Understanding the impact of the peaola microbiome on soil fertility, crop yield, and plant nitrogen content

Results

Land Equivalent Ratio and Yield Analysis

For our first objective, we found that the cropping system had a significant impact on the yield of pea and canola, and on the LER. Across the trials grown at the WSU research farm in 2021 and the trials grown near Colfax, WA in 2020, it was found that the average LER was 1.63 for peaola compared to the normalized value of 1 for the monocultures. The application of N fertilizers had no significant impact on the yield of peas and canola, or the LER for both of these locations. For the strip trials grown in Colfax, WA, the LER for peaola, at 1.37, was not significantly different from the monocultures. It was found that the yield of canola was not significantly different from when it was grown in monoculture, but the yield of pea was significantly reduced compared to when it was grown in monoculture at the Colfax, WA location. At the WSU research farm there was a significant increase in the LER of peaola from monoculture. However, there was a significant reduction in pea and canola yields from monoculture. At the Greene farm in 2021, no yield data was collected due to poor performance of the peas, so it was considered a loss. LERs were not calculated for the crops planted in 2022 at the WSU research farm and at the Greene farm as crops at the WSU research farm location were damaged significantly by deer and yield data was not collected at the Greene farm due to Dr. Isaac Madsen's move to Africa. The LERs that were recorded do match the range that has been recorded for peaola in the literature at low N application rates (Dowling et al., 2021).

Microbial Community Analysis

To accomplish our second, third, and fourth objectives, we collected 264 soil samples, 459 root and rhizosphere samples, and 333 plant shoots across 3 different locations from 2020 to 2022. Sequencing was done on all of soil, rhizosphere, and root samples collected in 2020, 2021, and 2022. Network analysis was successfully done on the sequences for our 2020 samples. Stable isotope analysis for C and N percentages was performed on the leaf tissue of the shoot samples collected in 2020 and 2022. Analysis of these results has been completed for both years. All results can be found bellow. Comparisons were not able to be performed on samples collected from Mark Greene's plots as a result of him only planting peaola as a result of spatial constraints.

Data analysis was done for the 2020, 2021, and 2022 soil and rhizosphere sequences. Through the inclusion of a microbial community standard, we determined we were able to detect bacteria at the appropriate abundance down to a relative abundance of 0.089%. When analyzing the 2020 soil using Shannon diversity index, Observed Features, and Evenness (measures of α-diversity) we did not find any significant differences (P < 0.05) or trends (0.05 ≤ P ≤ 0.1) between pea monoculture, canola monoculture, and peaola. We did find a trend towards the monoculture pea soil being richer than the monoculture canola soil using Faith's Phylogenetic Diversity index (Kruskal–Wallis Test, n1 = 12, n2 = 12, H = 5.60, p = 0.0537) (Figure 1). No other significant differences or trends were found using Faith's Phylogenetic Diversity index. For the 2020 rhizosphere, we found that the bacterial community in pea is significantly more even than canola (P < 0.05) and that there was a trend towards it being more even than intercropped canola (0.05 ≤ P ≤ 0.1) (Figure 2).

When looking at the β-diversity of the 2020 soil using Jaccard distance, Bray-Curtis distance, and unweighted UniFrac distance, we did not find any significant differences (P < 0.05) or trends (0.05 ≤ P ≤ 0.1) between pea monoculture, canola monoculture, and peaola. Under weighted UniFrac distance, we found that between pea and canola monoculture soils, and between peaola and canola monoculture soils, there was a trend toward there being a difference in the community composition (PERMANOVA, F = 2.06, P = 0.0705; PERMANOVA, F = 1.72, P = 0.0705) (Figure 3). This was not observed between pea monoculture and peaola soils. When looking at the 2020 rhizosphere, we found a significant difference in the β-diversity between canola and pea rhizosphere regardless of cropping system (PERMANOVA, P<0.05), and trends between like intercropped and monoculture crops (PERMANOVA, 0.05≤P≤0.1) based on Bray-Curtis distance (Figure 3). Similar observations were found using other measurements of β-diversity. Crop type was found to be the number one contributor to variance in the data accounting for 13%, while block accounted for 7% and cropping system accounted for 2%. Fewer differentially abundant amplicon sequencing variants (ASV) were seen in the rhizosphere between intercropped canola and intercropped pea than between monoculture canola and pea (Figure 4). The same held true for differentially abundant predicted KOMPs in the rhizosphere involved in nitrogen cycling processes (Figure 5).

