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INVESTIGATOR: Suseela, V.

PERFORMING INSTITUTION:
CLEMSON UNIVERSITY
CLEMSON, SOUTH CAROLINA 29634

INVESTIGATING THE PLANT-SOIL FEEDBACKS THAT MAINTAIN THE LEGACY EFFECT IN INVADED ECOSYSTEMS TO FORMULATE KNOWLEDGE BASED RESTORATION PRACTICES

NON-TECHNICAL SUMMARY: NON-TECHNICAL SUMMARYInvasive plant species pose a great threat to both agricultural and natural ecosystems leading to an estimated loss of $34 billion in the US, annually. Many invasive species create persistent changes in soil properties, which persists even after the removal of the invasive species (termed as the legacy effect). These changes in soil properties impedes the efforts to restore the invaded ecosystems. This is particularly true with invasive species that produce large quantities of biomass that contain chemical compounds different from that in the biomass of native species. These chemical compounds undergo microbial decomposition and are incorporated into soils leading to changes in the amount and chemistry of soil organic matter. Most of these invader-derived chemical compounds will persist in soil preventing the germination or growth of the native species even after the removal of the invasive species. The proposed research focuses on obtaining a detailed understanding of the mechanisms through which the tissue chemistry of invasive species creates the legacy effect in the invaded ecosystems. The influence of soil mineralogy in facilitating the creation and maintenance of the legacy effect will be evaluated at regional scales. Further the research team will test various management practices to successfully restore the invaded ecosystems to their pre-invasion status using various soil amendments. The study will utilize multiple invasive species that are prevalent in eastern United States that produces different litter chemistry. The proposed soil amendments would sequester the invader-derived compounds, and are environmentally sound methods that increase the soil health, which in turn would accelerating the natural recovery of the ecosystems in the invaded sites. The insight gained from this research would be instrumental in formulating knowledge-based management practices to remove the legacy effect of invasive species from invaded ecosystems.

OBJECTIVES: GOALS AND OBJECTIVESNon-native invasive plant species (NIPS) poses a serious threat to agricultural and forest ecosystems. The invasive species which are functionally and chemically different from the native species alter their biotic and abiotic (soil) environment through plant-soil feedbacks. The invasive species that input large quantities of chemically distinct litter that are rich in secondary metabolites have the potential to cause persistent changes in the chemistry of soil organic matter (SOM) in invaded ecosystems. The changes in SOM in invaded ecosystems can persist over longer time scales even after the removal of the invasive species (legacy effect). The proposed research will investigate the mechanisms through which NIPS induce persistent changes in SOM, which then would be used to formulate management practices that could reverse the legacy effect. The study will utilize multiple invasive species that are prevalent in eastern United States that produces different litter chemistry.The major goals of this project are 1) to test the plant secondary metabolite-mediated mechanisms through which NIPS engage in the creation of legacy effect by altering soil C cycling in invaded habitats; 2) to develop knowledge-based restoration practices to negate the effect of these chemical compounds, and successfully restore invaded habitats.Our specific objectives are to:Characterize the secondary metabolites in tissues of invasive plant species and their novelty in invaded ecosystems along eastern United States.Characterize the extractable and bulk soil carbon chemistry in soils invaded by the NIPS, and how it related to the tissue chemistry of the invader.Quantify the rate of nutrient mineralization, litter decomposability and enzyme activities in invaded and non-invaded soils across multiple sites across south eastern United States.Evaluate the role of soil minerology, specifically clay and Fe/Al oxide contents, in maintaining legacy effects at regional scales.Characterize the influence of NIPS in altering SOM in soils of different mineralogy classes across multiple sites that vary in the percent clay and Fe/Al oxide content.Evaluate the effectiveness of soil amendments to reverse the legacy effect by their ability to sequester or degrade the invader induced soil carbon compounds.

APPROACH: METHODSThe proposed project involves lab research, greenhouse studies, and field experiments to elucidate the mechanisms through which the invasive plant species alter soil organic matter cycling and thus create a legacy effect. The study will be conducted in five states along eastern United States using invasive species that differ in their tissue chemistry. The project team would characterize the labile and recalcitrant compounds in the tissues of invasive species that can create persistent changes in soil properties. The soils will also be characterized to elucidate the changes in extractable and bulk soil organic matter chemistry induced by the invasive species. The project team would utilize gas chromatography/liquid chromatography-mass spectrometry techniques to characterize both plant tissues and soil extractable soil organic matter chemistry. Spectroscopy techniques such as infrared spectroscopy and nuclear magnetic resonance spectroscopy would be utilized to characterize the bulk soil organic matter chemistry. The changes in soil nutrient cycling would be measured by analyzing nitrogen mineralization, microbial community, and extracellular enzyme activity and by following the litter decomposition of invasive plant tissues. The project team will analyze the suitability of various soil amendments such as activated carbon, sugar and biochar in degrading or sequestering invader derived soil carbon and thus their ability to reverse the legacy effect of invasive species.

