Effects of Durum Wheat (Triticum durum Desf.) Inoculation with PGPR (Azospirillum brasilense, Bacillus sp and Frankia CcI3) and Its Tolerance to Water Deficit

Drought is one of the major limiting factors that affect plant growth, biomass and production (Zhu et al., 2002). Climate model predictions indicate that global warming will gradually increase temperature, thereby escalating drought stress in the near future (Barrow et al., 2008).
       
Plant growth promoting rhizobacteria (PGPR), detected mostly in the rhizosphere of many cultivated plants (Kloepper,1993),  are not only able to fix nitrogen, increase root growth and bio-control against pathogens but also produce substances such as phytohormones (auxin) or proteins such as polyamines thus inducing plant tolerance to stress. Several important metabolites including osmolytes (such as proline and soluble sugar) and active molecules (such as polyamines and melatonin) accumulate substantially in drought-treated plants (Zhang et al., 2015). These molecules are involved in stabilizing membrane structures and regulating stress-responsive pathways. On the other hand, under stress, the ability of soil microorganisms to reduce stress can be improved (Miransari et al., 2008). These conditions are common in arid and semi-arid areas around the world.
       
The use of plant growth promoting rhizobacteria (PGPR) to induce plant drought resistance offers the following advantages (Coleman-Derr et al., 2014): (i) some PGPR benefit diverse plant hosts, including monocots and dicot plants; in addition, (ii) transferring the ability of one stress-resistant plant species to another can easily manipulate through microbial inoculation (Zhou et al., 2016). Some strains of B. megaterium (Bacillus megaterium) and Azospirillum can improve plant growth (Chakraborty et al., 2006) but no information about the effects of Azospirillum brasilense, Basillus. sp and Frankia CcI3 on durum wheat stress tolerance has been reported. For this purpose, the vegetative parameters including water content and its effects on the growth of durum wheat inoculated and not inoculated under stress were studied during this experiment. The objectives of this study were (1) to evaluate the inoculation effects of durum wheat under stress and (2) to test if the inoculation of durum wheat with PGPR strains under stress effect can restore its growth.

Seeds sterilization and germination
 
Seeds of wheat (Triticum durum Desf.) GTA- dur and WAHA with similar size and weight were washed with distilled water three times and soaked in alcohol of 70% for 20s. Seeds were then sterilized for 15 min using sodium hypochlorite 3% and rinsed with sterilized water 5 times. The sterilized seeds were soaked in distilled water for 3h and 15 of them were grown in sterilized Petri dishes for 48 h at 20°C in darkness (Benmati et al., 2013).

Seedlings inoculation with different strains
 
The bacterial suspension was prepared in LB (Luria-Bertani) medium for Azospirillum. b, Bacillus. sp and BD (Broughton et Dhlworth) for Frankia CcI3. After germination, wheat seedlings were inoculated with 3 ml of bacterial inoculums. After 3 h, the extra inoculums were removed from tubes and the tubes were rinsed with distilled water. After the phase of inoculation, the germination seed were transferred into the pots containing sterile soil and N-free nutrient solution (10 g Malat, 1 g KH₂PO₄, 0.2 g MgSO₄ 7H₂O, 0.2 g NaCl, 0.005 g FeSO₄ 7H₂O, 5 g Mannitol, 100 mg yeast extract) (Azlin et al., 2005) and uninoculated seeds are irrigated once a week also with nutrient solution.
 
Water stress study under greenhouse
 
The experiment was placed in the greenhouse under conditions controlled (temperature 25°C, 16 h photoperiod and humidity 30%).
       
The experiment was based on a device with 1 level of severe stress (12% of CC) and a portion was devoted to the witnesses. After three months of growth, water stress was applied by the irrigation stop until the level of stress was reached (12% of CC).
 