Analysis of the 2021 soil samples showed no significant differences across all measures of α-diversity and β-diversity between the different cropping systems. Nitrogen fertilizer treatment did increase the diversity of the soil microbial community alone both at 33 and 67 kg N ha^-1, however, compared to no fertilizer. In the rhizosphere, the intercropped pea microbial community was found to be more diverse than monoculture pea (Kruskal-Wallis, H = 4.57, P = 0.039), but not when considering the combined effects of cropping system and fertilizer treatment (Figure 6A, B). Significant differences remained between the microbial community compositions of canola and pea regardless of cropping system and fertilizer treatment (Figure 7A). This makes sense considering that 15% of the variance was explained by crop type, while block accounted for 8%, N treatment 3%, and cropping system 2%. Significant differences were seen in the community compositions of intercropped canola with no fertilizer and 67 kg N ha^-1 (PERMANOVA, F = 1.83, P = 0.018; Figure 7B). Again, ASVs were found to be differentially abundant between pea and canola, but no ASVs were found to be differentially abundant between intercropped canola and intercropped pea with nitrogen fertilizer addition. This pattern was also true for predicted KOMPs with no differentially abundant predicted KOMPs involved in nitrogen cycling found between intercropped canola and intercropped pea at 67 kg N ha^-1 and few at 33 kg N ha^-1.

The 2022 soil samples showed similar patterns to what we had observed in our other datasets, as no significant differences were found across all of the analyses we performed. Rhizosphere samples showed similar patterns to what we had observed in our other two datasets. We did see that the intercropped canola microbial community was more diverse than the intercropped pea microbial community (Kruskal-Wallis, H = 39.76, P < 0.0001) and
monoculture pea (Kruskal-Wallis, H = 6.02, P = 0.028). , however (Figure 8). Again, crop type was found to be the largest contributor to variance in the data, accounting for 16%. Block and seeding treatment accounted for 4% each, and cropping system accounted for less than 1%. Few patterns were observed to be associated with pea and canola seeding rates, although we did see differentially abundant ASVs did seem to be impacted by seeding rate. More specifically, we found that there were more differentially abundant ASVs at the largest and smallest pea:canola seeding ratios (Figure 9). The second largest and second smallest pea:canola seeding ratios had the fewest differentially abundant ASVs (Figure 9). Differentially abundant predicted KOMPs associated with nitrogen cycling processes did not show any clear pattern associated with pea:canola seeding ratios.

For roots, we saw similar patterns to what we saw in our rhizosphere samples, but differences were even more pronounced. Consistently across studies canola roots were found to be more diverse than pea roots (P < 0.05). The only  exception was observed in our 2022 samples where there was only a significant differences between intercropped canola and intercropped pea (Kruskal-Wallis, H = 11.87, P = 0.0034), with intercropped canola having greater diversity. Consistently, canola and pea roots had significantly different community compositions regardless of crop type and treatment. The only cases where differences were observed between roots from like crops (i.e. canola versus intercropped canola) were seen in our 2021 samples with intercropped canola at 67 kg N ha^-1 having a different community composition than monoculture canola (PERMANOVA, F = 1.63, P = 0.036) and intercropped canola with no nitrogen fertilizer (PERMANOVA, F = 1.88, P = 0.013). Like with our rhizosphere samples, crop type accounted for the majority of the variance observed within our samples accounting for 49%, 46%, and 18% for the 2020, 2021, and 2022 datasets respectively. Differentially abundant taxa were observed between pea and canola across cropping systems and treatments for all three studies. Unlike for our rhizosphere samples in 2021, we saw an increase in the number of differentially abundant bacteria between canola and pea with increasing nitrogen fertilizer application, although no differentially abundant bacteria were seen at 67 kg N ha^-1. Variation in the number of differentially abundant bacteria was observed across different seeding treatments in our 2022 samples, but more work needs to be done to determine if there is a pattern associated with this variation and pea:canola seeding ratio. Additionally, differences were observed in the number of differentially abundant predicted KOMPs involved in nitrogen cycling processes between canola and pea across cropping systems and treatments. More work needs to be done to determine if there are patterns associated with these results and the associated treatments.

