Plant Growth-promoting Rhizobacteria Mitigate Deleterious Effects of Salt Stress on Strawberry Plants (Fragaria 3ananassa)

The effect of selected plant growth-promoting rhizobacteria (PGPR) on the growth, chlorophyll content, nutrient element content, and yield of strawberry plants under natural field salinity conditions stress was investigated. Field experiments were conducted using a randomized complete block design with five PGPRs (Bacillus subtilis EY2, Bacillus atrophaeus EY6, Bacillus spharicus GC subgroup B EY30, Staphylococcus kloosii EY37, and Kocuria erythromyxa EY43) and a control (no PGPR) in 2009 and 2010. PGPR inoculations significantly increased the growth, chlorophyll content, nutrient element content, and yield of strawberry plants. PGPR treatments lowered electrolyte leakage of plants under saline conditions. The leaf relative water content (LRWC) of plants rose with bacterial inoculation. All nutrient element contents of leaves and roots investigated were significantly increased with PGPR inoculations with the exception of sodium (Na) and chlorine (Cl). The highest efficiency to alleviate salinity stress on the yield and nutrient uptake of strawberry plants was obtained from EY43 (228 g per plant) and EY37 (225 g per plant) treatment and the yield increasing ratio of plants was 48% for EY43 and 46% for EY 37 compared with the control treatment (154 g per plant). The highest nitrogen (N), potassium (K), phosphorus (P), calcium (Ca), magnesium (Mg), sulfur (S), manganese (Mn), copper (Cu), and iron (Fe) concentrations were obtained from EY43 and followed by E6, E37, and E30, and increasing ratio of leaves and root N, P, K, Ca, Mg, S, Mn, Cu, and Fe contents were 22% to 33%, 34% to 8.8%, 89% to 11%, 11.0% to 7.2%, 5.1% to 6.2%, 97% to 65%, 120% to 140%, 300% to 15%, and 111% to 9.0%, respectively. The results of the study suggested that PGPR inoculations could alleviate the deleterious effects of salt stress conditions on the growth and yield of strawberry plants under salinity conditions. Salinity is one of the most important stress factors limiting plant growth and productivity. Salinity stress conditions negatively affect nearly 100 million ha around the world (Ghassemi et al., 1995) and almost 1.5 million ha in Turkey (Dinc et al., 1993). High salt concentrations cause osmotic shock and ionic imbalance on plant cells (Zhu et al., 1997). Salt stress negatively affects plant physiology, both in the whole plant as well as at cellular levels, through osmotic and ionic stress. High salt concentrations in soil solution can negatively influence seed germination, growth, flowering, and fruit set, decreasing yield and the quality in crops (Arora et al., 2008). Strawberry cultivation (with 250,000 t production) (FAO Food and Agriculture Organization, 2009) has great importance in the horticulture sector in Turkey. Small family farms of 0.05 to 0.5 ha provide almost all of this production. Strawberry plants are considered to be a salt-sensitive crop and it was reported that salt stress conditions negatively affected plant growth and yield (Karlidag et al., 2009; Yildirim et al., 2009). Studies have been conducted to ameliorate the negative effect of salt stress conditions on growth and yield, most focusing on chemical amelioration. PGPR can be an alternative approach for mitigation of salt stress. Recently, PGPR has been used as an alternative biological approach. Salt-tolerant bacterial inoculants may be useful in developing strategies to facilitate plant growth in salinity conditions (Bacilio et al., 2004). The PGPRs have been reported to ameliorate the negative effect of salt stress on the plant growth of vegetable crops such as tomato (Mayak et al., 2004), eggplant (Bochow et al., 2001), squash (Yildirim et al., 2006), bean (Yildirim and Taylor, 2005), artichoke (Saleh et al., 2005), and radish (Yildirim et al., 2008). Karlidag et al. (2011a) have determined that PGPR root inoculations could mitigate of the deleterious effects of salinity conditions on plant growth of strawberry in pot experiments. However, no attempts have to date been made to study the effects of PGPR on growth, productivity, and ionic compositions in strawberry plants under natural saline field conditions. Therefore, this experiment focuses on the effect of the exogenous root application of five different PGPRs on the plant growth, some physiological variables, and the chemical content of strawberry plants under saline field conditions. Materials and Methods This study was conducted under field conditions at Ataturk University, Hamza Polat Vocational School, in Upper Coruh Valley (Ispir) in Turkey in 2009 and 2010. Ispir is located at latitude 40 29# N and longitude 41 01# E, 1200 m above sea level and had a total rainfall of 245 mm in 2009 and 310 mm in 2010, and average air temperature of 19.1 C in 2009 and 18.6 C in 2010 during the growing period (April to September). The mean minimum and maximum air temperatures were 11 and 27 C in 2009 and 12 and 28 C in 2010, respectively (April to September). Bacterial isolation, culture, and treatment From an initial set of 44 bacteria that were isolated from the rhizosphere of plants naturally grown in high