Physiological and growth responses of two wheat (Triticum aestivum L.) varieties inoculated with a new strain of Bacillus siamensis under Cadmium (Cd) stress


 Bioavailability of cadmium (Cd) metal in the soils due to scarcity of good quality water and industrial waste could be the major limiting factors negatively influencing the growth and yield of crops needs prompt solution to fulfil the requirement of food for increasing world population. In the recent time, variable range of plant growth promoting rhizobacteria (PGPR) are being used on large scale in agriculture to reduce the risk of abiotic stresses on plants and increase crop productivity. Among them, the Bacillus siamensis has a huge potential to enhance the plant tolerance against abiotic stress but limited evidences are reported about the putative role of B.s in crop plants under heavy metal stress. The current study was aimed to investigate the potential of a new metal tolerant strain of B.s on two wheat (Triticum aestivum L.) varieties (NARC-2009 and NARC-2011) grown in Cd contaminated soil at different treatments i.e Cd (0, 20, 30 and 50 ppm) and Cd (0, 20, 30 and 50 ppm) + B.s. Our results depicted that Cd stress decreased the wheat growth related attributes, biomass, and photosynthetic parameters (Chlorophyll a, b and a + b) which increased in both wheat varieties upon inoculation with B.s. Moreover, Cd stress caused significant membrane damage and negatively affected the water content, water potential, and osmotic potential of leaf. However, PGPR considerably increased the soluble sugars to reduce the Cd toxicity. Overall, the plants inoculated with B.s enhanced their tolerance index of root and shoot and found better in NARC-2009 than NARC-2011. Therefore, microorganisms efficiently increase the plant growth by reducing the metal toxicity.

. Cd enters the environment via geogenic and anthropogenic sources such as fertilizer, sewage slough dispersal, industrial waste, electroplating and atmospheric deposition ). The Cd firstly accumulated by root directly from the soil and caused reduction in root length, then transferred to aerial parts where it reduced the photosynthesis and resulted in stunted growth and reduced yield (Rizwan et al. 2017). The Cd led to the excessive production of ROS caused oxidative damage and negatively affected the antioxidant defence system of plants (Hussain et al. 2018).
The Cd has high mobility and bioavailability, and enters the food chain via consumption of different vegetables, cereals and cereal grains obtained from cd contaminated soil due to its efficient mobility and bioavailability (Rizwan et al. 2016b). The wheat (Triticum aestivum L.) is utilized as staple food by more than 50% population of the world and an important cereal crop worldwide (FAO and http://faostat.fao.org/). The demand for food from wheat is increasing globally day by day and requirement of wheat to feed the increasing population is getting more attention (Curtis and Halford 2014). Wheat has greater potential to accumulate Cd in its various parts as compared to other cereals, resulting the higher Cd compartmentalization in wheat (Naeem et al. 2016). However, the accumulation of Cd varies with wheat cultivars, type of soil and soil contamination level. But the uptake and transfer of Cd from root to shoot depends upon the xylem and phloem loading (Harris and Taylor 2013). Therefore, it is extremely important to reduce the intake and transfer of Cd to aerial parts which is an ultimate risk to humans and other living organisms that consume wheat (Keller et al. 2015).
Plant growth promoting rhizobacteria (PGPR) increase the root development which reflects the accumulation of more water and essential nutrients to a suitable concentration and consequently improve the plant growth by enhancing the photosynthetic apparatus efficiency, linked with chlorophyll concentration and PSII functionality (Mesa-Marín et al. 2018). PGPR are currently used to immobilize and resist the metal toxicity and improve the plant growth by reducing the heavy metal uptake and accumulation within plants (Mallick et al. 2018). PGPR increase the plant growth by restricting the heavy metal accumulation in roots and stopping its transfer toward aerial parts through shoot (Mesa et al. 2015). The higher uptake of heavy metals negatively impacted the photosynthetic carbon consumption during respiration by altering the mitochondrial and electron transport chain configuration however, inoculation with PGPR recovered the plant metabolism by limited translocation of metals in the roots of plants (Mesa-Marín et al. 2018). Previously, it has been reported that Bacillus megaterium limited the intake and transfer of Ni and improved the growth of Sorghum halepense, Luffa cylindrica and Brassica juncea (Rajkumar et al. 2013). Neorhizobium huautlense considerably increased the growth and biomass production of Chinese cabbage and radish by reducing the uptake surface sterilized with sodium hypochloride (2.6% active chloride) for three mins then properly washed with double distilled water. Afterwards, the half of the seeds of each wheat variety were inoculated with Bacillus siamensis (strain no. MH559649 obtained from the department of botany with adjusted concentration of bacteria at 1.2 × 10 8 cells/ml) for 24 hours at room temperature. Then the PGPR inoculated wheat seeds were air dried. The PGPR inoculated seeds were sown in twenty four (24) pots and rest of twenty (24) pots had un-inoculated seeds (pre-treated with distilled water over night). Eight seeds of each wheat variety were sown in each plastic pot containing 5 kg air-dried loamy soil (1:3) of sand and silt respectively. The soil analysis has given in Table.1. Before sowing the seeds, soil was subjected to Cd stress (CdCl 2 .

Measurement of Chlorophyll contents
For chlorophyll content, the fresh leaf samples were extracted with 85% v/v acetone at 4 °C for 24hours under dark conditions. Afterwards, the ready sample's wavelength was measured at 470, 647 and 664 nm by using a spectrophotometer. The chlorophyll contents were calculated according to method described by (Lichtenthaler 1987).