We analyzed the strict bacterial core microbiome in our study, consisting of bacteria found in 100% of our samples. Across all of our soil and rhizosphere sampling, we found that there was variation in the composition of the core microbiomes based on crop type, cropping system, and treatment (i.e. nitrogen fertilization rate or seeding treatment). In some cases, unique ASVs were found to be present in the core microbiomes of peaola, although some ASVs were always shared between all the core microbiomes analyzed. Both unique and share members were consistently found to identified as bacteria known to have plant growth promoting traits, or to be involved in nutrient cycling processes. For root samples, variation was still observed across cropping systems and treatment groups. Root core microbiomes shared fewer core members, with only one core ASV found to be shared across all core microbiomes in 2020 and none found to be shared across all the root core microbiomes in 2021 and 2022. Instead, root core microbiomes tended to be more heavily unique to a single sample set or crop type (i.e. canola and intercropped canola). Further work needs to be done to determine the roles of the ASVs identified in the root core microbiomes.

Network Analysis

Network analysis using SPIEC-EASI was performed successfully at the genus level on the 2020 soil samples. The peaola co-occurence network consists of 1016 nodes and 10,058 edges (Figure 10). The canola monoculture network consists of 965 nodes and 8330 edges and the pea monoculture network consisted of 984 nodes and 8674 edges (Figures 11 and 12).

This shows that there is a change in the structure of the microbial community from monoculture pea and canola to peaola, which can also be seen visually (Figures 10, 11, 12). Although there are nodes that are more highly connected than others, there does not appear to be any one node that is more highly connected than the others. Instead, there are clusters of highly connected nodes found throughout all 3 networks. These results affirmed that there are changes occurring in the composition and structure of the microbial communities from monoculture to peaola, which was shown by our microbial community analyses.

Stable Isotope Analysis

In 2020, we saw no significant differences in the leaf nitrogen content across all three crop types. In 2022, we saw that intercropped canola had significantly more nitrogen in its leaf tissue than monoculture canola (t(228) = -2.88, P = 0.026; Figure 13). No pattern was seen in association with differences in seeding treatment and leaf nitrogen content. Comparisons were not able to be performed on samples collected from Mark Greene's plots as a result of him only planting peaola as a result of spatial constraints.

Discussion

Land Equivalent Ratio and Yield Analysis

It is not surprising that the peaola system did not appear to benefit from increasing the rates of synthetic N fertilizer, as previous work done on legume-oilseed intercropping has found similar results (Porter et al., 2020). Despite these results, more work needs to be done to determine if the peaola cropping system can be grown independently of N fertilizer application. Largely, this is due to there being a potential that N was not a limiting factor in the crops grown in this study. Therefore, future research should address if N is being transferred from peas to canola via plant-plant-microbe interactions. This research will help us determine if these interactions are responsible for the increased LER with decreased synthetic N inputs of peaola. The relative yield of peas to canola, calculated based on monoculture pea and canola, showed that winter peaola did not strongly favor either pea or canola. Although some studies have found that pea is favored over canola in overyielding peaola, our data align with the majority of studies on peaola with an overall LER of 1.63 with neither pea nor canola being strongly favored (Fletcher et al., 2016). The crop grown in 2021 at the Greene farm likely experienced failure of its pea crop due to extreme weather conditions around the time of flowering. This is because heat is known to decrease yield in both pea and canola, especially around flowering (Mohapatra et al., 2020). Yield data was unable to be collected for 2022 as a result of herbivory damage from deer.