PROGRESS: 2019/06 TO 2020/06
Target Audience:Scientific community Graduate and undergraduate students Postdoctoral fellows Public Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided training to one graduate student, twopostdoctoral fellows, one research assistant and four undergraduate students in establishing experimental plots in the field, processing soil samples for soil chemistry and microbial enzyme analysis and data processing. How have the results been disseminated to communities of interest?The results have been disseminated through peer reviewed journals and national meetings. Min K J* and Suseela V. 2020. Plant invasion alters the Michaelis-Menten kinetics of microbial extracellular enzymes and soil organic matter chemistry along soil depth. Biogeochemistry; https://doi.org/10.1007/s10533-020-00692.(*Postdoctoral mentee). Zhang Z*, Bhowmik P, Suseela V. 2020. Effect of soil carbon amendments in reversing the legacy effect of plant invasion. Journal of Applied Ecology; DOI: 10.1111/1365-2664.13757.(*Postdoctoral mentee). Zhang, Z*., Bhowmik, P. C., & Suseela, V. (2020). Data from: Effect of soil carbon amendments in reversing the legacy effect of plant invasion. Dryad Digital Repository, https://doi.org/10.5061/dryad.crjdfn327 (*Postdoctoral mentee) Zhang Z*,Tharayil, N, Suseela V. 2020. Nitrogen availability modulates the impacts of plant invasion on the chemical composition of soil organic carbon. Annual Meeting of the Ecological Society of America, Utah, USA. (*Postdoctoral mentee). What do you plan to do during the next reporting period to accomplish the goals?Next steps for coming year(s). (1) Identify and regulate the processes through which invasive plants induce changes in soil organic matter in invaded ecosystems, (2) Predictive understanding of the potential interaction between the invasive species with soil mineralogy in creating legacy effects, (3) Understand the differential effect of roots vs aboveground tissues of invasive species in altering SOC chemistry and creating a legacy effect (4)Develop management practices that will restore the invaded ecosystems based on a mechanistic understanding of the invader-induced legacy effect.

