Effect of Azotobacter and Azospirillum on Growth and Physiological Characteristics of Barley (Hordeum vulgare) under Salinity Stress

Worldwide studies have shown that inappropriate land uses over the past 45 years have resulted in salinization of 6% of the world's land. Salinity has negative effects on soil physicochemical properties and microbial activities. The imbalance in nutrient uptake, ion toxicity and decreasing water consumption due to high osmotic pressure are resulted from high accumulation of solutes in soil solution. One of the strategies to mitigate soil salinity is the inoculation of crops with different types of beneficial soil bacteria and fungi. Plant growth promoting bacteria (PGPB) are a diverse group of bacteria capable of promoting growth and yield of many crops. The most important growth promoting mechanisms of bacteria are the ability to produce plant hormones, non-symbiotic nitrogen fixation, solubilization of insoluble phosphate and potassium, biocontrol of plants pathogens through producing hydrogen cyanide and siderophore production. Plant inoculation with growth promoting bacteria causes an increase in several indices such as shoot fresh and dry weight, root dry weight and volume as well as chlorophyll content. The synergetic effect of Azotobacter and Azospirillum on the plant has been documented by increasing the absorption of nutrients, production of hormones that stimulate plant growth such as auxin, and influencing the root morphology. Due to the wide area of saline soils, appropriate methods to reduce the negative effects of salinity are of great significance. Given the importance of using bacteria adapted with climatic conditions and soil ecosystems in each region, as well as the efficiency of the combined application of growth promoting bacteria, this study was conducted to investigate the effect of growth promoting bacteria as a single and combined application at two levels of salinity calculated based on the threshold of barley yield reduction (Karoon cultivar) and 50 % reduction in barley yield.

In order to record the Azotobacter isolates, 15 soil samples were collected from salt affected lands of Golestan province. Thirty two Azotobacter isolates were isolated by physiological and biochemical tests and cyst production in old culture. Then, their ability to grow in different concentrations of salinity, drought stress tolerance, polysaccharide production, auxin production, phosphorus and potassium solubilization, hydrogen cyanide synthesis and biological fixation of molecular nitrogen were investigated. Based on physiological and growth stimulation tests, Az13 isolate was selected as the superior isolate of Azotobacter for greenhouse test. Azospirillum superior isolate was then prepared from the microbial bank of Soil Science Department, Gorgan University of Agricultural Sciences and Natural Resources. A soil with 16 dS/m salinity was selected to determine the effects of experimental treatments at two threshold salinity levels of yield reduction and 50 % reduction of barley yield. Then, soil salinity was reduced to 8 dS/m (yield reduction threshold) by leaching. After reaching to the desired salinity, the soil was removed from the pots and air dried. The sample was sifted through a 2 - mm sieve and again transferred to the pots. The barley seeds, Karoon cultivar, were used. To prepare the inoculum, firstly the bacterial isolates were grown in the pre-culture nutrient broth medium, and then incubated at 120 rpm in a shaking incubator at 28°C for 48 hours. Afterwards, each seed was inoculated with one milliliter of the bacterial inoculant with a population of 109 CFU/ml. This experiment was conducted as factorial in a completely randomized design with three replications in the greenhouse at Gorgan University of Agricultural Sciences and Natural Resources. The treatments included four levels of bacteria (without inoculation, Azotobacter inoculation, Azospirillum inoculation, combined inoculation of Azotobacter and Azospirillum) and two levels of salinity (8 and 16 dS/m). After 70 days (late vegetative growth period), some growth and physiological indices and concentration of nutrients uptake were measured.

The results showed that salinity stress had a significant (p < 0.01) negative effect on growth and physiological traits and nutrient uptake of the plant. The combined application of Azotobacter and Azospirillum bacteria showed a positive significant influence (p < 0.01) on growth, dry weight, and root dry weight in the plant under salinity stress. The combined application of bacteria increased the chlorophyll a, b and a + b content at a salinity level of 16 dS/m by 136.49, 117.86 and 127.97 %, respectively. The combined application of bacteria resulted in a 65.39 and 55.94 % increase in proline amino acid content at salinity levels of 8 and 16 dS/m, respectively. The results revealed that nitrogen, phosphorus and potassium levels increased by 81.97, 80 and 66.67%, respectively, at 16 dS/m salinity level in combined application of both bacteria. Sodium ion accumulation in all bacterial treatments decreased in both salinity levels compared to control treatment and the highest reduction was observed in combined bacterial inoculation. These findings underline the positive effect of bacterial inoculation, particularly their combined application, on the growth and nutrients uptake of barley under salt stress.

Our results indicate that increasing salinity level significantly decreased shoot dry weight, root dry weight, plant height, chlorophyll content and nutrient concentrations of barley. Inoculation of salt-resistant bacteria, including Azotobacter and Azospirillum, reduced the adverse effects of salinity on growth and physiological traits, which was more pronounced in Azotobacter than Azospirillum. The combined application of Azotobacter and Azospirillum had a significant effect on root dry weight, plant height, chlorophyll content, increasing nutrient concentration efficiency (nitrogen, phosphorus, and potassium) and decreased sodium concentration at both salinity levels (8 and 16 dS/m) compared with the individually inoculated bacteria. Hence, the application of Azotobacter and Azospirillum isolates is an appropriate method for pot experiments with saline soils. To apply these results, field experiments in saline soils must be carried out to evaluate the effect of these bacterial isolates on the crop growth, yield and physiological characteristics.