Biopriming of durum wheat seeds with newly halotolerant PGPB bacterial isolates for improving their potential of plant growth under stressful conditions

Abstract


Introduction
Salinization is recognized as the main threats to environmental resources and human health in many countries, affecting almost 1 billion ha worldwide/globally representing about 7% of earth's continental extent.Plants face various biotic and abiotic stresses in adverse environmental conditions.Abiotic stress indeed is a complex process, which informs cells to adapt themselves at the molecular, biochemical, and physiological levels [1].
Salt stressed soils are known to suppress the growth of plants [2].Plants in their natural environment are colonized both by endocellular and intracellular microorganisms [3].For the enhancement of sustainable agricultural production under stressful conditions, use of bacterial inoculants or PGPB (Plant Growth Promoting Bacteria) is becoming a more widely accepted practice in intensive agriculture in many parts of the world.Rhizosphere microorganisms, particularly beneficial bacteria and fungi, can improve plant performance under stress environments and, consequently, enhance yield both directly and indirectly [4].Some plant growth-promoting rhizobacteria (PGPR) may exert a direct stimulation on plant growth and development by providing plants with fixed nitrogen, phytohormones, iron that has been sequestered by bacterial siderophores, and soluble phosphate [5,6].
Besides developing mechanisms for stress tolerance, microorganisms can also impart some degree of tolerance to plants towards abiotic stresses like drought, chilling injury, salinity, metal toxicity and high temperature.In the last decade, bacteria belonging to different genera including Rhizobium, Bacillus, Pseudomonas, Pantoea, Paenibacillus, Burkholderia, Achromobacter, Azospirillum, Microbacterium, Methylobacterium, Variovorax, Enterobacter etc. have been reported to provide tolerance to host plants under different abiotic stress environments [7,8].Use of these microorganisms per se can alleviate stresses in agriculture thus opening a new and emerging application of microorganisms [9].Microbial elicited stress tolerance in plants may be due to a variety of mechanisms proposed from time to time based on studies done.Therefore, the present study aims to (i) isolation of bacteria from rhizosphere of halophyte grown in saline sodic soils, (ii) identification of http://sciforum.net/conference/mol2net-07plant growth promoting (PGP) attributes, and (iii) analyze the seed germination ability through seed coating with potent PGPB.
Then, a 10-fold serial dilution was prepared, and 0.1 mL aliquots were spread in Petri plates in duplicate over the Burk's N-free medium.The plates were kept at 30 •C for 7 days.
The macerat of washed roots were inoculated in a culture flask containing Burk's N-free medium and incubated for 7days at 30°C.Then, a 6-fold serial dilution was prepared, and 0.1 mL aliquots were spread in Petri plates in duplicate over the Burk's N-free medium.The plates were incubated for 7 days at 30°C and morphologically different colonies appearing on the medium were isolated and subcultured for further analysis.

Diazotrophic potential of the isolates
Bacterial isolates were examined for their nitrogen-fixation (diazotrophic) potential first by testing their growth on the liquid and solid mineral nitrogen free medium [10] ( medium removing NaNO3) with oil as a sole source of carbon and energy.Isolates were then tested for nitrogenase, using the method of Quantification of N fixing capacity.

Quantification of N fixing capacity
The isolates which showed positive and predominant growth during the first 3 to 4 days were selected for quantification using the Kjeldahl N digestion and distillation system [11] (Kelplus system, Classic http://sciforum.net/conference/mol2net-07Dx[VA]).The selected isolates were incubated in 10 ml of Jensen's broth in a rotary shaker at 150 rpm for 5 days at 28° C. The amount of N fixed in the microbial tissues contained in the broth was determined by the method described by [12].From the quantification result, one potent N fixing isolates having the highest N fixing ability was selected for further characterization.

Effect of physiological conditions on the growth of potent N fixing Bacteria
Effect of various growth conditions such as temperature, salt tolerance and pH on the growth of the most potent N fixers were checked in nutrient broth.For studying the effect of temperature, potent bacteria were incubated at temperatures viz., 25 o C, 30 o C, 35 o C, and 40 o C for 24 h at 150 rpm.Nutrient broth supplemented with different concentrations of NaCl (ranging from 50-200mM) was used for salt tolerance studies and the hydrogen ion concentration in the range of 5-8 was selected for pH studies.
The flasks were incubated at 30 o C for 24 h in a rotary shaker at 150 rpm.The growth and activity of the potent N fixing bacteria in the given growth conditions were observed by taking the optical density of the medium.