IMPACT: 2019/06 TO 2020/06
What was accomplished under these goals? Project 1.Plant invasion alters the Michaelis-Menten kinetics of microbial extracellular enzymes and soil organic matter chemistry along soil depth Specific objectives: 1. Characterize the extractable and bulk soil carbon chemistry in soils invaded by the NIPS, and how it extracellular enzyme activity and soil organic matter decomposition. 2. Quantify the rate of enzyme activities in invaded and non-invaded soils across multiple sites. We did additional analysis and revisions of themanuscript related to this project and is now published in the journal Biogeochemsitry (impact factor: 4.61). Results:The Vmax of peroxidase, the oxidative enzyme that degrades lignin, increased in the invaded soils (0-5 cm) compared to the non-invaded soils. Among the hydrolytic enzymes, the Vmax of N-acetyl-glucosaminidase which degrades chitin from fungal cell walls increased in the invaded soils (0-5 cm). However, there was no associated change in the km of peroxidase and Nacetyl-glucosaminidase under invasion, suggesting that microbes modified the enzyme production rates, not the types (isozyme) of enzymes under invasion. The Vmax of all enzymes decreased with depth, due to the reduced substrate availability. These results highlight that the addition of relatively recalcitrant substrates due to plant invasion altered the kinetics of microbial extracellular enzymes with implications for SOM chemistry in the invaded soils. Project 2.Effect of soil carbon amendments in reversing the legacy effect of plant invasion Objectives: Evaluate the effectiveness of soil amendments to reverse the legacy effect by their ability to sequester or degrade the invader induced soil carbon compounds. In this study, we hypothesized that the management practices that can restore soil C and nutrient cycling in invaded ecosystems can facilitate the rapid restoration of the invaded sites. We predicted that adding soil carbon amendments can revert the microbial composition and functional activity leading to changes in C and nutrient cycling similar to the pre-invasion stages. The manuscript related to this project is revised and is now published in the Journal of Applied Ecology (impact factor: 5.84). The abstract is as follows Abstract: We conducted this study in an old-field in Massachusetts, USA that has been invaded by Japanese knotweed (Polygonum cuspidatum) for >20 years. We chose knotweed as a model system as it alters soil chemistry and microbial community through the input of polyphenols such as tannins and creates a legacy effect. We investigated the effect of two soil carbon (C) amendments (biochar and activated carbon) on the growth and establishment of native and prairie species that were seeded after removing the knotweed above- and below-ground biomass. We measured the percent plant cover and above-ground biomass to assess the establishment of the native and prairie species. We also measured soil and microbial characteristics including nutrient availability, extracellular enzyme activities, and fungal biomass to elucidate the effect of carbon amendments in reversing the legacy effect. Our results revealed that activated carbon and biochar amended plots had 80% more biomass of the prairie species than the control plots. The nitrate content of C amended plots was five times higher than the non-amended plots indicating an increased nitrogen mineralization in the C amended plots. This could be potentially due to the sorption of phenolic compounds by activated carbon and biochar, which makes them unavailable. The phenol peroxidase activity also increased in the activated carbon and biochar amended plots potentially due to the less inhibition by phenolic compounds such as tannins. The fungal biomass decreased in C amended plots that may have resulted in faster nutrient cycling and increased availability of soil nitrogen. Synthesis and applications: Our results revealed the potential of soil C amendments in reversing niche construction and legacy effects of polyphenol-rich invasive species and indicated that biochar could be a more economically feasible alternative to activated carbon in restoring invaded ecosystems. These results also emphasize that understanding the mechanisms through which invasive species create a legacy effect is pivotal in formulating suitable knowledge-based practices for the restoration of invaded ecosystems. Project 3.Nitrogen availability modulates the impacts of plant invasion on the chemical composition of soil organic carbon Objectives:Plant invasion can impact soil carbon (C) cycling and sequestration. Meanwhile, other global change factors such as nitrogen (N) deposition is predicted to promote plant invasion. However, questions remain as to whether the chemical composition of soil organic C (SOC) alter with plant invasion and how N availability modulates the invasion effects on SOC. We carried out a 10-year mesocosm experiment simulating the invasion of Polygonum cuspidatum into a fallow soil, coupled with an N fertilization scheme for the invasive plants. Weinvestigated the invasion effects on the chemical composition of various SOC components and examined how the effects of plant invasion respond to changes in soil N availability. We investigated the invasion effects on the chemical composition of various SOC components (i.e., plant- and microbial-derived C) at the molecular level. We further examined how these effects of plant invasion responded to altered soil N availability. We also quantified the soil microbial biomass, community composition, and enzyme activities to elucidate potential mechanisms driving the variation of SOC compositions. Abstract: In this study, we carried out a 10-year mesocosm experiment simulating the invasion of Polygonum cuspidatum (Japanese knotweed) into a fallow soil, coupled with a contemporary N fertilization scheme for the invasive plants. Using paired invaded and noninvaded treatments as well as paired invaded and invaded + fertilized treatments, we investigated the invasion effects on the chemical composition of various SOC components at the molecular level. We further examined how the effects of plant invasion responded to changes in soil N availability. Compared with noninvaded soils, knotweed-invaded soils exhibited a 17% increase in the microbial-derived C, mainly through the accumulation of fungal residue in the form of amino sugars.N fertilization increased the retention of plant-derived compounds in knotweed-invaded soils, but also stimulated the degradation of lignin monomers. Moreover, knotweed-invaded soils accumulated 46% more microbial-derived C under N enrichment, primarily due to the altered microbial biomass and community composition. Collectively, our findings suggest that plant invasion has the potential to influence SOC chemical composition, with microbial-derived C fractions showing a higher sensitivity relative to plant-derived C. Furthermore, N fertilization could modulate the invasion effects on the molecular composition and accrual of SOC. Our results also highlight the need to understand the impacts of biological invasion in the context of other global change drivers that both affect invasion and modulate their effects. The manuscript issubmitted to Soil Biology and Biochemistry. Project 4. Objective: To understand the effect of root vs above ground tissues in altering SOC chemistry in invaded ecosystems. We have planted Lonicera japonica under greenhouse settings to collect the aboveground tissues and roots. These tissues will be analyzed for tissue chemistry using LC and GC based mass spectrometry analysis. The root tissues and aboveground tissues will be added to soils with different minerology in an incubation study to understand the effect of root tissues and aboveground tissues on the plant-derived and microbial derived soil biomarker and hence on soil organic carbon chemsitry. This study is ongoing.

