# Sustaining Rice Production through Biofertilization with N\({}_{2}\)-Fixing Cyanobacteria
## 3 Results and Discussion
### Morphological Description of the Cyanobacterial Isolates  
We selected a representative strain from each of the five phylogenetic groups and further characterized it using morphological traits, such as cell dimensions, shape, color, structure of the trichomes and cell differentiation (Figure 4) [15,26]. We complemented this analysis with a genetic characterization of the 16S of these strains in order to corroborate the previous phylogenetic analysis (Table 1). \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline
\begin{tabular}{c} **Phylogenetic** \\ **Group (Strain)** \\ \end{tabular} & **Culture Color** & **Cells Per Trichome** & **\% Heterocysts** & **Akinetes** & **Hormogonia** & **Closest Related** \\ \hline PG1 (G10) & Brown & 24.58 \(\pm\) 2.17 & 7.48 \(\pm\) 0.40 & Yes & Yes & _Nostoc punctiforme_ \\ PG2 (G04) & Green & 24.31 \(\pm\) 1.79 & 9.18 \(\pm\) 0.35 & Yes & Yes & _Nostoc_ sp. \\ PG3 (G16) & Blue-Green & 31.62 \(\pm\) 1.12 & 5.38 \(\pm\) 0.25 & No & No & _Wollea salina_ \\ PG4 (G15) & Green & 41.13 \(\pm\) 1.53 & 4.98 \(\pm\) 0.26 & Yes & No & _Anabaena cylindrica_ \\ PG5 (G17) & Dark-Green & 12.47 \(\pm\) 0.59 & 10.51 \(\pm\) 0.67 & No & Yes & _Calothrix membraneacea_ \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Morphological characters of 5 cyanobacterial strains used in this study. Figure 4: Morphological illustrations of 5 strains used in this work. (**A**) Strain G10 from PG1, (**B**) strain G04 from PG2, (**C**) strain G16 from PG3, (**D**) strain G15 from PG4, and (**E**) strain G17 from PG5. H: Heterocysts; A: Akinetes; and Horm: Hormogonia. Strain G10 was selected as representative of PG1. A 16S rRNA analysis of this strain revealed a high similarity to _Nostoc punctiforme_ MBDU 009. _Nostoc punctiforme_ is a terrestrial cyanobacteria with a broad symbiotic competence with fungi and terrestrial plants, including bryophytes, gymnosperms, and angiosperms, providing fixed nitrogen to the plant partner [27]. It has been shown that _Nostoc punctiforme_ PCC 73102 enhances rice growth [12]. Strain G10 formed brown colonies on a solid medium and growth forming rough mats in liquid BG11\({}_{0}\). The G10 strain showed long curved trichomes (24.58 \(\pm\) 2.17 cells per trichome). Intercalary and terminal heterocysts were found at a percentage of 7.48 \(\pm\) 0.40 heterocysts per vegetative cell. Akinetes and hormogonia were easily found in the culture (Figure 4A). Strain G04 was selected as representative of PG2. This group was most closely related to _Nostoc_ sp. PCC 7524. They formed green colonies on a solid medium and grew uniformly in liquid BG11\({}_{0}\). The trichomes were slightly curved and were composed by rounded vegetative cells with an average of 24.31 \(\pm\) 1.79 cells per trichome. However, long trichomes containing more than 100 cells were occasionally observed. Intercalary and terminal heterocysts were found at a percentage of 9.18 \(\pm\) 0.35 heterocysts per vegetative cell. Akinetes and hormogonia were easily found in the culture (Figure 4B). Strain G16 was the only component of PG3. This group was most closely related to _Wollea salina_ L38, which was first isolated from Thailand in wet, salty soils [28]. Soil salinization is an environmental stressor that causes a significant reduction in crop productivity [29]. Thus, the use of salt-resistant cyanobacteria in salty paddy fields could have positive effects on rice productivity. G16 formed blue-green colonies on a solid medium and grew with parallel and densely packed trichomes, forming rough mats in liquid BG11\({}_{0}\). Trichomes were formed by rounded vegetative cells with conical terminal cells. One intercalary heterocyst was found per trichome at a percentage of 5.38 \(\pm\) 0.25 heterocysts per vegetative cell. This strain did not differentiate akinetes or hormogonia (Figure 4C). Strain G15 was selected as representative of PG4. This group was most closely related to _Anabaena cylindrica_ PCC 7122, a planktonic cyanobacterium that has been evaluated previously as a biofertilizer in lettuce crops [30].