# Sustaining Rice Production through Biofertilization with N\({}_{2}\)-Fixing Cyanobacteria
## 3 Results and Discussion  
Isolation and Genetic Characterization of \(N_{2}\)-Fixing Cyanobacteria from the Rice Paddies of the Andalusian Marshes  
The Dofana marshes in the south of Spain are one of the most important wetland complexes in Europe and in the Mediterranean region (Figure 1). Part of this region was transformed in the early 1970s from the original natural marshes to one of the most productive and largest areas of rice cultivation in Spain, where specific farming practices and rice varieties are used. Intensive rice cultivation, which is highly dependent on N fertilization, has been associated with the environmental pollution of the area, causing nitrate leaching and emission of nitrous oxide (N\({}_{2}\)O) [4]. Because of that, this region has been included in the list of the \"nitrate vulnerable zones\" by the Andalusian Government (Decree 36/2008), according to the Nitrate Directive (EEC, 1991). Thus, it is a priority to reduce the impact of the ongoing nitrate pollution due to the intensification of agriculture in the watershed. Natural wetlands provide a favorable environment for the growth of N\({}_{2}\)-fixing cyanobacteria, meeting their requirements for light, water, elevated temperature, and nutrient availability [17; 18]. The ability of these phototrophic microorganisms to fix nitrogen in the soil makes N\({}_{2}\)-fixing cyanobacteria promising biofertilizers for rice paddies. Thus, we aimed to isolate indigenous N\({}_{2}\)-fixing cyanobacteria from the Andalusian paddies, then re-introduce these beneficial microorganisms, and restore the health of plants and soil to its natural balance. Soil samples from seven different plots were taken in March 2018, just before initiation of the rice culture (Figure 1). N\({}_{2}\)-fixing cyanobacteria strains were isolated by a classical serial dilution technique described by [19] using BG11\({}_{0}\) as a growth medium (without combined nitrogen) [15]. A total of 470 different colonies were randomly picked based on their diversity in morphology, color, and micromorphology as observed under a light microscope. The strains were isolated by repeatedly streaking on fresh dishes that were supplemented with cycloheximide in order to inhibit the growth of microalgae and fungi. This strategy has been used in other studies to isolate N\({}_{2}\)-fixing cyanobacteria from their natural habitats, including rice paddies from India, Vietnam, and Pakistan [20; 21; 22]. However, to our knowledge, this is the first report describing the isolation of N\({}_{2}\)-fixing cyanobacteria from the Andalusian rice paddies. To characterize the isolated cultures, we selected 97 strains to be further studied by a genetic approach. Genetic classification of cyanobacteria has been traditionally performed by PCR-denaturing gradient gel electrophoresis (DGGE). Differential DNA fingerprint patterns are ascribed to different strains [18; 23; 24]. However, this method does not provide information about the genetic classification of the samples. We designed a new PCR-based approach that was successfully used for the genetic characterization of our samples. It is based on the amplification of a genomic region of the _sep_] gene, which is required for filament integrity and only found in filamentous cyanobacteria [25]. The _sep_] gene contains a hypervariable region that is flanked by two conserved regions, based on which we have designed degenerate primers (Figure 2). We set up a colony PCR approach to amplify the genomic region of each of the 152 strains (see Materials and Methods). PCR amplification bands were sequenced and aligned, and identical sequences were grouped. This analysis provided 18 different genotypes, containing 1 to 26 strains in each one. In order to identify these genotypes, they were subjected to a phylogenetic analysis against other reference sequences obtained from the GenBank database (Figure 3). We concluded that the isolated strains could be grouped into five major phylogenetic groups (PG01, PG02, PG03, PG04, and PG05).