PUBLICATIONS (not previously reported): 2019/06 TO 2020/06
1. Type: Journal Articles Status: Published Year Published: 2020 Citation: Min K J* and Suseela V. 2020. Plant invasion alters the Michaelis-Menten kinetics of microbial extracellular enzymes and soil organic matter chemistry along soil depth. Biogeochemistry; https://doi.org/10.1007/s10533-020-00692.(*Postdoctoral mentee).
2. Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhang Z*, Bhowmik P, Suseela V. 2020. Effect of soil carbon amendments in reversing the legacy effect of plant invasion. Journal of Applied Ecology; DOI: 10.1111/1365-2664.13757.(*Postdoctoral mentee).
3. Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Zhang Z*,Tharayil, N, Suseela V. 2020. Nitrogen availability modulates the impacts of plant invasion on the chemical composition of soil organic carbon. Annual Meeting of the Ecological Society of America, Utah, USA. (*Postdoctoral mentee).
4. Type: Other Status: Published Year Published: 2020 Citation: Zhang, Z*., Bhowmik, P. C., & Suseela, V. (2020). Data from: Effect of soil carbon amendments in reversing the legacy effect of plant invasion. Dryad Digital Repository, https://doi.org/10.5061/dryad.crjdfn327 (*Postdoctoral mentee)

PROGRESS: 2017/06/15 TO 2018/06/14
Target Audience:Scientific community Graduate and undergraduate students Postdoctoral fellows Park services Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided training to onegraduate student, one postdoctoral fellow, one research assistant and twoundergraduate students in establishing experimental plots in the field, processing soil samples for soil chemistry and microbial enzyme analysis anddata processing. How have the results been disseminated to communities of interest?The results have been dissimiated to the scientific community through the SSSA meeting and a publication in the journal Global Change Biology Suseela, V. Tharayil. N. 2017. Using Biomarker Approaches to Predict the Chemical Attributes of Organic Matter That Facilitates Soil Carbon Sequestration. Symposium "New Insights on Biogeochemical Processes in Terrestrial Ecosystems As Revealed By Isotopic and Biomarker Approaches II" Crop Science Society of America Annual Meeting. October 24, 2017, Tampa FL. M Tamura*, V Suseela*, M Simpson, B Powell, N Tharayil . 2017. Plant litter chemistry alters the content and composition of organic carbon associated with soil mineral and aggregate fractions in invaded ecosystems. Global Change Biology, 23: 4002-4018. (*co-first authors) The results have also been incorporated into the undergraduate/ graduatecourses taught by the PI and Co-PI at Clemson University. What do you plan to do during the next reporting period to accomplish the goals?Next steps for coming year(s). (1) Establish more invaded sites along eastern US that differ in native vegetation and soil mineral properties, (2) Identify and regulate the processes through which invasive plants induce changes in soil organic matter in invaded ecosystems, (3) Predictive understanding of the potential interaction between the invasive species with soil mineralogy in creating legacy effects, (4) Identify the suitability of soil amendments in reversing invader-induced changes in soil organic matter cycling, (5) Develop management practices that will restore the invaded ecosystems based on a mechanistic understanding of the invader-induced legacy effect.

