) and ZM4 (24.6%), respectively.
The ability of microbes to release metabolites, such as organic acids, can be used as an index for determining phosphate-solubilizing activity 23 . The ability of bacteria to secrete organic acids is a function of mechanisms that are controlled by bacterial gene expression patterns which, in turn, can be influenced by environmental factors. Many phosphate-solubilizing bacteria (PSB) can forage Fe from the mineral complex into soluble Fe 3+ which takes form through mechanisms of active transport carriers 24 . Siderophore production by PSB could improve the availability of P indirectly. Since siderophores are ligands that can extract Fe from ferric phosphate and ferric citrate 25 , PSBs tend to produce organic acid compounds that help to transform metal species into chelates, thereby reducing metal toxicity 26 .
The ability of plants to produce auxin is one of the main traits by which plant growth promoters are generally measured. Auxins are a group of hormones which act as molecular signals that regulate plant growth, prolong cell proliferation, cell division and differentiation. While coexisting with plants, different bacteria produce various amounts of auxin and release them into the rhizosphere. Auxins are reportedly produced by Rhizobium, Bradyrhizobium and Nostoc species, as well as by other species which occur in the rhizosphere 27 . Among the strains in the current research, the ability to produce auxins differed significantly among the bacterial species. Bacillus strains Cha41 (24.7 μg mL −1 ), Cha21 (24.1 μg mL −1 ) and ZM7 (22.7 μg mL −1 ) had the highest performance in producing auxin under normal conditions. Under drought stress, however, IAA formation increased in the Bacillus strains Cha43 (29.4 μg mL −1 ), ZM39 (28.7 μg mL −1 ) and Cha21 (26.9 μg mL −1 ) ( Table 3). The current results are in agreement with those previously reported by Beneduzi et al. 28 regarding the ability of Bacillus strains to produce auxin in Luria and Berthani Bruce media. Similarly, Lwin et al. 29 and Kaur and Sharma 30 showed that rhizobacteria produced auxin (53.1-71.1 μg mL −1 ) under optimal growth conditions, whereas Husen et al. 31 reported lower amounts of bacterial auxin production (33.28 μg mL −1 ). Shobha and Kumudini 32 reported a wide range of IAA (35-217 μg mL −1 ) produced by bacteria, since IAA production by PGPB strains can be affected by different factors, including the species of the microorganisms, the conditions in which plants and bacteria coexist, the specificity of each growth stage in plants, and the availability of suitable substrates 9,33 .
The current study showed significant differences in the amounts of GA 3 which were produced by the different strains. The highest gibberellin production was observed in strain Haw20 (80.9), followed by Cha28 (78.9) and Haw14 (77.9) (μg mL −1 ). In contrast, drought-stressed strains produced significantly higher amounts of GA 3 , as observed in Cha41 (94.3), Haw20 (86.7) and ZM39 (85.4) (μg mL −1 ) ( Table 3). Previous studies reported that the maximum amount of gibberellin (65.3 μg mL −1 ) was produced by Pseudomonas sp. when the bacteria were isolated from the wastes of processed olive fruits in a culture medium of NB 34 .
One of the secondary metabolites produced by the PGPR is hydrogen cyanide, which plays an important role in the biological control of pathogens. In this study, all of the evaluated strains produced HCN, although their levels of production were not similar. Strains ZM39 and Cha43 produced the highest amounts of HCN, so much that the color of filter papers changed from pale yellow to brown. Also, these two strains showed starch Table 1. Physio-chemical characteristics and mineral elements of soils from the different rhizospheres of walnut trees. AECC active equivalent calcium carbonate, OC organic carbon, FC field capacity, pH acidity of soil, EC electrical conductivity. *Each measurement is the mean of three replications. All of the nutrients were measured as the absorbable form.     www.nature.com/scientificreports/ hydrolysis activity along with other 134 strains (Fig. 1). Earlier research in the available literature suggested that HCN production by PGPR can promote plant growth by inactivating pathogens, but recent findings have argued that HCN indirectly increases P availability by metal chelation 35 . Increasing of soluble sugar contents in the leaves of Brachypodium distachyon (switchgrass) treated by some Bacillus strains has been reported 39 . Based on morphological, biochemical and biological assessments, while considering plant response to drought stress, two promising strains were identified, ZM39 and Cha43, which caused high levels of resistance to drought. Thus, both strains were selected for further genetic identification by molecular markers. Upon completing the amplification and sequencing of their 16S rDNA gene sequences, and after using the BLAST-N program (NCBI), a complete identification showed > 99% similarity of their partial sequence with the available sequences from the NCBI database. The obtained sequences were submitted to NCBI and, together with other relevant information, their accession numbers were provided (Table 4). According to molecular assessments, ZM39 and Cha43 strains were identified as members of B. velezensis and B. amyloliquefaciens, respectively (   www.nature.com/scientificreports/ Table 3. Multiple plant growth promoting activities of the promising rhizobacterial strains, isolated from different rhizospheres of walnut trees, under control (0 MPa) and PEG 6000 -induced drought stress (− 1.5 MPa) treatment. *Value represent mean ± SEM; ** Least Significant Difference (LSD; p < 0.001) for mean comparison of strain-drought interaction effects.

Rhizobacteria strains
Phosphate-solubilizing activity (mg L −1 ) Siderophore production (% Siderophore unit) IAA production (μg mL −1 ) Gibberellic acid (GA 3 ) production (μg mL −1 ) HCN production    36 . Among the strains of the B. velezensis clade, secondary metabolites are diversely created by bacterial cells, which could exhibit antibacterial and anti-stress activities 37 . In the current study, with respect to the 16S rDNA gene sequence, phenotypic and molecular characteristics of B. velezensis were similar to those of B. amyloliquefaciens. In previous research, gyrB gene sequences confirmed that B. velezensis and B. amyloliquefaciens had similarities in heterotypic terms 38 .
Strains of the Bacillus genus have reportedly enhanced drought-tolerance in switchgrass through upregulation of drought-responsive genes and the modulation of the DNA methylation process 39 . Other strains can also make close associations with host plants and produce phytohormones, along with several well-characterized lipopeptide toxins 40 . These characteristics suggest that these strains have strong potential to act as bio-inoculants and can increase biomass in fruit trees, while assisting the defense system in plants against abiotic stress. PGPR can ultimately contribute to the production of plant growth regulators such as gibberellins 41 , auxins 42,43 , cytokinins and ABA 44 , thereby mitigating the adverse effects of abiotic stress on the physiological and biochemical processes of plants. Hormone levels in plant tissues could be modulated by microbial regulators via mechanisms that mimic the modes of exogenous phytohormone application 45,46 .

Conclusion
A large outlook of research potential is envisaged to explore walnut trees in terms of microbial populations in their rhizospheres. In this work, a significant diversity of PGPRs was confirmed in walnut rhizospheres. In addition, drought stress induced the ability of the identified PGPRs to solubilize phosphates and produce siderophore, IAA, gibberellic acid and HCN. Thus, there is scope that these PGPRs can be used as bio-fertilizers for sustainable crop production. Such improvements in the capabilities of plants may protect them against various forms of biotic and abiotic stress. Two promising strains (ZM39 and Cha43) were identified based on the current morpho-biochemical assays upon drought stress treatment. These strains were molecularly identified as B. velezensis and B. amyloliquefaciens, respectively. An ongoing project involves specifying their roles in improving tolerance against drought stress in walnut seedlings.  F: 5′-AGA GTT TGA TCT TGG CTC AG-3′  R:5′-AAG GAG GTG ATC CAG CCG  CA-3′