Parameters studies
 
Relative water content (RWC %)
 
The relative water content of the leaves is determined by the method described by Barrs, (1968). According to this method, the leaves are cut at the base of the blade, weighed immediately t obtain fresh weight (FW). Then put into test tubes filled with distilled water and placed in the dark in a cool place. After 24 hours, the leaves are removed, passed through blotting paper to absorb water from the surface and then weighed again to obtain the weight of full turgor (FT). The samples are finally placed in the oven set at 80°C for 48 hours and weight to their dry weight (DW). The relative water content is calculated by the formula of Clark and Mac-Caig (1982):
               
RWC % = (FW-DW) ÷ (FT-DW) × 100
 
Stomatal resistance measurement
 
Stomatal resistance at leaf level is measured using a Delta devices MK3 type Porometer. Reading is done by inserting the middle part of sheet into the forceps (Herbinger et al., 2002). Stomatal resistance data is stored in the meter and then transferred to a computer for processing.
 
Determination of Proline
 
The method followed is that Rasio et al., (1987). It consists of taking 100mg of fresh material in test tubes containing 2 ml of 40% methanol. The whole is heated to 85°C in bain marie for 60 minutes. After cooling, 1ml extract is taken to which are added: 1 ml of acetic acid, 25 mg ninhdrin, 80 ml of orthophosphoric acid. The solution obtained is brought to the boil for 30 minutes at 100°C which then begins to turn red, after cooling 5 ml of toluene are added to the solution which is stirred, two phases separate. After removing the lower phase, the upper phase is recovered and dehydrated by adding anhydrous Sodium Sulfate. The determination of the optical density (Do) is then carried out using a spectrophotometer on a wavelength of 528nm.
 
The total chlorophyll content
 
The chlorophyll level in the leaves is measured using a SPAD 502 chlotophyll meter from Minolta (Nouri, 2002). The values conventionally found are between 0 and 50 (SPAD units). The measured values consist in making three measurements at the level of the sheet on three different parts (top, middle and base). The average of the three values is displayed on the screen at the end in SPAD units.
 
Determination of soluble sugars
 
Total soluble sugars are determined by the phenol method of Dubois et al., (1956). It consists of taking 100 mg of fresh material, placed in test tubes, to which are added 3ml of 80% ethanol to extract the sugars, the whole is left at room temperature for 48 hours in the dark. At the time of dosing, the tube are placed in the oven at 80°C to evaporate the alcohol, thereafter, 20 ml of distilled water are added to extract in each tube, this is the solution to be analyzed. In this solution, 1 ml of phenol is added and 5 ml of sulfuric acid are the added. The tubes are left as they are for 10 minutes and then place in a bain-marie for 10 to 20 minutes at a temperature of 30°C. Absorbance measurements are made at a wavelength of 485 nm with the spectrophoto-
-meter.
 
Statistical analysis
The results were statistically interpreted by an analysis of variance (ANOVA) and principal component analysis (PCA) using XLSTAT software (2014). The Newman-Keuls test was used at a significance level of 5 %.

Relative water content
 
A comparison of changes in water content of control and inoculated durum wheat plants showed that relative water content decreases as the moisture deficit increases (Table 1). The highest water contents were recorded in controls, with a maximum value of 75.30 ± 0.19% recorded in GTA- dur without inoculation and stress (Fig 1) and the lowest were recorded under the influence of water stress.
 

 
@figure1
       
The highest water contents were obtained in inoculated plants with a maximum value of 87.48 ± 0.17% recorded in GTA / DUR inoculated with A.brasilense (Fig 1). On the other hand, recorded water contents were lower under water stress. At the 12% CC stress level, the minimum value was observed in control plants of 46.11 ± 0.13% and 47.59 ± 0.26% recorded in GTA and WAHA, while the minimum value was recorded in inoculated plants of 72.32 ± 0.07% in GTA/ DUR inoculated with A.brasilense.
       
The NEWMAN-KEULS test at the 5% threshold classifies the genotype factor into two homogeneous groups (Table 1), group A (GTA- dur) and B (WAHA), the NEWMAN-KEULS test with a 95.00% confidence interval.
       
