Relative Effects of Glyphosate on Glyphosate-Tolerant Maize Rhizobacterial Communities is Not Altered by Soil Properties

The rhizosphere is the narrow region of soil that is directly influences by root secretions and soil microorganisms. Much of the nutrient cycling and disease suppression needed by plants occurs immediately adjacent to roots. In this study, researchers compared the effects of glyphosate (Roundup Plus), a post-emergence applied herbicide, a pre-emergence applied herbicide (GTZ), and untreated soil. The effects of these variables was monitored by sequencing the soil DNA encoding 16S rRNA and high-throughput DNA pyrosequencing of the bacterial DNA coding for the 16S rRNA hypervariable V6 region. Previous research suggests that PCR amplification and sequencing of the full-length 16S rRNA genes from soil DNA samples combined with massive parallel pyrosequencing of the SSU rRNA V6 hypervariable region proves to be very useful for studying changes in the diversity of the bacterial communities associates with various habitats (Acosta-Martinez et al. 2010). In total, three different methods were used to analyze the herbicide effect on the rhizobacterial communities of genetically modified NK603 glyphosate-tolerant maize. These results were compared with the diversity data obtained when glyphosate was grown in soil with different characteristics. Barriuso and Mellado found that both herbicide treatments decreased the bacterial diversity in the rhizosphere, especially the Actinobacteria taxonomic group.
Barriuso J., Mellado R. Relative Effects of Glyphosate on Glyphosate-Tolerant Maize Rhizobacterial Communities is Not Altered by Soil Properties. 2011. Journal of Microbial Technology 22 (2), 159–165
                  Previous study has indicated that the absorption of glyphosate by the leaves of glyphosate-resistant plants can alter the root exudation and thereby affect the rhizosphere communities (Kremer et al. 2005). The study by Barrius and Mellado was aimed at testing if the effects of herbicides on the rhizobacterial communities of genetically modified NK603 glyphosate-tolerant maize varies according to different soil locations. First, to determine the chemical properties of the soils, the rhizosphere samples were ground using a mortar and dried at 60°C for 12 hours. The total mean values for the carbon, nitrogen, and hydrogen content from three independent measures were determined using a Leco CHSN-932 autoanalyzer.
                  Rhizosphere samples were taken from commercial maize fields of glyphosate tolerant maize, of the NK603 variety, located in Calera y Chozas in Toledo, Spain. There, plants were treated in pre-emergence with 450 g/l acetochlor or 214 g/l terbuthylazine (4 l/ha), in post emergence with 360 g/l of glyphosate as isopropylamine salt (0.72 kg/ha), or untreated with herbicides. The maize and both herbicides were obtained from Monsanto. The plants were harvested from the three experimental fields at two different growth stages. The first was 7 days after glyphosate application and the second was just before crop harvesting at final growth, approximately six months after seeding. Each plot was divided into subplots, and three samples were taken from each subplot at collection times. Therefore, a total of nine subplots were sampled from each maize field at each collection time and equal amounts of soil were pooled from all 27 samples.
                  The rhizospheres from each collection time were pooled and three independent DNA extractions were conducted. The DNA was then subjected to PCR amplification to amplify the 1.5–kb-long fragment encompassing the 16S rRNA coding sequence. The DNA was then subjected to pyrosequencing, resulting in 750–nucleotide long sequences corresponding to the 16S rRNA protein. A phylogenetic tree showing the highest degree of homology between the 16S rRNA sequences was creating using ClustalX, a multiple sequence alignment computer program.
                  The chemical properties of the soils showed carbon values of 1.81%, 1.78%, and 1.75% and nitrogen values of 0.21%, 0.22%, and 0.22%, respectively corresponding to soils not treated with herbicide, treated with glyphosate, and treated with GTZ for the samples taken 7 days after glyphosate application. Furthermore, carbon values of 2.35%, 2.31%, and 2.35%, and nitrogen values of 0.23%, 0.22%, and 0.22%, were obtained at the final sampling times. There were no significant differences ascribed to the treatment with either of the two herbicides.
                  Pyrosequencing of the V6 region of the 16S rRNA subunit were processed in a taxonomic breakdown and a diversity distribution was obtained. Among the sequences obtained from the untreated soil, 41% belonged to the Proteobacteria phylum, 20% to the Acidobacterium phylum, and 13% belonged to the Actinobacteria phylum. This bacterial distribution was similar to that of the 8,345 sequences analyzed from the glyphosate-treated soil (Proteobacteria was slightly lower, and Acidobacteria and Actinobacteria were slightly higher). Conversely, the Proteobacteria and Actinobacteria were lower in the 11,277 sequences analyzed from the GTZ-treated soil, and the Acidobacteria were slightly higher. At the final sampling time, Proteobacteria represented 38% of the rhizobacterial composition in the untreated soil, whereas Acidobacteria ccounted for 30% and Actinobacterial for 11%. These results were similar for the glyphosate-treated soil, but differed from those of the GTZ-treated soil, where the Proteobacteria and Actinobacteria were lower, whereas the percentage of Acidobacteria was higher.
                  The rhizobacterial communities were more similar within each sampling time than when comparing the herbicide treatments, and the supporting values for all the nodes were above 90%. Furthermore, the species richness was determined using the ACE and Chao1 estimators, the results showed a reduction in diversity in bother herbicide-treated soils at both sampling times. In addition, diversity was lower at the second sampling time when compared to the first sampling time. The relative differences observed at 3%, %%, and 10% dissimilarity levels were qualitatively equivalent for al the soil treatments. In conclusion, Actinobacteria was the phylum most significantly affect by the GTZ herbicide. Actinobacteria are an important component of soil bacterial communities playing a major role in organic matter turnover in soils, and are able to decompose organic materials by means of hydrolytic enzymes they synthesize and secrete. However, the diversity analysis showed a reduction of species richness in both herbicide-treated soils at both sampling times, with the values from GTZ-treated soil at the final sampling time being the lowest.

                  The results of this study unveils the effects of herbicidal treatments on the structure of bacterial communities present in the maize rhizosphere. Although, glyphosate treatment revealed a less aggressive impact than GTZ treated soil, further research is need to determine the extent and rate to which chronic herbicidal use may deteriorate the composition of rhizobacterial communities, depending on the particular soil characteristics and applied herbicide. 

Works Cited
Acosta-Martinez, V., S. E. Dowb, Y. Sun, D. Wester, and V. Allen. 2010. Pyrosequencing analysis for characterization of soil bacterial populations as affected by an integrated livestock cotton production system. Appl. Soil Ecol. 45: 325.
Kremer, R. J., N. E. Means, and S. J. Kim. 2005. Glyphosate affects soybean root exudation and rhizosphere microorganisms. Int. J. Environ. Anal. Chem. 85: 11651174.

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