salty soils in Upper Coruh Valley (latitude 40 29# N, longitude 41 01# E), Erzurum, Turkey. Five bacteria of them were selected for their ability to grow in a saline culture medium [10% sodium chloride (NaCl)]. Bacillus subtilis EY2, Bacillus atrophaeus EY6, Bacillus spharicus GC subgroup B EY30, Staphylococcus kloosii EY37, and Kocuria erythromyxa EY43 (with MIS Received for publication 27 Dec. 2012. Accepted for publication 18 Mar. 2013. We are grateful to Ataturk University for generous financial support. To whom reprint requests should be addressed; e-mail [email protected]. HORTSCIENCE VOL. 48(5) MAY 2013 563 similarity index of 0.760, 0.710, 0.580, 0.580, and 0.600, respectively, based on fatty acid methyl ester analysis using Sherlock Microbial Identification system and confirmed with Biolog) were selected as inoculants. For this experiment, the bacterial strains were grown on nutrient agar. A single colony was transferred to 250-mL flasks containing nutrient broth and grown aerobically in flasks on a rotating shaker (95 rpm) for 24 h at 27 C. Inoculation of bacterial treatments was performed using a dipping method in which plant roots in celled trays were inoculated with the bacterial suspensions of the concentration of 10 colony-forming units/mL in sterile water for 30 min before planting. Control plants were dipped into sterile water. The bacterial strains were able to grow in N-free basal medium indicating their N-fixing potential. In the present study, the P-solubilizing activities of the five PGPRs were measured according to the qualitative methods (Mehta and Nautiyal, 2001) (Table 1). Growth conditions and plant materials Cold-stored bare-rooted strawberry plants with one well-developed crown of diameter 8 to 10 mm were planted in celled-trays containing peat [pH: 5.5, electrical conductivity (EC): 0.25 dS·m, N: 300 6.6 kg·ha, P2O5: 6.6 kg·ha, K2O: 8.8 kg·ha , organic matter: 2%]. Twenty days after well-rooted and inoculated strawberry crowns were planted in late August of 2008 for the 2009 experiment and in 2009 for the 2010 experiment. The soil physical and chemical properties of the experimental area are presented in Table 2. At the beginning of each growing season, 160 kg·ha N and 180 kg P205 kg·ha –1 as ammonium nitrate and triple superphosphate, respectively, were applied in between the rows (Hochmuth and Albert, 1995). Electrical conductivities of experiment soil were determined during experiments. The EC ranged from 1.30 dS·m at the beginning of the experiment to 3.50 dS·m at the end of the experiment. EC was measured in saturation extracts according to Rhoades (1996). Strawberry plots consisted of six rows spaced 30 cm apart and were 3 m in length with 30 cm within-row spacing. There were 18 plots. Plants were irrigated with natural saline water as furrows. The EC of the water was 3.16 dS·m. There were no insecticide and fungicide treatments in either experiment. Weeds were kept under control by hand-weeding. In both years, regular cultural practices were applied uniformly through all plots. Chlorophyll measurements (SPAD readings) A portable chlorophyll meter (SPAD-502; Konica Minolta Sensing, Inc., Japan) was used to measure the leaf greenness of the strawberry plants. The SPAD-502 chlorophyll meter can estimate the total chlorophyll amounts in leaves of a variety of species with a high degree of accuracy, which is a nondestructive method (Neufeld et al., 2006). For each plant, measurements were taken at four locations on each leaf, two on each side of the midrib on all fully expanded leaves, and then averaged (Khan et al., 2003). Measurement of electrolyte leakage (membrane permeability) For measurement of electrolyte leakage, 10 leaf discs (10 mm in diameter) from the young fully expanded leaves from two plants per replicate were placed in 50-mL glass vials and rinsed with distilled water to remove the electrolytes released during leaf disc excision. Vials were then filled with 30 mL of distilled water and allowed to stand in the dark for 24 h at room temperature. The EC (EC1) of the bathing solution was determined at the end of the incubation period. Vials were heated in a temperature-controlled water bath at 95 C for 20 min and then cooled to room temperature and the EC (EC2) was measured. Electrolyte leakage was calculated as a percentage of EC1/EC2 (Shi et al., 2006). Leaf relative water content LRWC is a useful measure of the physiological water status of plants (Gonzalez and Gonzalez-Vilar, 2001). Two leaves were collected from the young fully expanded leaves of two plants per replicate. Individual leaves were first detached from the stem and then weighed to determine the fresh weight (FW). To determine the turgid weight (TW), leaves were floated in distilled water inside a closed petri dish. Leaf samples were weighed periodically after gently wiping the water from the leaf surface with tissue paper until a steady state was achieved. At the end of the imbibition period, leaf samples were placed in a preheated oven at 80 C for 48 h to determine the dry weight (DW). Values of FW, TW, and DW were used to calculate LRWC using the equation subsequently (Kaya et al., 2003): LRWC % ð Þ 1⁄4 FW DW ð Þ= TW DW ð Þ 1⁄2