Some strains of PGPR play crucial roles in assisting plants to cope with unfavorable conditions, including drought (Bresson et al., 2013). Among these PGPR strains, Pseudomonas chlororaphis O6 induces systemic tolerance to drought stress in plants through a salicylic acid-dependent signaling pathway (Cho et al., 2008).
 
Stomatal resistance
 
A comparison between the stomatic resistance evolution of the control plants and the studied inoculated hard wheat plants showed that the stomatic resistance changes a lot under the effect of stress (Table 1). The highest value was noted in the control groups, with a maximum of 1.05 ± 0.05 S /CM recorded in WAHA control plants without inoculation and stress. In inoculated plants, the highest stomatic resistance was 0.77 ± 0.02 S /CM recorded in the WAHA plants inoculated with A.brasilense.
       
In the case of 12% CC stress, there was a very large increase in stomatic resistance in the control plants; the values   were between 62.27 ± 0.04 S /CM and 77.66 ± 0.05 S /CM recorded in GTA and WAHA respectively. In inoculated plants, the maximum value was 6.55 ± 0.03 S /CM recorded in the WAHA inoculated with Bacillus.sp. The results in Fig 2 show stomatic resistance in durum wheat (GTA- dur, WAHA) of the control groups and of the inoculations (A.brasilense, Bacillus sp and Frankia CcI3) as well as stressed and stressed inoculated control plants.
 
@figure2
       
The NEWMAN-KEULS test at the 5% threshold classified the genotype factor into two homogeneous groups (Table 1) group A (GTA- dur) and B (WAHA).
       
In contrast, the protein and nitrogen content of the plant depends on the nitrogenase activity of the nitrogen-fixing bacteria and the type of fertilizer contained in the soil (Rodrigues et al., 2008). When wheat is subjected to salt or moisture stress, the leaf responds primarily by changing the volume and number of stomata to maintain enough water to restore photosynthetic activity. The leaf must therefore keep its surface intact to ensure the proper development of the plant.
 
Determination of proline
 
A comparison between the evolution of the proline of the control plants and the studied inoculated hard wheat plants showed that the proline level changes a lot under the effect of stress (Table 1). The highest value of proline noted in control plants, with a maximum value of 35.60 ± 0.35µg /100mg MF, was recorded in WAHA plants without inoculation and stress. In inoculated plants, a maximum value of 34.65 ± 0.23 µg /100mg MF was recorded in GTA- dur plants inoculated with Bacillus.sp. In the case of 12% of CC stress, the levels of proline increased a lot in the plants without inoculation one notes values of 164,30 ± 1,41 µg /100mg MF; 164.63 ± 3.30 µg /100mg MF respectively at GTA- dur and WAHA. The results in Fig 3 show the proline levels in control durum wheat (GTA- dur, WAHA) and in inoculated wheat (A. brasilense, Bacillus.sp, Frankia CcI3), the control plants stressed 12% of CC and inoculated stressed have a high proline level with maximum values   obtained in stressed plants.
 
@figure3
       
The NEWMAN-KEULS test at the 5% threshold classified the genotype factor into two homogeneous groups (Table 1), group A (GTA- dur) and B (WAHA).
   
The effect of PGPR on the restoration of wheat plant growth in the presence of stress is a summary of the reduction of the effect of stress on plants and provides a source of nitrogen to the plant. Proline and glycine betaine improve cereal growth in the presence of various abiotic stresses (Ashraf and Foolad, 2007). Furthermore, the close relationship between water content, proline accumulation and exposure of plants to high levels of water stress causes the activation of the repressor and inhibition of proline in flowers and seed (Verbruggen et al., 1996).
 
The total chlorophyll content
 
A comparison between the chlorophyll evolution of the control plants and the studied hard wheat inoculated plants showed that chlorophyll did not change much under stress in the case of inoculated plants (Table 1).
   