Determination of total soluble sugars content 7
The leaf tissues were taken into 10 ml centrifuge tube with 80% ethanol (5 ml). The reaction mixture was incubated in water bath with shaking for 30 min at 80 °C and centrifuged for 5 min at 4000 rpm to get the supernatants. Pallets were treated with 80% ethanol for two more extractions.
Supernatants were collected and diluted with 80% ethanol and mixed to form whole volume upto 25 ml, and kept at -20 °C for further analysis. The total soluble sugars were measured by following the method of (Seifter et al. 1950).

Determination of Membrane Stability Index (MSI)
The leaf from each sample was cut into small pieces (100 mg) and washed with double distilled water.
Afterwards, leaf pieces were inserted in test tubes and placed in a water bath at 40 o C for 30 min.
Then, (C 1 ) electric conductivity was measured by using EC meter. Again the samples were placed in a water bath at 100 o C for ten mins and electric conductivity (C 2 ) was measured. the MSI was calculated according to formula given by (Sairam et al. 2005).

Determination of Osmotic and Water Potential
To calculate the osmotic potential and water potential under water deficit conditions, the pressure chamber was utilized with pressure measurement value of 6.0 MPa (Turner and Begg 1981). A fully expanded leaf was taken to determine the osmotic potential with vapour pressure osmometer.
Osmotic potential was obtained by measuring the difference at 100% relative water content with water scarcity relative water content. According to the (Turner 1986), the equation was used to calculate osmotic potential; OP100 = OP (RWC-Assumption of apoplastic water)/100 -Assumption of apoplastic Water OP stands for osmotic potential and RWC stands for the relative water content of the leaf.

Relative water content and tolerance index of root and Shoot
To measure the leaf relative water content (LRWC), the procedure given by (Turk and Erdal 2015) was used. Immediate after harvesting the plants, fresh weight (FW) of seventh leaf of wheat plant was calculated. Then leaf was cut into segments and dipped in distilled water over night to get the turgid weight (TW). Afterwards, the samples were subjected to an oven at 70C and measured the dry weight (DW). The RWC was measured according to given formula as; To find out the tolerance index of root and shoot, the formula of (Turner and Marshall 1972) was used as given below;

Statistical analysis
The analysis of data was accomplished by using SPSS. The significance of data was analysed with one-way analysis of variance ANOVA. All values are given as mean of three replicates. The 5% level of probability was used to compare the mean with least significance difference (LSD) test.

Plant morphological traits and leaf area
The results of current study depicted that inoculation with B.s positively improved the growth of both wheat varieties grown in Cd contaminated soil ( Figure.  However, more decrease in Chl contents were observed in NARC-2011 due to Cd toxicity as compared to NARC-2009. Results showed that 28%, 45%, 48% decrease in Chl a, 28%, 47%, 65% in Chl b and 27%, 46%, 55% in Chl a + b was noticed in plants exposed to Cd at 20, 30 and 50 ppm over the control. In addition, B.s application improved the Chl a, b and a + b contents by 8%, 17%, 12% over the control.

Total soluble sugars
The Cd treatment negatively affected the total soluble sugars in both wheat varieties at all levels however, the application of B.s enhanced the soluble sugars and improved the plant growth significantly (Figure.5). In the wheat plants, the maximum reduction in soluble sugars was 35% and 32% at Cd-50 ppm while the maximum production was 13% and 14% when plants were inoculated with B.s for NARC-2009 and NARC-2011, respectively.

Determination of Water Potential and Osmotic potential
The Cd at different levels of drastically effected the water potential and osmotic potential however, the seed inoculation with B.s positively impacted the water and osmotic potential in wheat as

Discussion
The current study depicted that Cd treatment impaired the growth of wheat plants with respect to all morphological traits and Cd had severe impact at highest level of Cd-50 ppm ( Figure.1 and 2). In contrast, the seed inoculation with B.s positively affected the plant growth profile exposed to Cd stress. The detailed molecular mechanism of Cd toxicity is poorly understood yet however, few researchers explained the damaging effects that Cd may destroy the soil microbial communities, reduce the water and nutrients uptake, and impair the cell division and elongation process ultimately  In our study, the Cd treatment significantly decreased the water potential, osmotic potential and LRWC in both wheat varieties while the highest values for these parameters was observed under PGPR application. Cd stress in the soil decreased the microbial community and damaged the root tips to reduce the uptake of water and disturb the water balance of cells in leaf resulting in the reduction of stomatal conductance and transpiration rate (Qadir et al. 2014). Consequently, this is directly linked with decline in chloroplast amount as well as cell enlargement and ultimately reduced the plant growth and biomass formation (Rucińska-Sobkowiak 2016). In addition, Cd reduced the surface area of cells that absorb water indicating the disturbance of water balance (Sun et al. 2016). However, the PGPR improves the LRWC and water potential in different plant species exposed to different types of environmental stresses (Naveed et al. 2014). It is reported that PGPR improves the stomatal aperture to uptake more water via roots and enhances the stomatal conductance as compared to non-PGPR  Overall, it is estimated that Cd stress at all levels severely impacted the growth of both wheat varieties while the inoculation with PGPR reduced the Cd toxicity and improved plant growth attributes. However, further studies are needed to find out the actual mechanism of Cd toxicity in plants at molecular level with the application of PGPR.

Ethics approval and consent to participate
This article does not contain any studies with human and animal participants. Consent to participate: Not applicable.

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Availability of data and material
All the analysed data for this study are included in this article. Conclusions of the current study is included in this article.

Competing interests
Authors declare that there is no conflict of interest.