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. Crops planted in 2022 at the WSU research farm and at the Greene farm still have not been harvested, so yield data is not available at this time.

Microbial Community Analysis

To accomplish our second, third, and fourth objectives, we have collected 264 soil samples, 459 root and rhizosphere samples, and 333 plant shoots across 3 different locations from 2020 to 2022. Substantial delays were experienced in regard to accomplishing our second and third objectives due to there being initial issues with our DNA extraction and quality control pipeline. As a result, we are slightly behind schedule in regard to our sequencing timeline, but we are quickly getting back on schedule. To date, we have extracted DNA from all of the soil and rhizosphere samples we collected in 2020 and 2021. DNA extraction is in progress for the samples collected in 2022. Additionally, since we are working on piloting our method for our root samples, we have only extracted DNA from 8 root samples that were collect in 2020. Sequencing has been done on all of the samples collected in 2020, and samples collected in 2021 are currently being sequenced at the Michigan State University Genomics Core along with the 8 root samples. Due to delays in our optimization of our lab's protocol of the SmartChip Real-Time PCR system, we have not performed qPCR for the nifH gene on any of our samples. Once we have optimized this protocol, results should be produced quickly. Network analysis has been successfully done on the sequences for our 2020 samples. Stable isotope analysis for C and N percentages has been performed on the leaf tissue of the shoot samples collected in 2020 and 2022. Analysis of these results has been completed for the 2020 samples, and analysis is ongoing for the 2022 samples. All results can be found bellow.

Data analysis has been done on the 2020 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 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 rhizosphere, we found no significant differences (P < 0.05) or trends (0.05 ≤ P ≤ 0.1) between pea monoculture, canola monoculture, and peaola using Shannon diversity index, Observed Features, and Faith's Phylogenetic Diversity index (Figure 2).

We did find that the rhizosphere of the canola monoculture was significantly more even than pea monoculture as determined by Evenness (Kruskal–Wallis Test, n1 = 21, n2 = 22, H = 7.01, P = 0.0485). No other significant differences or trends were observed under Evenness. When looking at the β-diversity of the 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 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 and Jaccard distance (Figure 4). Under weighted UniFrac distance, we found that the trends between like intercropped and monoculture crops were not observed. Using unweighted UniFrac distance, we did not observe the trend between monoculture and intercropped canola, and we saw that only a trend existed between intercropped pea and intercropped peaola (PERMANOVA, F = 1.229370, P = 0.084).

Analyses of the bacterial core microbiomes of our soil and rhizosphere samples were performed. For soil, we analyzed the strict bacterial core microbiome, consisting of bacteria found in 100% of our samples. We found that the peaola core microbiome consisted of 34 members, the pea core microbiome consisted of 23 members, and the canola core microbiome consisted of 29 members. When comparing those microbiomes, we found that 13 bacteria were shared across them. From the canola core microbiome, 8 bacteria were shared with peaola. Out of the pea core microbiome, 3 bacteria were shared with peaola. Despite the peaola microbiome having shared members with monoculture pea and canola, it does have 10 core members that were unique to its own microbiome (Figure 5). When analyzing the rhizosphere, we did not analyze the strict core microbiome because it was not found across all 3 cropping systems. Instead, we analyzed core microbiomes consisting of bacteria found across 90% of our samples since a core microbiome was found across all 3 cropping systems at this level. At this level, we found that the peaola core microbiome consisted of 23 members, the pea core microbiome consisted of 14 members, and the canola core microbiome consisted of 18 members. When comparing those microbiomes, we found that 8 bacteria were shared across them. From the canola core microbiome, 4 were shared with peaola, and from the pea core microbiome, 1 was shared with peaola. Again, we found that despite peaola having shared core members with the microbiomes of pea and canola monoculture, we did find 10 unique members of the peaola core microbiome (Figure 5).

Network Analysis

Network analysis using SPIEC-EASI has been performed successfully at the genus level. It should be noted, however, that identification to the genus level is rare when performing analyses on Illumina amplicon sequences due to the small size of the sequences. The peaola co-occurence network consists of 1016 nodes and 10,058 edges (Figure 6). The pea monoculture network consisted of 984 nodes and 8674 edges and the canola monoculture network consists of 965 nodes and 8330 edges (Figures 7 and 8).

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 6, 7, and 8). 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.

Stable Isotope Analysis

After performing statistical analyses on the carbon and nitrogen percentages for the 2020 samples, we determined that there were no significant differences (P < 0.05) or trends (0.05 ≤ P ≤ 0.1) between the intercropped and monoculture pea and canola, and between pea and canola regardless of cropping system (Figure 9).

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 will be addressing 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 input 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).

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. Despite this finding, we did find that peaola did have a unique core microbiome compared to pea and canola when grown in monoculture. Taken with our findings for α-diversity and β-diversity, it does show why we did not see a significant difference here as in the core microbiome there does appear to be a combining of the pea and canola microbiomes despite peaola's unique members. Therefore, it would make sense that the diversity would not be significantly different from pea and canola if there were not many unique members added to the peaola microbiome overall. Despite this, our findings that peaola does have unique core members does suggest to us that it is possible that peaola is creating a unique soil environment from pea and canola, potentially due to changes it is making to the soil chemistry. Further work will need to be done in order to confirm this. In addition, since changes were observed in the core microbiome, it is possible that some members of the microbial community are impacted by peaola in terms of their overall abundance. Therefore, we will be performing differential abundance analysis on our sequencing data in order to determine if there are any bacteria that are changing significantly in their overall abundance between cropping systems. This will be done in R version 4.2.1 in RStudio version 2022.07.0+548. Additionally, data on the abundance of the nifH gene across cropping systems from qPCR will also be insightful into how the microbial community is changing across cropping systems, as it will show us if there is a change in the abundance of bacteria with the ability to fix nitrogen.

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. Although there is useful information that can be extracted from these networks, we have decided to remake our networks with bacteria at the phylum level instead. This will allow patterns to become clearer in the networks, as this reduces the number of nodes. Further investigation will then be done into phyla that are highly connected in the network to determine the microbial members present in each phylum and their predicted ecological role.

Stable Isotope Analysis

From our analyses of our stable isotope data showing C and N percentages, it is difficult to make any conclusions on how pea and canola intercropping is impacting nutrient transfer between plants. However, it is still unknown if our results will be the same for our analysis on our 2022 leaf samples. With that said, even though these results show no significant difference in the N content of the leaf tissue across cropping systems and between plants, this data does not give us any information on where the N those plants have incorporated came from. Preliminary results from a greenhouse experiment we performed measuring the dry shoot mass of pea and canola across cropping systems suggests that pea is providing N to canola while experiencing no negative consequences to its own success.

This is because we saw that there was no significant difference in the dry shoot mass of pea across cropping systems both with and without a N treatment (Figure 10). When looking at canola, we did see that the dry shoot mass of canola when grown with pea was significantly greater than canola grown in monoculture with no N treatment (Kruskal–Wallis Test, n1 = 11, n2 = 20, P = 0.0366), but was not significantly different from canola grown in monoculture with a N treatment (Kruskal–Wallis Test, n1 = 11, n2 = 20, P = 0.445) (Figure 11). Therefore, further investigation will need to be done in the greenhouse to determine how pea and canola are interacting in order to confirm these results were the results of N nutrient transfer between plants.

Sources

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