The highest chlorophyll values were recorded in inoculated plants with a maximum value of 48.0 ± 0.2 SPAD, recorded in GTA- dur inoculated with Frankia CcI3 and without stress. In the case of stress at 12% CC, there was a decrease in total chlorophyll; these were between 40.8 ± 0.4 SPAD and 40.3 ± 0.3 SPAD measured at GTA- dur and WAHA respectively. On the other hand, they reached a maximum value of 42.7 ± 0.4 SPAD in GTA- dur inoculated with Frankia CcI3. Fig 4 shows the results of total chlorophyll in durum wheat (GTA- dur, WAHA) control and inoculated (A.brasilense, Bacillus.sp, Frankia CcI3) as well as control plants stressed 12%CC and inoculated stress. The NEWMAN-KEULS test at the 5% threshold classified the genotype factor into two homogeneous groups (Table 1).
 
@figure4
       
The chlorophyll content and photosynthetic efficiency are generally taken as the primary indexes of plant stress tolerance (Zhou et al., 2015) Azospirillum improves grain yield (Bashan and Levanony, 1991). The effect of Azospirillum is thought to be related to the production of phytohormones that are essential for plant growth.
       
The results obtained on the chlorophyll and proline contents agree with those of some authors (Reddy and Veeranjaneyulu, 1991) who reported the existence of a probable connection between the synthetic pathways of synthesis, chlorophyll pigments and those of proline.
 
Determination of soluble sugars
 
The highest value was noted in the controls, with a maximum value of 53.05 ± 0.16μg /100mg MF recorded in GTA- dur without inoculation and stress. In the inoculated durum wheat the maximum value 53.37 ± 0.41μg /100mg MF recorded in GTA / DUR inoculated with A. brasilense (Table 1). In the case of 12% CC, we noticed a considerable increase in sugars. Thus, in the control plants, the values   between 155.09 ± 0.50 μg/100mgMF and 119.46 ± 0.48 μg/100mg MF were recorded at GTA- dur and WAHA, respectively. In addition, in inoculated plants the maximum values   are 66.17 ± 0.40 μg /100 mg MF and 64.92 ± 0.07 μg /100 mg MF recorded in WAHA inoculated with A. brasilense and Bacillus.sp respectively. In Fig 5 shows the soluble sugar content in the control group of durum wheat (GTA- dur, WAHA) and in inoculated plants as well as stressed control plants (12% of CC) and inoculated stressed ones. The NEWMAN-KEULS test at the 5% threshold classifies the genotype factor into two homogeneous groups (Table 1) the first group A (GTA- dur) and B (WAHA).
 
@figure5
   
The adaptation of plants to stress is associated with a metabolic adjustment that leads to the accumulation of organic solutes such as sugars, polyols, betaines and proline. Soluble sugars play an important role in the regulation of cells during germination in the face of water deficit (Gill et al., 2002). The decrease in sugar content in the presence of PGPR (A. brasilense, Bacillus.sp and Frankia CCI3) could be explained by the elimination of the effect of stress.
 
On the other hand, Rhizobacteria produces exo-polysaccharides (EPS) in the presence of stress (Azospirillum brasilense) (Fischer et al., 1999). Sarig et al., (1988) revealed that in addition to mineral input, inoculation with Azospirillum of certain cereals improves soil water content in the event of water deficit.

In conclusion, the use of PGPR in agriculture can be considered as interesting and efficient strategy for enhancing crop growth under water deficit. In this study, our results showed that strains Frankia CCi3 have the potential to improve durum wheat growth under water deficit. The overall results suggest that inoculation with PGPR could be an effective approach to induce water deficit tolerance and improve growth and yield of durum wheat under salt affected conditions compared to the uninoculated plants. Taking into account the search for more conservative and biological agricultural systems, crop inoculation with PGPR seems to be a promising tool leading to increased agricultural sustainability. Further microbial and physiological studies are required under field conditions and plant growth promotion should be studied at molecular level.

The authors are grateful to Bouldjadj Ryma, engineer of the laboratory (Laboratory of Plant Genetics Biochemistry and Biotechnology-Constantine, Algeria) and Boudaoud Rania for her help in the writing of this paper.