Ammonia Oxidizing Prokaryotes Respond Differently to Fertilization and Termination Methods in Common Oat's Rhizosphere

Abstract

Cover crops (CC) have demonstrated beneficial effects on several soil properties yet questions remain regarding their effects on soil microbial communities. Among them, ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) have a key role for N cycling in soil and their responses in the rhizosphere of terminated CC deserve further investigation. A greenhouse experiment was established to assess N fertilization (with or without N) and termination methods (glyphosate, mowing, and untreated control) of common oat (Avena sativa L.) as potential drivers of AOA and AOB responses in the rhizosphere. The abundance of amoA genes was determined by quantitative real-time PCR (qPCR), the community structure was assessed with Illumina amplicon sequencing of these genes, while the function was assessed from potential nitrification activity (PNA). While N fertilization had no influence on AOA, the termination method significantly increased amoA gene copies of AOA in mowed plants relative to glyphosate termination or the untreated control (1.76 and 1.49-fold change, respectively), and shifted AOA community structure (PERMANOVA, p<0.05). Ordination methods indicated a separation between AOA communities from control and glyphosate-terminated plants relative to mowed plants for both UniFrac and Aitchison distance. Converserly, N fertilization significantly increased AOB abundance in the rhizosphere of mowed and control plants, yet not in glyphosate-treated plants. Analyses of community structure showed that AOB changed only in response to N fertilization and not to the termination method. In line with these results, significantly higher PNA values were measured in all fertilized samples, regardless of the termination methods. Overall, the results of this study indicated that bacterial and archaeal nitrifiers have contrasting responses to fertlization and plant termination methods. While AOA were responsive to the termination method, AOB were more sensitive to N additions, although, the stimulative effect of N fertilization on amoA _AOB abundance was dependent on the termination method.

Keywords: Avena sativa L; amoA; cover crop management; glyphosate; inorganic nitrogen.

Figure 1

Non-metric multidimensional scaling (NMDS) using Aitchison distance (Stress=0.14, R²=0.98) for AOA. The SE of mowing termination, glyphosate termination, and untreated control is indicated with green, red and blue ellipses, respectively. The letter M in the labels of samples indicates mowing termination, letter G desiccation with glyphosate and letter U untreated control. Letter N indicates N fertilization and the number identifies the replicate of each treatment.

Figure 2

Compositional biplot of AOA communities using centered log-ratio transformed data (Aitchison transformation). The letter M in the labels of samples indicates mowing termination, letter G desiccation with glyphosate and letter U untreated control. Letter N indicates N fertilization and the number identifies the replicate of each treatment.

Figure 3

Phylogenetic tree of AOA with the 13 operative taxonomic units (OTUs) obtained after clustering amplicon sequence variants (ASVs) at 90% identity threshold. Green and red labels indicate OTUs highly correlated with PC1 in Figure 2. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura 3-parameter model (lowest Bayesian Information Criterion index, BIC) in MEGAX. The tree with the highest log likelihood (−3195.09) is shown. Bootstrap values (>65%) are shown at branch nodes. A discrete Gamma distribution was used [five categories (+G, parameter=0.9061)]. The rate variation model allowed for some sites to be evolutionarily invariable [(+I), 46.46% sites]. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 42 nucleotide sequences, including 13 OTUs and 29 database sequences. All positions with less than 95% site coverage were eliminated (partial deletion option). There were a total of 261 positions in the final dataset. Interactive Tree of Life (iTOL) tool was used for tree visualization.

Figure 4

Non-metric multidimensional scaling of AOB using (A) Aitchison distance (Stress=0.13, R²=0.98; B) generalized UniFrac distance (Stress=0.08, R²=0.99). The SE of fertilized and unfertilized groups is indicated with red and black ellipses, respectively. The letter M in the labels of samples indicates mowing termination, letter G desiccation with glyphosate and letter U untreated control. Letter N indicates N fertilization and the number identifies the replicate of each treatment.

Figure 5

Phylogenetic tree of AOB with the six OTUs obtained in this study after clustering ASVs at 90% identity (bold letter). The different Nitrosospira clusters commonly found in soil are indicated in colours (light blue: cluster 0; light purple: cluster 4; light red: cluster 2; light green: cluster 3b and green: cluster 3a). Nitrosomonas lineage is shown in gray. The evolutionary history was inferred by using the Maximum Likelihood method based on Hasegawa-Kishino-Yano model (lowest Bayesian Information Criterion index, BIC) in MEGAX. The tree with the highest log likelihood (−4633.71) is shown. Bootstrap values (>65%) are shown at branch nodes. A discrete Gamma distribution was used [five categories (+G, parameter=0.2979)]. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 50 nucleotide sequences. All positions with less than 95% site coverage were eliminated (partial deletion option). There were a total of 410 positions in the final dataset. iTOL tool was used for tree visualization.