Antifungal assay
The agar well diffusion method as adopted earlier [13] was used with minor modification.The bacterial isolates tested for their antifungal activity were fully grown in LB medium.
Wells of 8mm diameter of test fungus were punched into in the Potato dextrose agar (PDA) slants and filled with 200 ml (2. 10 7 CFU/ml) of bacterial culture.Potato dextrose broth was taken as negative control.The plates were incubated for 5-6 days at 28°C.The antifungal activity was evaluated by measuring the growth inhibition zone against test fungi.

Screening of phosphate solubilizing Bacteria
Modified Pikovskaya agar plates were prepared and test isolates were streaked on plates, then the plates were the incubated at 37°C and observed for 2-7 days [14].The strains forming zone of clearance were maintained by streaking on nutrient agar slants and stored at 4°C.IAA production http://sciforum.net/conference/mol2net-07Indole acetic acid (IAA) production was detected as described by Patten et al, (2002) [15].Bacterial cultures were grown for 7 days in halophilic medium containing supplement of 20g NaCl at 37°C.Fully grown cultures were centrifuged at 3,000 rpm for 30 min.The supernatant (2 mL) was mixed with 2 drops of orthophosphoric acid and 4 mL of Salkowski reagent (50 mL, 35% of perchloric acid and 1 mL 0.5 M FeCl 3 solutions).Development of pink color indicated IAA production.

Siderophore production
Production of siderophore was detected by standard method [16] using chrome azurol S (CAS) as indicator.The isolates were spot inoculated at the center of the plate and incubated for 7 days.The change in the colour of the medium around the bacterial spot was an indication of siderophore production.

Hydrogen cyanide (HCN) production
HCN production was determined by color change of filter paper [17].Loopfull of bacterial suspension was inoculated on nutrient agar medium (Merck, Germany) containing 4.4 g L-1 glycine.Filter papers were soaked in a reagent solution (sodium carbonate 2% and picric acid 0.5%) and placed in the upper lid of Petri dishes.To prevent volatilization, the plates were sealed with parafilm and incubated at 37°C for 7 days.One plate without inoculation of bacterium was considered as control.If HCN was produced, yellow filter papers changed to cream, light brown, dark brown and eventually turn into reddish-brown.

Production of Ammonia
Bacterial isolates were tested for the production of ammonia in peptone water.Freshly grown cultures were inoculated in 10 ml peptone water in each tube and incubated for 7 days at 37°C.Nessler's reagent (0.5 ml) was added in each tube.Development of brown to yellow colour was a positive test for ammonia production [18].

Exopolysaccharide production
The qualitative determination of exopolysaccharide production was performed according to Paulo et al.

Preparation of inoculum and seed coating:
Seeds of wheat variety "aouija" were obtained from Agriculture Research Station in Tunisia.Bacterial strains were grown overnight in LB broth at 28±2°C with constant shaking.Cells were harvested by centrifugation and re-suspended in normal saline to get an optimum growth (OD 10 8 cells per mL at λ600).Seeds were constantly shaken along-with the bacterial suspension with continuous addition of the sterile carrier material until the seeds become coated with a thin film of bacterial suspension and carrier material.Coated seeds were air-dried before sowing.

Seed germination test
After seed coating with potent PGPB strains, uniform seeds were sowed in pots and germination of seeds was observed after 5 th day.

