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Salt and Alkali Stresses Reduction in Wheat by Plant Growth Promoting Haloalkaliphilic Bacteria

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Salt and Alkali Stresses Reduction in Wheat by Plant Growth Promoting Haloalkaliphilic Bacteria Journal of Soil Science and Plant Nutrition, 2017, 17 (4), 1058-1073 RESEARCH ARTICLE Salt and alkali stresses reduction in wheat by plant growth promoting haloalkaliphilic bacteria Mehrnoush Eskandari Torbaghan1, Amir Lakzian1*, Ali Reza Astaraei1, Amir Fotovat1 and Hossein Besharati2 1Department of Soil Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Iran. 2Soil and Water Institute, Agricultural Research, Education and Extension Organization, Karaj, Iran. Corresponding author: [email protected] Abstract Haloalkaliphilic bacteria have plant growth promoting characteristics that can be used to deal with different environmental stresses. To study the effect of haloalkaliphilic bacteria to reduce salinity and alkalinity stress in wheat, 48 isolates were isolated and grouped into halophiles, alkaliphiles and haloalkaliphiles based on growth characteristics. The ammonia, 3-indole acetic acid and ACC (1-aminocyclopropane-1-carboxylate) deaminase production were studied. Wheat yield was evaluated in the presence of six plant growth promoting activity supe- rior isolates in the greenhouse condition. Definitive identification was performed by 16S rRNA gene sequencing. All three groups had plant growth promoting characteristics. Halophilic, alkaliphilic and haloalkaliphilic strains increased wheat yield about 20.6%, 42.7% and 58.2% in comparison with the control, respectively. The econom- ic grain yield was maximum in H7 (3366 Kg ha-1), A11 (5530 Kg ha-1) and HA6 (2672 Kg ha-1) strains. The 16S rRNA gene sequencing indicated that all of them were owned to Bacillaceae family. These bacteria belonged to four genera including, Alkalibacillus, Bacillus, Haloalkalibacillus and Virgibacillus. H1 and H7 belonged to Virgibacillus genera and Virgibacillus pantothenticus species, respectively. Both superior alkaliphilic isolates were classified to Bacillus clausii. The haloalkaliphilic isolates (HA1 and HA6) were allocated to Virgibacillus marismortui and Alkalibacillus haloalkaliphilus species, respectively. All three extremophilic native groups of bacteria had the ability to aid the wheat plant to deal with high stresses, but plants inoculated with haloalkaliphi- lic strains had higher yield compared to the other groups due to better root growth and development. Keywords: ACC deaminase, Bacillaceae, haloalkaliphilic bacteria, 3-indole acetic acid, root to shoot ratio (R/S), wheat yield 1058 1059 Torbaghan et al Salt and alkali stresses reduction in wheat by plant growth 1. Introduction One of the biggest limiting agricultural production fac- yield begin to decline have been reported to be 8.6 dS tors in the world is soil salinity and alkalinity. Finding m-1 for bread wheat. Each unit increases in soil salin- a suitable and economic method to manage salinity ity more than 10 dS m-1 at 0 to 50 cm of the soil layer, stress is a major challenge beyond researchers. Recent thereby causing wheat grain and straw yield reduc- studies have demonstrated that microorganisms can tion equal to 217 Kg ha-1 and 723 Kg ha-1, respectively help plants to overcome salinity and alkalinity stress (Siadat and Sadat, 1998). (Venkateswarlu and Shanker, 2009). Studies have Study of bacterial populations in the Sambhar salt lake shown that there is an application potential for halo- in India led to 93 haloalkaliphilic bacteria isolation that alkaliphilic microorganisms and if these microorgan- have optimum growth in 2-25% salt and 6-12 pH in the isms have plant growth promoting haloalkalitolerant environment (Sahay et al., 2012). Isolates based on 16s (PGPHA), it will help to cope with flooding stress, rRNA gene sequencing were classified into 32 groups drought stress, soil salinity and alkalinity, improving with each group representing a different taxa belonging plant resistance to pathogens and even more heavy to 3 phyla (Firmicutes, Proteobacteria and Actinobac- metal tolerance in plants (Siddikee et al., 2011). teria). The majority of the isolates (53.12%) showed Soil halophilic bacteria are a group of microorganisms similarity with phylum Firmicutes which was followed that able to live and increase in environments with high by Proteobacteria (40.63%) and Actinobacteria (6.25%). concentration of salt and adapted to grow in such envi- Among Plant Growth Promoting (PGP1 ) characteristics, ronments (Zanjirband et al., 2013). Alkaliphilic bacteria ammonium producers isolates were maximum with 56% retain their internal pH of about 9.5 in the 9 to 11 exter- ammonium production compared with 53% ACC-deam- nal pH. These bacteria maintain their activities with the inase2 producers, 50% IAA3 producers, 28% HCN4 proton transfer