Comparison of the plant growth-stimulating ability of epiphyton and epiphyton microbial communities in some rice fields of Guilan province

-Abstract

-Background and objectives: Rice paddies provide over half of the world’s food, yet chemical fertilizers used in them are inefficient with high losses. Hence, biological approaches to enhance rice growth are gaining importance. Epipelon and epiphyton microbial communities form a diverse and dynamic system in paddies, consisting of phototrophs (such as algae and cyanobacteria) and heterotrophs (such as bacteria and fungi). Their biodiversity boosts microbial activity, increasing stress tolerance and production of growth-promoting substances. Nutrient accumulation and gene transfer within biofilm layers further strengthen microbial resilience. However, the role of periphytic biofilms in promoting plant growth remains unstudied.

-Materials and methods: Sampling was conducted in July 2022 from rice paddies located in the villages of Khaleh sara, Gileh sara and Vajargah, as well as from fallow soils in Masal, Guilan Province with three replications. In this study, 21 experimental treatments were sampled. Epipelon (Ep) samples were taken from the surface soil of the rice paddies using a rectangular box-shaped device (fully open at the front and 1 cm open at the back), while epiphyton (Eph) samples were collected from suspended periphytons in the water that used larger algae as an attachment surface, using sterilized bottles. Fallow field (FF) samples were also taken from the surface of dry soil. The collected samples were transported to the laboratory in iceboxes, revived in BBM liquid medium, and maintained in a greenhouse at a temperature of 28–30°C under a light intensity of 5000 lux. The quantitative potassium solubilization assay was carried out in a modified BBM culture medium containing muscovite and phlogopite over a 7-day period and was read using a flame photometer. Quantitative assays of mineral and organic phosphate solubilization were conducted in Sperber and modified Sperber media containing inositol, respectively, and measured using a spectrophotometer at 470 nm. For siderophore evaluation, CAS-agar medium was prepared, and the drop plate method was used; the diameter of the orange halo around the colony was considered as the evaluation criterion. To assess auxin (IAA) production capability, salkowski reagent was used, and absorbance was measured by a spectrophotometer at 535 nm. Hydrogen cyanide production was tested in TSA medium, with a change in the color of filter paper placed on the lid of the Petri dish as the indicator. Salt stress was induced in both liquid and solid BBM media with NaCl concentrations of 0% and 8%. Drought stress testing was performed using polyethylene glycol to create osmotic pressures of 0, -2, -5, -10 and -15 bar in BBM medium, and absorbance was measured using a spectrophotometer at 630 nm.

Statistical analysis of the results of all experiments (except for the water stress test) was performed using a completely randomized design with three replications, utilizing SAS software. The water stress experiment was conducted using a factorial design with two factors: microbial treatments at 22 levels and osmotic pressure treatments at 4 levels.

-Results: The results showed that the highest potassium dissolution was in treatment PF3 Eph(3) (Vajargah Epiphytic sample), with a 3.4-fold increase compared to the control within 7 days. The next ranks were treatments PF1 Eph(1) and PF3 Eph(2), which were in the same statistical group as treatment PF3 Eph(3). The values of these treatments were higher in samples containing phlogopite than in those containing muscovite. For the phosphate dissolution test from the tricalcium phosphate source, treatment PF3 Eph(2) showed a value of 1121 mg/L. Following that, treatments PF2 Eph(2) and PF3 Eph(3) added 989 and 935 mg/L of soluble phosphate to the culture medium, respectively. The lowest dissolution power of insoluble mineral phosphorus was measured in the fallow soil sample, with an average of 266 mg/L. All epiphytic samples had a significantly higher phosphorus dissolution rate compared to the epipelon samples from the same sampling point, showing a statistically significant difference. The phosphate dissolution test from the inositol hexaphosphate source, yielded 427.25 mg/L. The lowest organic phosphate dissolution was reported in treatment FF (1), which pertains to the fallow soil. This treatment, along with the other fallow soil treatments (FF(2) and FF(3)), was placed in the same statistical group. The highest hydrogen cyanide production was observed in epiphytic treatments in the Vajargah rice paddy during the 5-day incubation. The highest siderophore production was measured in periphyton samples number PF1 Eph(2) and then PF1 Eph(1), with halo to colony diameters of 1.62 and 1.41, respectively. These treatments pertain to the Khale Sara rice paddies, and both are from epiphytic samples. However, comparing the halo-to-colony diameter of other treatments shows that epiphytic samples had a higher siderophore production capability. The results of the indole acetic acid test indicate that the highest amount was observed in Gile Sara epipelic sample. This treatment shows a statistically significant difference compared to all other treatments. The results of the salinity test showed that under natural culture conditions, microbial growth was completely normal and turned the culture green, while culturing periphyton in a medium with 8% salt made the culture turbid. Reculturing the periphyton on solid medium showed that in 0% salt conditions, algae, bacteria, and fungi grew, whereas in the solid medium with 8% salt, only fungi were observed. It is noteworthy that no microorganisms grew in the fallow soil samples and treatment number PF3 Eph(3). Comparison of the average results of drought stress showed that the highest density was recorded at zero osmotic pressure, followed by osmotic pressures of -2 and -5, which were in the same statistical group. The highest optical density measured was in periphyton treatment PF2 Ep(3) at zero pressure, which was in the same statistical group as periphytons PF3 Eph(3), PF2 Ep(2), PF3 Ep(2), and PF3 Ep(1). In all experiments except for the drought stress test, the lowest values were recorded from the fallow soil treatment

-Conclusion: The overall conclusion of this research indicates that periphyton, including epipelon and epiphyton, has a high potential for growth-promoting characteristics. It is recommended that more greenhouse and field studies be conducted on periphyton, so that if confirmed, it can be utilized in the development of biofertilizers.