IMPACT: 2017/06/15 TO 2018/06/14
What was accomplished under these goals? Project 1 Objectives: The project aims to obtain a comprehensive understanding of the mechanisms through which invasive plants change soil carbon cycling in their invaded ranges resulting in legacy effects. Further, based on these identified plant-soil feedbacks, knowledge based management practices will be formulated that reverse the legacy effect and restore theinvaded ecosystems. Methodology: We selected two sites along the eastern US that were subjected to prolonged invasion by exotic species that input contrasting litter chemistry compared to the resident native species. Japanese knotweed (Polygonum cuspidatum) that produce recalcitrant litter was invading an old-field ecosystem dominated by grasses and forbs that produced relativelylabile litter, whereas Pueraria lobata (kudzu) that produce labile litter was invading a Pinus forest with recalcitrant-rich litter. Soils were collected to 30 cm depth both in invaded and adjacent non-invaded stands. To understand the influence of litter chemistry on finer-level sequestration of soil carbon, the bulk soils were separated into different size fractions such asmacroaggregates (250-2000 μm), microaggregate (53-250 μm), and silt-clay (<53 μm) fraction. We analyzed the chemistry of different fractions of soil carbon associated with these size-fractions using FTIR and NMR spectroscopy, base hydrolysis and CuO oxidation followed gas chromatography- mass spectrometry analysis to obtain the plant and microbial biomarkersand lignin monomers, respectively. Notable findings: At the knotweed site, the higher C content in soils under P. cuspidatum, compared with non-invaded soils inhabited by grasses and forbs, was limited to the macroaggregate fraction, which was abundant in plant biomarkers. The non-invaded soils at this site had a higher abundance of lignins in mineral and microaggregate fractions and suberin in the macroaggregate fraction, partly because of the greater root density of the native species, which might have had an overriding influence on the chemistry of the above ground litter input. At the kudzu site, soils under P. lobata had lower C content across all size fractions at a 0-5 cm soil depth despite receiving similar amounts of Pinus litter. Contrary to our prediction, the non-invaded soils receiving recalcitrant Pinus litter had a similarabundance of plant biomarkers across both mineral and aggregate fractions, potentially because of the higher surface area of soil minerals at this site. The plant biomarkers were lower in the aggregate fractions of the P. lobata invaded soils, compared with non-invaded pine stands, potentially suggesting a microbial co-metabolism of pine-derived compounds.These results highlight the complex interactions among litter chemistry, soil biota, and minerals in mediating soil C storage in natural ecosystems; these interactions are particularly important under global changes that may alter plant species composition and hence, the quantity and chemistry of litter inputs in terrestrial ecosystems. Project 2 We aim to understand soil carbon, nitrogen, and phosphorus cyclings in the invaded ecosystems in the context of plant-microbe-soil interaction. The knowledge about biogeochemical processes under plant invasion obtained from our field and lab studies will be used to develop management practices to reverse legacy effects of plant invasion. We selected three Japanese Knotweed invaded sites in Amherst, MA. Soils were collected from 0-5, 5-10, 10-15, and 15-30 cm from invaded and adjacent non-invaded stands. We chose five microbial extracellular enzymes targeting common organic compounds in soil: beta-glucosidase for cellulose, N-acetylglucosaminidase for chitin, acid phosphate for organic P, protease for proteins, and peroxidase for lignin. The Michaelis-Menten parameters were determined for each enzyme using fluorogenic and colorimetric methods. Notable findings: Japanese Knotweed invasion differentially influenced distinct soil microbial extracellular enzyme activities and their catalytic efficiencies at 0-5 cm depth. Peroxidase and N-acetylglucosaminidase exhibited lower activities in the invaded stand than those in the non-invaded stand. In contrast, activities of acid phosphatase and beta-glucosidase did not differ between the non-invaded and the invaded stands. Plant invasion reduced catalytic efficiencies of peroxidase and beta-glucosidase compared to the adjacent non-invaded stand. Similar catalytic efficiencies of N-acetylglucosaminidase between the non-invaded and the invaded stands suggest that the apparently low activity of N-acetylglucosaminidase in the invaded stand is due to decreases in total enzyme production, not due to reductions in enzyme performance. In contrast, beta-glucosidase with low catalytic efficiency but similar enzyme activity under invasion compared to the non-invaded stand implies that soil microbial communities in the invaded stand produced more beta-glucosidase. These results highlight that invasion modifies soil microbial biogeochemical processes via changes in the amounts of extracellular enzyme production and enzyme performance, which in turn may distinctly alter the stability of individual compound in soil organic matter under plant invasion.

PUBLICATIONS: 2017/06/15 TO 2018/06/14
1. Type: Journal Articles Status: Published Year Published: 2017 Citation: M Tamura, V Suseela, M Simpson, B Powell, N Tharayil . 2017. Plant litter chemistry alters the content and composition of organic carbon associated with soil mineral and aggregate fractions in invaded ecosystems. Global Change Biology, 23: 4002-4018.
2. Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Suseela, V. Tharayil. N. 2017. Using Biomarker Approaches to Predict the Chemical Attributes of Organic Matter That Facilitates Soil Carbon Sequestration. Symposium "New Insights on Biogeochemical Processes in Terrestrial Ecosystems As Revealed By Isotopic and Biomarker Approaches II" Crop Science Society of America Annual Meeting. October 24, 2017, Tampa FL.
3. Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Tharayil, N. 2017. Organismal- & ecosystem-level roles of plant metabolites in facilitating resilience in weedy and invasive species. Dept. of Plant science. University of California Davis.