systems in their cytoplasmic membrane producers, 21% siderophore producers and 34% sol- (ATP pump and sodium-proton antiporter) (Horikoshi, uble phosphorous producers. Isolates showed PGP 1999). Another group of bacteria that can grow in the activities and enzymatic producer characteristic, and presence of salt and alkaline conditions are known as these can be utilized for plant growth improvement in haloalkaliphiles. This dual haloalkaliphilic characteristic saline area (Sahay et al., 2012). Five inoculation halo- makes them interesting for basic studies of different as- tolerant bacteria for improving the reduction of salin- pects of biotechnology (Feng et al., 2005). ity effects (80, 160 and 320 mM sodium chloride) in Many physiological processes are affected by salinity wheat seedling increased 71.1% root length compared stress in plants and the most important of them are cell with non-inoculated plants (Dhanushkodi et al., 2013). growth and leaf area reduction, biological yield de- Especially, Hallobacillus sp. SL3 and Bacillus halode- crease and ultimately grain yield decline. Soil salinity nitrificans PU62 bacteria increased more than 90% in decreases plant establishment in the soil, as wheat seed- root length and 17.4% in dry weight compared to ling emergence percentage may be reduced to 50% in 8.8 dS m-1 salinity level. Soil salinity also reduces pri- 1Plant growth promoting characteristics mary and secondary tillers, number of leaves in main 2ACC(1-aminocyclopropane-1-carboxylate) deaminase stem and number of spikelets per spike in wheat (Asadi 3Indole-3-acetic acid and Khademi, 2013). The salinity threshold at which 4Hydrogen cyanide Journal of Soil Science and Plant Nutrition, 2017, 17 (4), 1058-1073 Salt and alkali stresses reduction in wheat by plant growth 1060 non-inoculated wheat seedlings in 320 mM NaCl sa- promoting characteristics, including ammonia, 3-in- linity level which represented a significant reduction dole acetic acid and ACC deaminase were measured in the adverse effects of sodium chloride. This study on them. Afterward, the performance of each group showed that the use of halotolerant bacteria can act as of bacteria was investigated in the greenhouse condi- a bio-inoculant in saline soil conditions (Dhanushkodi tions to reduce damage caused by salt stress on wheat et al., 2013). (Triticum aestivum L.) resistant cultivar (Bam) to soil The family Bacillaceae, which belongs to the phylum salinity. Finally, the genetic identification of all three Firmicutes, class Bacilli and order Bacillales compris- halophilic, alkaliphilic and haloalkaliphilic native iso- es 15 genera that include halophilic or haloalkaliphi- lates that could decrease the effects of salinity and al- lic, aerobic endospore-forming species isolated from kalinity stress for wheat plants was done through 16S soil or sediment samples. These genera are Alkaliba- rRNA gene sequencing. cillus, Bacillus, Gracilibacillus, Filobacillus, Halal- kalibacillus, Halobacillus, Lentibacillus, Ornithini- 2. Materials and Methods bacillus, Paraliobacillus, Salirhabdus, Salsuginiba- cillus, Tenuibacillus, Terribacillus, Thalassobacillus In order to isolate halophilic, alkaliphilic and haloal- and Virgibacillus. Bacilluses produce ammonia from kaliphilic native isolates, soil sampling was carried protein analysis and widely spread in nature. Most out on six different Khorasan Razavi saline soils from of them are mesophiles but some of them are psy- 0-25 cm soil depth (Figure 1). Then samples were chrophiles, thermophiles, halophiles and alkaliphiles transferred to the laboratory in sterile tubes with area (Marquez et al., 2011). recording by GPS less than 48 hours. The samples Irrigated lands in Iran are approximately eight million were kept at 4 °C during this interval and then were hectares and about 90% of agricultural production is stored at this temperature in the laboratory. Isolation produced on them, 6.6 million hectares of them are sa- and purification of each halophilic, alkaliphilic and line soils, 4.5 million hectares have only salinity limi- haloalkaliphilic isolates from soil samples was done tation, while 2.5 million hectares also have other soil- by their specific medium culture. For isolation, a 1:1 related restrictions (texture, depth, flow coefficient, soil: water suspension (one gram of soil to 1 ml of alkalinity, and permeability), erosion, topography sterile distilled water) was prepared. Five hundred and groundwater limitations in addition to the salinity micro-liter (500 µL) of this suspension was spread stress. Twenty percent of Khorasan Razavi province on the specific agar media culture. Medium cultures soils have high salinity and alkalinity limitations be- were incubated in the proper temperature (35 to 37 cause they have been located on irrigated agricultural °C) for 3 to 7 days depending on the type of micro- lands
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