# Sustaining Rice Production through Biofertilization with N\({}_{2}\)-Fixing Cyanobacteria
## 3 Results and Discussion
### Plant Growth Promotion in Field Trials  
Long-term application of nitrogen fertilizers changes the composition and structure of soil and negatively affects the species richness of plants and soil microbes [34]. In the case of the Andalusian paddy fields, synthetic nitrogen fertilizers are the main factor in producing cropland degradation and decreasing soil microbial biomass and ecosystem services [4]. Novel fertilization approaches involving long-term sustainable farming practices are required for the restoration of these fields. The use of biofertilizers containing soil beneficial microbes has been adopted as an effective method for restoration of degraded soil due to agricultural practices, providing nutrient cycling and improving plant health [3; 35]. The application of cyanobacterial biofertilizers in rice paddies would be an eco-friendly approach, contributing to the development of sustainable agriculture. We studied whether re-inoculation of the cyanobacterial isolates from the five phylogenetic groups influenced plant health and rice productivity in field trials. We used a microbial consortium containing the six representative strains selected upon morphological examination. We chose a representative plot of the Andalusian rice paddies where a conventional mode of rice cultivation was applied (Figure 1). This plot was maintained under continuous flooding and was fertilized with the recommended dose of urea (145 units N-ha-1). Within this area, we then selected three smaller sub-plots of 130 m2, each of which were inoculated with the cyanobacterial consortium containing representatives of all the cyanobacterial isolates from the area. The three sub-plots were managed as the main plot, except for the inoculation with the consortia. We performed two inoculations: 1 month after planting, coinciding with filler initiation, and 3 months after planting, coinciding with panicle formation. The effect of biofertilization on plant growth was evaluated in plants, 1 month, 2 months and 4 months after seeding, and the yield was measured at the ripening state. In 2-months-old plants (1 month after the first treatment), we did not observe any significant difference in length (Figure 5A). However, we found a positive significant effect on plant growth in 4 months (1 month after the second treatment), recording a significant increase in plant length of 127% (\(p\) = 1.22 \(\times\) 10\({}^{-26}\)) (Figure 5A,B). Plants that were treated with the bioinoculant were greener and healthier with respect to the untreated control. At the ripening state, we collected panicles and measured the weight and number of grains per panicle. We recorded a significant increase for both parameters (\(p\) = 3.79 \(\times\) 10\({}^{-6}\) and \(p\) = 4.93 \(\times\) 10\({}^{-13}\), respectively) (Figure 5B,C). Taken together, these results point to a promising application of cyanobacterial biofertilizers to sustain rice production. Cyanobacterial bioinoculants have been used previously in Asian countries, such as China, Vietnam, India, etc., for paddy cultivation as an alternative to nitrogen fertilizers. Several cyanobacterial species, such as _Anabaena variabilis_, _Nostoc muscorum_, _Aulosira fertilsima_, and _Tolypothrix tenuis_, have been described as effective biofertilizers [3]. Our results were obtained with a group of strains particularly well adapted to local climatic and agronomic conditions. To the best of our knowledge, this is the first report using autochthonous cyanobacteria as biofertilizers for the Andalusian paddy fields, the largest region for rice cultivation in Spain and one of the most productive for the Indica variety in Europe. Under laboratory conditions, it has been shown that the model cyanobacterium _Nostoc punctiforme_ enhances the plant shoot/root length and weight of rice plants [12]. The molecular mechanism involved in cyanobacteria-induced plant fortification remains unsolved, yet it is likely that nitrogen fixed in heterocysts and plant phytohormones are involved.