Biochemical characterization of PGPB strains
Accordingly, our strains (MA9, MA14 MA9, MA14, MA17 and MA19) were found to be able to metabolise sucrose, mannitol, glucose, starch, citrate, and nitrate. The isolates were also evaluated for their tolerance to different concentration of salt and growth at different temperature and pH. As a result it was observed that almost all of them were able to grow under NaCl concentrations ranging from 0 to 7%, with optimal growth at of 30 oC and pH 6.5 (Fig. S2).
In addition, the strains were positive for β-glucanase, protease, chitinase as well as for important plant growth promoting (PGP) properties including production of siderophores (Fig. 1), antimicrobial compounds and hydrolytic potential of 1-aminocyclopropane-1-carboxylate (ACC deaminase +) (Table 1). The majority of the tested strains were found to produce variable amounts of auxins (Fig. 2) ranging from 35 to 376 µg/mL except for MA17 and MA19, which produced relatively less auxin with and without tryptophan at 56/109 and 35/125 µg/mL, respectively. Particularly, MA14 was found to produce indole acetic acid (IAA) more than the rest of tested strains and even higher yield than values reported in literature . The isolates also showed phosphate-solubilizing activity as evidenced by the formation of clear zones on NBRIP agar plates. In the liquid NBRIP medium, bacterial strains solubilized variable amounts of phosphates ranging from 78 to 476 µg/ml (Fig. 2).
Antifungal activity for the different isolates was evidenced by the formation of inhibition zones between the bacterial isolates and the fungal isolates (Table 1). Among which, MA17 showed the highest antifungal activities against F. oxysporum (Table 1). All of the tested diazotrophic bacteria, except MA14, showed antagonistic effects to both R. solani, and F. graminarum.
Molecular profiling of the PGPB starins
The analysis of the 16S rRNA gene-based sequences for the different PGPB strains was done using BLASTn of the sequences against the NCBI’s database. We, accordingly, found that the sequences of isolates MA9 and MA17 showed 99% similarity with Bacillus pumilus Bp02 (DQ910855.2) and Bacillus pumilus ML270 (KC692158.1), respectively. While isolate MA19 showed 98% similarity and MA14 showed 84% similarity with Bacillus tequilensis BPR061 (KU161291.1) and Virgibacillus sp. SKW19 (KU132375.1), respectively.
Diazotrophic potential of the isolates
The results in Table 2 confirms the diazotrophic activity of the PGPB strains. Most of the tested strains were able to grow in the mineral medium without any nitrogen source as 1-Aminocyclopropane-1-carboxylate (ACC) and glucose as sole carbon source (Table 2; Fig. S2). The MA14 and MA17 strains were the most efficient ones in terms of biofilm formation after growing in a NFMM medium without nitrogen source, but MA14 exhibited the lowest capacity of acetylene reduction. Both strains (MA14 and MA17) were also found to produce ethylene after 72 h of incubation (data not shown) based on the acetylene reduction test and the CPG analysis of gases. These satrins were shown to have nitrogenase activities ranging from 0.452 to 3.125 μmol C2H4 mg protein/h based on the acetylene reduction assay (ARA). In addition, MA17 isolate showed maximum ARA and Kjeldhal levels of nitrogen but this strain had the least potential of growth on an N-free medium. The ARA test was verified by measuring N2 by the Kjeldhal method of bacterial culture in NFMM medium. Thus, the results imply that the strains had functional nitrogenase activity. The growth test revealed that the three strains MA9, MA14 and MA17 grew very well in the ACC medium, while, MA19 grew equally on NFMM and NFMM+ACC media (Fig. S2).
Evaluation of mechanisms regulating plant homeostasis
ACC deaminase activity assays in vitro in a chemically defined medium were carried out to determine wether different strains might play a role in regulating plant stress homeostasis.
In fact, bacterial culture in NFMM medium with 5 µg/mL of 1-aminocyclopropane-1-carboxylate (ACC) suggested active metabolic pathways of ACC degradation for the PGPB strains (MA9, MA14, MA17 and MA19) (Fig. 3). As known, the ACC metabolic pathway promotes plant growth under stress conditions by reducing the ethylene concentration in plant cells. In this context, numerous PGPB strains that are able to produce AcdS were found to promote plant growth under various stress factors [31-33].
