My name is Erin Brennan, and I'm a Restoration Ecologist at RES. In 2024, I received an Innovation Leadership Program (ILP) grant to study a question that sits at the heart of restoration ecology: why do some wetland restoration sites establish successfully while others, planted under nearly identical conditions, struggle to meet performance standards? Working with Dr. Sarah Emery of Bowling Green State University, we examined soil health, bacterial diversity, and mycorrhizal fungi — the vast underground network of fungal filaments that most land plants have relied on for millions of years — to determine whether what happens beneath the surface determines what survives above.
This study was inspired by two wetland restoration sites located just a few miles apart. One is a struggling flatwoods forested wetland restoration, planted in a former crop field. On paper, this site checked all the boxes for a wetland restoration site: crop fields with hydric soil, evidence of wetland hydrology, and adjacent to reference forested wetlands. However, this site has struggled for years to develop a diverse native plant community, and has experienced stunted tree growth, high tree mortality, and severe invasions of non-native species. Just a few minutes down the road is a successful flatwoods wetland restoration completed in the same year, also in a former crop field with hydric soil, and planted with a similar planting list. However, this site has consistently met performance standards for tree growth and survival. There are plenty of native volunteer trees, understory vegetation, a diverse array of native herbaceous wetland plants, and virtually no invasive species. If the restoration plans were so similar, and the planting and environmental conditions were nearly identical, how did the restorations end up so different? Was it something in (or missing from) the soil contributing to this difference? And if so, is there something that we could do to improve the odds of restoration projects being successful?
Soil is a below-ground ecosystem that supports most of Earth's biodiversity. A 2023 study found that 59% of all biodiversity is in soil, from microbes to mammals (Anthony et al.). According to Kate Scow, a professor of soil science and soil microbial ecology at UC Davis, there are billions of bacterial cells and miles of mycorrhizal fungi filaments found in a single gram of soil. These microbes, especially mycorrhizal fungi, turn sediment into soil by aggregating soil particles, breaking down organic matter, and making nutrients available to plants. Over millions of years, almost all land plants have formed symbiotic relationships with mycorrhizal fungi that live not only in the soil but also on and even inside plant roots. These soil microbes are the real ecosystem engineers, taking lifeless sediment and creating living soil, giving soil structure, aerating it, and providing space for plant roots to move and grow, and making nutrients accessible to plants. It is this below-ground ecosystem that supports and sustains life above ground.
Many wetland restorations occur on former agricultural land, but studies have shown that agricultural practices such as tilling and fallow are damaging to soil health, destroy indigenous fungal communities, and alter bacterial community composition. Trees form symbiotic relationships with ectomycorrhizal fungi that help with the uptake of soil nutrients, transfer of substances throughout the mycelial network, and the movement and storage of water. Tisdale and Oades (1980) found that 50 years of crop rotation not only decreased the amount of mycorrhizal fungal hyphae in soil but also decreased the stability of soil aggregates and the length of plant roots. Miller and Jastrow (1992) found that it can take as long as six years in an undisturbed pasture for enough fungal hyphae in the soil to recover, allowing stable soil aggregates to form again. Mycorrhizal fungi are extremely important for the health of the soil itself, the health of the native plant community, and the growth and survival of trees. Ectomycorrhizal fungi, the primary fungal symbionts of trees, are not likely to be found in the soil in crop fields because they are mostly associated with woody species. Could this absence explain the problems with tree survival and growth at some restoration sites?
We hypothesized that due to deforestation and agricultural practices, soil in wetland restorations on former agricultural land is probably deficient in ectomycorrhizal fungi, the primary fungal associates of oak trees. This deficiency likely leads to two common problems in wetland restorations: tree mortality and stunted tree growth. We wanted to see whether inoculating oak trees with mycorrhizal fungi would increase survival and growth rates in wetland restorations and to rank the effectiveness of commercially available mycorrhizal fungi products. I was also really interested in whether soil transfer from a mature forested wetland reference site would be comparable to a purchased product.
While we can learn a lot from greenhouse and laboratory studies, the conditions in a real wetland restoration site vary greatly and cannot be controlled. Everything from previous land use to weather to wildlife can have major implications for the success of a restoration project. It made sense to set up a field study at an actual restoration site as trees were being planted. The field study is located at Fourteen Mile Stream and Wetland Restoration Bank in southeastern Indiana. This site was selected for its high-quality on-site forested wetlands with large, mature trees and vernal pools, against which we could compare the development of the restored wetlands in the former crop fields.