Results and Discussion
To date, soil salinity becomes a huge obstacle for food production worldwide since salt stress is one of the major factors limiting agricultural productivity.To embark upon this harsh situation, numerous strategies such as plant breeding, plant genetic engineering, and a large variety of agricultural practices including the applications of plant growth-promoting rhizobacteria (PGPR) and seed biopriming technique have been developed to improve plant defense system against salt stress, resulting in higher crop yields to meet human's increasing food demand in the future [20].Biopriming of durum wheat seeds with the potent PGPB is the main approach studied for improving the germination rate in this mini-scientific research.Four bacteria were selected from 22 halotolerant strains isolated on minimal medium devoid of any nitrogen source.These bacteria were subjected to an ecophysiological study in vitro in order to determine their resistance and ability to survive under different physiological stressors such as tolerance to osmotic pressure (salt), temperature and growth pH.http://sciforum.net/conference/mol2net-07 The growth study shows that these bacteria have the ability to survive to different degrees under all used salt concentrations and the same for growth at different temperatures and pH (Table 1).The MA11 bacterium shows a good ability to grow under all the conditions tested.While MA2 is resistant to osmotic pressure, sensitive to temperature greater than 30°C and a pH less than or greater than 7.All bacteria have an identical growth optimum of 100 mM, 30°C, and 7 respectively for salt concentration, temperature and pH except for MA13 of 150mM (Table 1).The ameliorative functions of PGPR consist of three aspects, namely, the ability to protect themselves against hyperosmotic conditions and abnormal NaCl concentrations, the capacity to aid plant tolerate better to elevated salinity, and to improve soil quality [21].Regarding the alleviating roles of PGPR in promoting plant salinity tolerance, PGPR exhibit beneficial traits in mitigating the toxic effects of high salt concentrations on morphological, physiological, and biochemical processes in plants, resulting in the significant rescue of yield loss.The bacterial growth of the isolates was put under their optimum salt at 30°C and pH 7 under two media: one rich LB and the other poor lacking a nitrogen source to enhance the growth capacity of the strains to support stressful and non-stressful conditions.Growth on LB as well on N-free medium shows that MA13 exhibits the best growth ability.While MA5 shows good growth on N-free medium than MA11 unlike their growth on LB medium (Fig. 1 A; B).Furthermore, PGPR can also mitigate salt stress symptoms by producing Na + binding exopolysaccharides (EPS), improving ion homeostasis, decreasing ethylene levels through enzyme 1aminocyclopropane-1-carboxylate (ACC) deaminase, and synthesizing phytohormones [22][23][24].The most reported mechanism predominantly used to explain the positive PGPB effects on plant growth is their ability to produce auxin.The production of indol-acetic acid of the strains was studied on a medium with and without tryptophan (Fig. 2).All strains tested produced IAA at concentrations ranging from 0.092 to 0.194 mg / ml.Strain MA13 is a strain dependent on tryptophan to produce growth hormone IAA, whereas the strains produce IAA even in the absence of tryptophan.We note that these three strains produce IAA at the same non-variable concentration.And the production of IAA in the presence of tryptophan is 0.139; 0.092 and 0.161 respectively for MA2, MA5, and MA11 (Fig. 2).Auxins function in geotropism and phototropism, vascular tissue differentiation, apical dominance, root initiation (lateral and adventitious), cell division, stem and root elongation [25].