Biocontrol effect of PGPB strains against Fusarium graminarum head blight in wheat
Biocontrol experiments were conducted to evaluate the bioprotection effect of two selected PGPB strains (MA17 and MA14) against Fusarium graminarum in wheat. The data obtained revealed that head blight severity was reduced in samples inoculated by the two strains compared to the untreated control. It is worth mentioning that strain MA14 did not exhibit an in vitro antifungal activity against F. graminarum despite the bioprotection it rendered to the plant, which means indirect mechanisms might have been involved (induced systemic resistance, ISR) or nich exclusion in its in planta activity. MA17 strain provided a more efficient bioprotection against the disease in the soil without fertilizer compared to MA14 with the disease index of 36 and 76%, respectively. Moreover, MA17 was found to render the plant growth promotion as well as bioprotection against fusarium head blight (Table 4, 5). The biocontrol efficiency was also refletcted by enhanced morphological parameters of the explant such as root length, shoot length, total length, total dry weight, total survival plants after treatment, and disease index. Thus, seed biopriming with both MA14 and MA17 provided a significant resistance to head blight in wheat explants, nevertheless, MA17 was more efficient than MA14 in terms of disease reduction efficiency (Fig.S3 - S8).
Effect of seed bacterization by PGPB on wheat growth under salt stress
PGPB seed bacterization enhanced germination rate
Tolerance level of selected PGPB strains to different salt concentrations was measured after seven days of culture. The results indicated that both MA17 and MA19 strains were halotolerant as they showed greater tolerance tol salt stress compared to both MA9 and MA14.
The sensitivity of durum wheat cv. Aouija was assessed at different NaCl concentrations in order to determine the effective salt concentration on limiting wheat plant growth to be used in the entire experiments. Accordingly, plant growth was affected at 50 mM NaCl and severely retarded at 120 mM NaCl. Thus, the later concentration (120 mM NaCl) was chosen for the further assays.
Regarding germination assays, we found that all PGPB strains enhanced wheat seed germination under salt stress compared to the negative control (non-bacterized but at 120 mM NaCl) (Fig. 4). The MA9, MA14, MA17 and MA19 significantly improved the germination rate by 47, 50, 57 and 40% under 120 mM NaCl treatment, respectively. Besides, most of PGPB strains significantly improved the germination rate of wheat seeds under normal conditions (positive control or no salt stress) (Fig. 4).
Biomass promotion of wheat plants by PGPB seed biopriming
The treatment of durum wheat seeds with halotolerant strains significantly improved root length under stress (125 mM NaCl) as well as under normal growth conditions except for MA19 strain, which reduced root length by 71% in the absence of stress. The growth promotion under stressful condition was 40, 131 and 70% for MA9 MA14 and MA17, respectively. While under normal growth condition the growth promotion was 48, 459, 27 and 70% for MA9, MA14, MA17, and MA19, respectively. However, the plant biomass was markedly increased after seed biopriming with MA17 and MA19 strains. The latter showed a significant plant development under 25 and 120 mM, as opposed to biopriming by MA9 and MA14 whose biopriming improved the length of the roots. However, it had no remarkable effect on the plant biomass which was reduced by 10% with MA9 under normal and by 7% with MA14 under stress conditions. The development of root length can be explained by an aggressive nutrient deficiency of the plant after inoculation by these two strains which have an inefficient biological activity spectrum under stress. The highest growth promotion effect on under stress was observed in MA14 strain (459%) in the length of roots and MA17 in the vegetative mass (98.94%) (Table 4) (Fig. S9).
Therefore, biopriming of seed with diazotrophic strains (MA9, MA14, MA17 and MA19) increased the dry weight of durum wheat var "Aouija". This induction effect was more pronounced under salinity conditions than under control conditions (Table 5) (Fig. S10). In fact, the percentage of improvement under (stress and non-stress), for MA9, MA14, MA17 and MA19 was (145, 51), (162, 82), (637, 200) and (166,114), respectively. The highest plant growth promotion percentage was observed in MA17 bioprimed-seeds under stress with a plant dry weight and 100-seed mass of 63.7 and 77%, respectively (Fig. S12).