While mycorrhizal fungi inoculum products are widely available – you can purchase a variety from home and garden stores and online retailers – research has shown that many of these products are either dead or vary in effectiveness (Salomon et al. 2022, Maltz and Treseder 2015). I wanted to see whether products available to the public, designed for and tested in greenhouses, horticulture, agroforestry, and home gardens, could make a difference in wetland restoration. I chose products that contained ectomycorrhizal fungi species in addition to endomycorrhizal fungi and beneficial bacteria, but no added fertilizers or plant foods. This simplified making comparisons and ensured that all variables stayed the same, except for the introduction of microbes.
Oak saplings of five different species were treated at the time of planting with either a commercially available mycorrhizal fungi inoculum or a soil transfer of soil, humus, and fine roots from an onsite reference forested wetland. Commercial inoculums were applied according to package instructions. Soil transfer treatments were applied according to the methods used by Amaranthus and Perry (1987). Amaranthus and Perry examined whether soil transfer could increase the activity, survival, and growth of ectomycorrhizal fungi in conifer seedlings planted in nonreforested clear-cut areas, but the idea is the same. Just as our former agricultural fields had been deforested and lacked sufficient ectomycorrhizal fungi to support planted trees, the clear-cut areas were deforested, treeless, and had low levels of ectomycorrhizal fungi. The soil in a mature forest should contain the indigenous bacterial and fungal communities that newly planted trees need to survive, and the soil transfer should provide a site-specific mix of beneficial bacteria and mycorrhizal fungi, rather than a generic mix that a commercially available product would provide. Uninoculated oak saplings were selected as control trees and received no treatment. Tree height and diameter at ground level were measured at the time of planting in the dormant season. During the next growing season, in 2025, tree survival, height, diameter, and leaf count were recorded, and root samples were collected for analysis of potential differences in fungal colonization between inoculated and uninoculated trees.
Results after the first growing season look promising, with greater tree survival and growth in inoculated saplings. Incredibly, 100% of inoculated trees survived, compared with 92% of uninoculated trees. The difference in tree growth was significant, with a median growth of 10cm for uninoculated trees and 30cm for inoculated trees. We also examined whether there were differences in tree height between treatments and the uninoculated group. Two treatments, the soil transfer and one of the commercially available products, were found to have significantly higher growth than the uninoculated group. These comparisons will continue through the second growing season, with a second set of measurements taken this summer. These preliminary results indicate that inoculation, even with a soil transfer, has a positive effect on oak tree establishment and is a relatively quick, easy, and low-cost solution to some of the most common problems wetland restoration projects face.
Young trees in wetland restorations face a range of stressors in their first few years. Deer browsing and rubbing can be an issue because these restorations are natural areas with wildlife, and the trees are not protected by tree tubes or fencing. Pathogens and disease can also be problematic, especially when trees are under stress. Prolonged flooding can also be an issue. Flatwoods and bottomland hardwood wetlands experience periodic and seasonal inundation. While oak trees are well adapted to short-term inundation, most cannot survive longer-term flooding. Drought stress is also a big issue that impacts restoration projects, especially if a drought occurs in the first year following planting. Kentucky and southern Indiana have experienced a spring drought this year, so I went to Fourteen Mile to check on the study trees. Signs of drought stress in the spring include reduced budding, delayed leaf emergence, and shriveled stems. Just 8% of inoculated trees showed signs of drought stress, compared to 40% of uninoculated trees. The hope is that the mycorrhizal fungi network, which acts as an extended root system for the trees, will allow the inoculated saplings to have greater tolerance to these and other stressors that they face in the first couple of years.
After additional data are collected & analyzed, treatments will be ranked by effectiveness based on tree survival, growth, and root colonization. There is the potential to collect and analyze data from this site long term, as wetland monitoring could continue through 2036. Extensions of this research include examining the effects of inoculation across additional restoration project types, including stream and floodplain restorations; assessing impacts on the herbaceous plant community adjacent to inoculated and uninoculated trees; and eDNA analysis of soil biodiversity to examine changes over time. Extensions of the research would allow us to compare restoration types and regions across the US and examine additional impacts and potential benefits of inoculation.
Improved establishment and increased growth aren’t just ecological wins; they directly impact financial and operational aspects of a restoration project. Sites that struggle with high tree mortality and stunted tree growth often incur additional expenses and require replanting, extended monitoring, or even credit reductions.
As restoration practitioners, we must consider soil health and biodiversity. Below-ground soil biodiversity not only accounts for a majority of all biodiversity but also supports and sustains above-ground biodiversity. Factoring soil health into restoration planning means testing soil, applying amendments prior to planting, and inoculating with mycorrhizal fungi and beneficial bacteria, especially at sites where ectomycorrhizal fungi are missing, and soil is depleted. Restoring soil health can improve overall ecosystem health and biodiversity, reduce planting costs, prevent replanting, accelerate tree growth, and shorten monitoring periods.
If you're working on a restoration project and want to explore how soil health considerations could improve outcomes on your site, contact the RES team.