Isolate
Bacteria that solubilize phosphorus are referred to as phosphate solubilizing bacteria [26].They supply phosphate in a more acceptable way to the plants and are not deleterious to the environment.They convert insoluble organic and inorganic phosphate to a form which can be readily accessible to plants.
Environmental conditions, plant and soil conditions, and bacterial strains all affect the actions of phosphate solubilizers [27,28].The principal mechanism of inorganic phosphate solubilization is the use of mineral-dissolving compounds like hydroxyl ions, organic acids, protons, siderophores, and carbon dioxide (CO2) [29].The phosphate solubilization pathway is significantly activated in MA5 by the solubilization of 0.4 μg/ml, whereas this pathway has minimal activity in MA13 by the solubilstion of 0.0515 μg/ml.While MA2 and MA11 solubilize 0.2485 and 0.18475 µg/ml respectively (Fig. 3).
. Ethylene synthesis in a particular plant is affected by the presence and concentration of other plant hormones, temperature, gravity, light, nutrition, and the presence of various degrees of biotic/abiotic stress which the plant may be subjected [30].Its production more than its threshold level by the action of ACC oxidase enzyme in plant tissues causes "stress ethylene" which affects the root and shoot development in plants.Colonization of "stress ethylene" plant rhizosphere by ACC deaminase producing PGPB help to alleviate this situation and restores normal plant development.In order to test the ability of strain to assimilate 1-aminocyclopropane-1-carboxylate (ACC), precursor of ethylene responsible of senescence and stress hormone in plants, bacterial growth is measured in the presence of ACC and another source of inorganic nitrogen ((NH 4 ) 2 SO 4 ) in the minimum medium.Our results show that the growth of MA2, using both sources, is minimal for ACC and maximal for (NH 4 ) 2 SO 4 and contradictory in MA11 whose activity is maximal for ACC and minimal for (NH 4 ) 2 SO 4 .Whereas in MA5 the use of (NH 4 ) 2 SO 4 is better than ACC and MA13 assimilates ACC more than (NH 4 ) 2 SO 4 .The strain which able to assimilate In this regard, bacteria that express ACC deaminase, by lowering plant ACC levels (and subsequently plant ethylene levels) can decrease the detrimental effect on plants from different stresses [31].The ACC is being converted by ACC deaminase in the PGPB to α-Ketobutyrate and ammonia On average, various plant diseases reduce plant yields by around 10%/year in more developed countries and by about 20% /year in less developed countries of the world (http://www.fao.org/home/en/).In an effort to decrease the widespread use of chemicals as a means of preventing phytopathogen damage to plants, scientists have been developing the use of certain environmentally friendly PGPB as biocontrol agents The antifungal activity of all strains was checked against fusarium solani, fusarium oxysporum, fusarium graminearum and rhizoctonia solani using PDA medium (Table2).The antifungal activity of the tested strains varied according to PGPB and phytopathogenic fungal strain whose MA11 and MA13 as the most effective against all fungal strains.
It should be noted that only with MA11 and MA13, this activity was efficient against fusarium oxysporum.Moreover, no antifungal activity was noticed with MA5 for fusarium solani and fusarium oxysporum and with MA2 for fusarium oxysporum and rhizoctonia solani.However, with MA11 and MA13, the antifungal activity was highly effective against rhizoctonia solani and fusarium graminearum respectively, but less effective against fusarium solani and fusarium oxysporum.Pgpb as

Conclusion
In vivo studies showed that MA2, MA5, MA11 and MA13 strains are PGPBs seen that all these tested strains can promote wheat germination through some important biochemical traits.All these strains are able to grow under differents growth parameters, to produce auxin with differents levels, to solubilize phosphoric matter, to assimilate ACC, and to secrete antimicrobials compounds against phytopathogenic species.The germination test after seed biopriming demonstrated that, after five days of obscurity incubation, MA2 and MA11 can germinate all tested seeds than other tested strains.It is interesting to check the potentiel of these strains to contribute in plant production in the arid and semiarid regions, especially the salt affected sites, besides its potential role in improving healthy plants production under biotic and abiotic stresses.

Figure 2 :
Figure 2: (A) IAA production, (B) P-solubilization of selected strains in the presence of 2% NaCl.Error bars

Table 1 :
Bacterial growth of selected isolates under different temperature, pH, and salt concentrations.

Table 2 :
Bioprotection ability of PGPB strains against wheat fungal wilt caused by fusarium species.Effect of seed biopriming with PGPB strains on germination rate of durum wheat under salt stress conditions.The seeds were incubated in a suspension of 10 8 bacteria on 150 mM NaCl at room temperature for 30 min.
According to the FAO (http://faostat3.fao.org), after sugarcane the next three first crops in terms of production (million tons) in the world are the cereal maize (Zea mays), rice (Oryza sativa) and wheat (Triticum aestivum L. subseq.durum).Wheat represents a major renewable resource for food, feed, and industrial raw material and it is the most widely grown worldwide crop.For this, it is interesting to Climate change impacts on soil salinity in agricultural areas.European Journal of Soil Science, 2021.72(2): p. 842-862.2. Egamberdieva, D., et al., Salt-tolerant plant growth promoting rhizobacteria for enhancing crop productivity of saline soils.Frontiers in microbiology, 2019.10: p. 2791.3. Gray, E. and D. Smith, Intracellular and extracellular PGPR: commonalities and distinctions in the plant-bacterium signaling processes.Soil biology and biochemistry, 2005.37(3): p. 395-412.4.