In addition, seed bacterization with MA14, MA17 and MA19 strains induced an increase in root length by 48, 45.9, 27, and 70%, respectively, and in shoot length by 15, 7, 99 and 39%, respectively as compared to controls (untreated seeds) subjected to NaCl. However, treatment with MA19 decreased root length by 71% and enhanced shoot length by 48% in comparison with the controls (untreated seeds). In contrast, the strain MA14, displayed no significant impact on wheat plant growth parameters under non-saline conditions. Moreover, seed biopriming with PGPB strains, except MA9, offered greater growth promotion in comparison to control plants, whereas under natural and saline treatments, they showed a promotion in shoot length and total dry weight parameters (Table 5, 6). In fact, plant dry weight was increased at 25 and 120 mM NaCl for all the PGPB strains. However, at 120 mM NaCl, except of MA14, inoculated plants showed significant increase in root length. At 25 mM NaCl, the root length of inoculated plants with MA17 was markedly increased compared to uninoculated and other PGPB-inoculated plants (Fig. S9 - S13). These results indicated and the usefulness of MA19 to enhance wheat plant growth under salt stress.
Effect of PGPB treatments on nitrogen (N) and protein contents in wheat seeds
The N and protein contents in wheat seeds in response to PGPB isolates under both stress and control conditions are shown in Fig. 5. Both N and protein contents in wheat seeds were significantly (P < 0.05) affected in response to stress treatments. In fact, the total nitrogen content in untreated seeds was 1.06 and 1.71 g.100g-1, while, it ranged from 1.796 to 1.93 g.100g-1 and from 1.799 to 1.85 g.100g-1 for bioprimed-plants under stress and natural conditions, respectively. Among the different amendments, seed biopriming with the different isolates before germination provided an enhancement of these parameters compared to both untreated seeds under stress and natural conditions. The highest total N content of 1.93 g.100g-1 was recorded in plants bioprimed with MA17 under stressful conditions (125 mM NaCl) followed by those with MA19, MA14 and MA9 with 1.826, 1.807 and 1.796 g.100g-1, respectively. The comparative upsurge in total nitorgen content in plants bioprimed with MA9, MA19, MA14 and MA17 compared to the negative control (salinized soil) was 69, 72, 70 and 82%, respectively, and 4.6, 8.2, 7 and 8.2%, respectively compared to the control (soil without NaCl). A such increase in N content by PGPB relied on by their capacity to colonize the soil colonization and to grow stress environments. Nitroginase activity was recorded in all PGPB treatments (Table2), indicating the interplay between nitogen and protein contents in plant and N2-fixation (Fig. 5). Application of different PGPBs under stressful condition significantly (P < 0.05) increased plant protein contents in the range of 10.25 and 11.07 g.100 g−1 literally doubling the content as compared to the non-treated control. The highest protein content of 11.07 g.100 g−1 was recorded for MA17 under salt stress followed by that for MA17 under normal conditions (10.61 g.100 g−1). In the controls (without biopriming and without NaCl), the total protein content was 10.02 g.100 g−1. Moreover, there was a relative increase in protein content in plants treated with PGPB strains MA9, MA14, MA17 and MA19 compared to the control by 2.29, 3.4, 10.47 and 3.6 %, respectively.
Seed biopriming enhanced the total mineral content
The total mineral content in positive control plants (no PGPB, no NaCl) decreased by 72 % compated to those subjected to NaCl treatment. In addition, a reduction by 40% in dry weight was noted in negative control plants (with NaCl, no PGPB) when compared to controls (Fig. 6). The plants inoculated with selected PGPB showed an increase in their total mineral content either in the presence or absence of NaCl treatment. The plants inoculated with MA9 showed an increase in their mineral content by 14.6 to 441 %, in salted and unsalted soils compared to negative controls (Table 5). While plants inoculated with MA17 (B. pumilus) showed 24 and 56% higher total mineral contents compared to those of the control plants under primary and secondary salinity, respectively. The total mineral content in MA14-treated plant was greater than those measured in MA9-treated ones.