Isolation of Silicate Minerals-Solubilizing Bacteria from Tobacco (Nicotiana tabacum L.) Rhizosphere and Evaluation of their Efficiency on Soil Potassium Release

Potassium (K) is one of the major essential macronutrients for plant growth. Soil has rich reserves of K, among which only 1–2% can be directly absorbed by plants. It may be more economically viable to transform the fixed slow-release K into available K that can be absorbed by plants. The ability of some microorganisms to dissolve soil K-bearing minerals, such as micas is an important feature for increasing the yield of high-K-demand crops such as tobacco. Also, these microorganisms have both economic and environmental advantage. A large number of saprophytic bacteria such as Bacillus mucilaginosus and fungal strains such as Aspergillus spp. are known for their potential in releasing insoluble native K-source in soil into a plant available nutrient pool. Tobacco (Nicotiana spp.) is one of the most important industrial crops. K plays a vital role in increasing the tobacco yield and controlling quality parameters such as leaf combustibility that is one of the key criteria taken into account by the tobacco industry for assessing quality. Thus, high ranges of K fertilizers are applied in tobacco fields based on plant K requirement to build up soil K in tobacco producing countries. Increasing cost of the fertilizers and environmental risks necessitates alternate means to fertilizers such as application of microorganisms. The use of chemical K fertilizers can be reduced by exploiting the potential of bio-inoculants which are inexpensive and eco-friendly. Information related to K-solubilizing microorganisms in tobacco rhizosphere and their suitability in increasing the available K in tobacco-cultivated soils is not well-documented. Hence, the present study was conducted to screen the KSB isolates from tobacco-cultivated soils and evaluate their potential in dissolving K bearing silicate minerals and increasing soil available potassium.

Soil samples were randomly collected from the rhizosphere of tobacco from 25 different locations in northwest of Iran. The serial dilutions of the soil samples were made up to 10-4 and 5 µl of diluted soil suspension plated on Aleksandrov medium plates (on the agar-based culture medium). Aleksandrov medium contained 5.0 g Glucose, 0.5 g MgSO4.7H2O, 0.1g CaCO3, 0.006 g FeCl3, 2.0 g Ca3PO4, 2.0 g insoluble mica powder as potassium source and 20.0 g agar in 1 liter of deionized water. The plates were incubated at 28±2°C in incubator for 10 days. Finally, nine isolates of potassium silicate solubilizing bacteria were isolated and purified. Solid and liquid Aleksandrov media were applied for qualitative (Solubility Index = Diameter of zone of clearance/ Diameter of growth) and quantitative (K content) evaluation, respectively, based on the completely randomized design (CRD) with three replication. Liquid Aleksandrov medium containing 2 g L-1 of mica and feldspar mixture, was inoculated with bacterial isolates. Bacterial isolates creating high solubility index and releasing more K from K-bearing minerals into liquid medium, were selected as effective isolates. In order to evaluate the efficiency of the potent bacterial isolates for increasing soil available K, an experiment was conducted with three replication and eight potent bacterial isolates along with a control (non-inoculated soil). Sterilized soil samples were inoculated with bacterial isolates separately and incubated at 25°C, with 75% field capacity moisture levels for 90 days. After incubation, available K in soil samples were extracted with Ammonium Acetate 1M. Variance of solubility index, K concentration into liquid Aleksandrov medium and soil available K were analyzed using SPSS (Statistical Package for the Social Sciences). Student-Newman-Keuls (SNK) test comparisons were also used to compare available soil K using SPSS 16.0.

Eight KSBs isolates, including KSB20, KSB30, KSB40, KSB22, KSB42, KSB90, KSB92 and KSB10, were isolated and purified as effective isolates for dissolving mica and feldspar minerals. Most isolates were gram-positive, rod-shaped, and white in appearance. The studied isolates, except KSB22, KSB40 and KSB20, had α-amylase enzyme activity. Bacterial isolates, including KSB20, KSB30, KSB42 and KSB10, were significantly superior in sucrose and glucose hydrolysis. The isolate of KSB10 also had fluorescence properties. The highest solubility index (2.8, 2.7 and 2.5) was obtained from the activity of KSB22, KSB42 and KSB10 isolates in solid Aleksandrov medium, respectively. The highest concentration of potassium into liquid Aleksandrov medium was found for the KSB42 and KSB10 isolates (9.40 mg L-1). The KSB42 and KSB10 isolates increased medium K concentration approximately three times more than non-inoculated medium. In addition, KSB42 and KSB10 isolates were more effective in releasing potassium from soil potassium-bearing minerals. The amount of available potassium in soil incubated with KSB42 and KSB10 isolates increased by 44 and 46 mg kg-1 compared to the control, respectively.

Among bacterial isolates purified from the tobacco rhizosphere, the KSB42 and KSB10 isolates increased more significantly the solubility of potassium minerals and potassium availability in soil compared to other isolates. These bacteria isolates increased potassium concentration into Aleksandrov liquid medium by more than three times and also increased soil available potassium by about 44 to 46 mg kg-1 compared with the control. As a result, these isolates (KSB42 and KSB10) can be used as a bio-fertilizer to reduce potassium fertilizer application and increase the quality of tobacco after field experiments