3.1. Physico-chemical soil characteristics
The analyses revealed that the saffron soil studied is a sandy clay loam according to the USDA texture classification and a Luvisol according to the WRB soil classification. It’s an alkaline soil with a low percentage of nitrogen (0.04%) and total carbon (0.89%), a high C/N ratio (22), and a very low amount of available phosphorus (18 mg/kg). All these parameters are indictors of low soil fertility (Chamkhi et al., 2019).
3.2. Isolation of bacterial strains
A collection of 88 isolates was obtained from rhizospheric soil of saffron by using 3 selective media: PVK, Modi, and YEM-try. Among them, 34 isolates were obtained on PVK medium, 22 isolates on Modi medium, and 32 isolates on YEM-try medium.
3.3. Evaluation for PGP activities
3.3.1. Auxin production
The colorimetric method of Salkowsky demonstrated that saffron isolates tested produced different amounts of IAA, classified at 7 different levels from Group1: Interval [125.3 µg/mL; 123.83µg/mL] to Group7: Interval [14.9µg/mL; 1.1µg/mL] (Fig. 1). The best auxin producing strains were S11S2 (125.3 µg/mL), S11A1a (124. 4 µg/mL) and S11P12 (123.8 µg/mL) respectively group in interval 1 (Grp1).
3.3.2. Siderophore production
The qualitative test by using the chrome-Azul S reagent (CAS) detects the siderophores produced by the isolates, which can be classified the isolates into 4 categories according to the time and the color change of CAS reagent from blue to orange color. Percentage of 48.31 % of isolates produce highly the siderophores (+++), 23.60 % produce siderophores moderately (++), 11.24 % (+) weakly produce siderophores and only 16.85 % (-) of isolates were unable to produce these chelators (Fig.2).
The ratio A/Aref (semi-quantitative test) was calculated to the best strains screening on the first qualitative test of siderophores production. The results of siderophores production in decreasing order as follows: S12S4, S11P12, S11A11b, S13P5, S11A10a, S12S1, S13P9 represented in table 1, were the best siderophore producing isolates according to the ratio A/ Aref (Table 1).
3.3.3. Phosphate solubilization
The qualitative test of phosphate solubilization on solid Pikovskaya medium revealed that almost all the bacteria isolated on selective PVK medium were surrounded by transparent halos, what indicates the capacity of these bacteria to solubilize rock phosphate in vitro. The halo of phosphate solubilization is caused by the production of organic acids in the medium which turns the color of the bromophenol blue from mauve towards white/yellow. As a result, the strains S13P4 (19 mm), S12P9 (17 mm) and S13P11 (13 mm) have the biggest halo diameter size on solid Pikovskaya medium (Fig.3).
The quantification of soluble phosphate released confirmed that all of the tested bacteria possessed the ability to solubilize inorganic phosphate (Fig. 4). The measured concentrations were very close to each other for the majority of isolates, with the high amount of soluble phosphate released by strains S12A7 (65.13 mg/L), S11A8 (63.60 mg/L) and S11A5 (63.30 mg/L) (group 1).
3.3.4. Biological nitrogen fixation
Obtained results of atmospheric nitrogen fixation showed that only 9 % of the isolates had a high capacity of nitrogen fixation, 29% showed a medium capacity of nitrogen fixation, while 27% were low nitrogen fixers and 14% were unable to fix nitrogen (Fig. 5).
3.3.5. Aminocyclopropane -1-carboxylate (ACC) deaminase activity
Among the isolates screened only 55% of isolates growth in the minimal medium by measuring absorbance at 600 nm, where they have the ACC as a sole source of nitrogen. While, 9 % produce ACC deaminase highly, 22% produced ACC deaminase moderately, 24% isolates produced ACC deaminase lowly. Nevertheless, 45 % isolates not able to produce ACC deaminase (Fig. 6).
3.3.6. Cellulose activity
From all the collection of saffron, 11.36 % formed a clear halo around bacterial spots on plates containing carboxymethylcellulose (CMC-Na) degradation selective medium, which is indicative of cellulose activity. The isolates S12S3 (7mm), S13A4 (5mm) show the biggest halo which indicates high cellulose activity, then the isolates S13A6b, S13A2, S11P13, S11P10, S11A1b, S13P2, S11P5, S13S1 respectively go after Fig. 7.
3.3.7. Antagonistic activity against pathogenic fungus
The screening of antagonistic activity demonstrates that the percentage of 20.45 of the isolates shows an antagonistic effect against F. oxysporum. The maximum inhibition percentage in radial growth of F. oxysporum caused by strains was S11A3 (79 %), S12P3 (70%), S12P4 (67.4%), S11A2a and S12S4 (66.7%), S12A4b (65.2%), S12P1 (63%) and the minimum inhibition percentage was 35.6% caused by strain S13P9 (Fig. 8).
3.4. Molecular identification
The 16S rRNA gene sequences of 88 isolates obtained were analyzed using bioinformatic tools and deposit under NCBI gene bank accession number, from KU569610 to KU569698. Bacterial isolates belonged to 13 different bacterial species namely Pseudomona putida, Pseudomona fluorescens, Pseudomonas sp., Rahnella aquatilis, Variovorax paradoxus, Bacillus simplex, Bacillus subtilis, Rhizobium radiobacter (Agrobacterium tumefaciens), Rhizobium rhizogenes, Pantoea sp., Luteibacter sp., Delftia sp., and Rhizobium sp. Among the 8 genera identified, Pseudomonas is the predominant genus with 34.83%, followed by the genus Rahnella with 24.71%, Variovorax with 15.73% and Delftia with 11.23%. The other genera are weakly represented in the saffron rhizosphere (Fig. 9).
3.5. Correspondence analyses
The analyses showed a relationship between the biological activities and the genera of the isolated bacteria. At this respect genera Luteibacter, Pantoea, and Rahnella have presented the ability of solubilizing inorganic phosphate (Fig.10A), while Rahnella and Pantoea were the best producers of auxin (Fig.10B). Five genera, Variovorax, Pseudomonas, Rahnella, Luteibacter, and Pantoea are siderophore producers (Fig.10C), while biological nitrogen fixers were found in two genera, Rhizobium and Rahnella (Fig.10D). While Pseudomonas was the only genus showed cellulose activity (Fig.10E), however, the ACC deaminase activity includes Pseudomonas, Rahnella and Variovorax genera (Fig.10F) and finally, the antagonistic activity Bacillus was the only genus manifested this activity (Fig.10G).