Microbial Community Analysis

Through our analyses of the α-diversity and β-diversity of the microbial communities in the soil and rhizosphere, we did not find that intercropping pea and canola had a large impact on the diversity of the microbial community alone. Changes do seem to be occurring more on an individual level (i.e. to specific ASVs) within the community as demonstrated by our differential abundance analysis. Additionally, there does seem to be changes in predicted KOMPs associated with nitrogen cycling processes. Further work should be performed to confirm the presence of these KOMPs and if they are differentially abundant within the comparisons we performed for 2020, 2021, and 2022. The results from the sample taken in 2021 and 2022 do show that crop management practices do have a significant impact on microbial communities, although these impacts are likely mediated by plant responses to changes in abiotic and biotic factors resulting from these management practices. Overall, it appears that nitrogen fertilizer application is causing a homogenization of the rhizosphere microbial communities. In root communities, the opposite seems to be occurring with there being an increase in differentially abundant bacteria up until 67 kg N ha^-1. Differences in pea:canola seeding ratios do not appear to have predictable impacts on the microbial community of peaola, although there does appear to be some sort of pattern when looking at differentially abundant ASVs across pea:canola seeding ratios. Further work should be done to analyze this pattern. Additionally, a yield study should be conducted on peaola at different pea:canola seeding ratios in order to determine the impact this has on peaola LER in order for us to make any determinations on which method is the most productive.

Network Analysis

Based on the changes in the number of nodes and edges found in the networks from pea and canola to peaola, it is clear that there is a change in the structure of the microbial community. There is also the potential that this is demonstrating the combining of the pea and canola microbial communities in peaola as we saw through our microbial community analysis. As this analysis did not show us new information about changes occurring in the microbial community from our community analysis, we did not perform this analysis on any other samples.

Stable Isotope Analysis

Generally, not significant differences were observed in the nitrogen content of leaves despite changes in cropping system and management practices. This could be as a result of plants receiving all of the nitrogen they need in order to successfully reproduce and set seed. We did see in our 2022 study that intercropped canola had significantly greater leaf nitrogen content than monoculture canola, which does provide support to our hypothesis that nitrogen is more available to canola in the peaola intercropping system. As we did not observe this result in our other two studies, it is possible that variation in abiotic and biotic factors may impact whether or not this is true. Further studies should be conducted to answer this question and determine the conditions under which canola is observed to have great leaf nitrogen content than monoculture canola.

Sources

Dowling A, Victor SO, Penny R, Ashlea D, Yi Z, Matthew DD. Legume-oilseed intercropping in mechanised broadacre agriculture – a review. Field Crops Research. (2021) 260:107980. doi: https://doi.org/10.1016/j.fcr.2020.107980

Fletcher AL, Kirkegaard JA, Peoples MB, Robertson MJ, Whish J, Swan AD. Prospects to utilise intercrops and crop variety mixtures in mechanised, rain-fed, temperate cropping systems. In: Crop and Pasture Science, Vol. 67. CSIRO (2016). p. 1252–67. doi: 10.1071/CP16211

Mohapatra C, Chand R, Tiwari JK, Singh AK. Effect of heat stress during flowering and pod formation in pea (Pisum sativum physiology L). Mol Biol Plants. (2020) 26:1119–25. doi: 10.1007/s12298-020-00803-4

Porter MJ, Pan WL, Schillinger WF, Madsen IJ, Sowers KE, Tao H. Winter canola response to soil and fertilizer nitrogen in semiarid mediterranean conditions. Agron J. (2020) 112:801–14. doi: 10.1002/agj2.20119