DOI: 10.5061/dryad.fj6q57401
title: Pathways of glyphosate effects on litter decomposition in grasslands

description: 1. Grasslands store a third of global terrestrial carbon but are
 vulnerable to carbon loss due to inappropriate livestock grazing.
 Grasslands management can be improved with a mechanistic understanding of
 biogeochemical processes that determine carbon storage, such as plant
 litter decomposition. 2. Herbicides, such as glyphosate, are used to
 improve the quantity and quality of the forage. In the Flooding Pampa, the
 most extensive cattle-grazed natural grassland and one of the few remnant
 temperate grasslands in South America, glyphosate is applied to promote
 Lolium multiflorum, a forage grass associated with a fungal endophyte
 non-toxic for cattle. 3. We studied five mechanistic pathways in which the
 application of glyphosate can alter litter decomposition. We grouped them
 into single application pathways, through effects on living plants (1),
 leaf litter (2) and bare soil (3), and repeated annual application
 pathways, through legacies on ecosystem properties (4) and through the
 growth of an annual forage grass with a fungal endophyte (5). Single
 application pathways were tested in a greenhouse experiment using leaf
 litter of L. multiflorum and of a native dominant grass. Repeated annual
 application pathways were tested through a field experiment with 3-year
 annual glyphosate application using leaf and root litter of L. multiflorum
 with and without endophyte association. 4. Glyphosate application on
 living plants produced leaf litter with 70% higher nitrogen content and
 140% higher decomposition constant than naturally senesced litter. In
 contrast, glyphosate application on naturally senesced leaf litter reduced
 decomposition constant by 20%. Glyphosate application on the soil did not
 affect the decomposition of naturally senesced leaf litter but accelerated
 the decomposition of the glyphosate-killed plants even more. 5. Legacies
 of repeated annual application of glyphosate resulted in a notable
 reduction in plant cover (45%) and potential soil respiration (57%), with
 a consistent acceleration of leaf (53%) and root (18%) litter
 decomposition. Furthermore, the association of endophytes in L.
 multiflorum plants reduced leaf litter decomposition by 22%. On the
 contrary, the association of endophytes did not alter root litter
 decomposition. 6. Glyphosate application on living plants and legacies of
 repeated application on the ecosystem stimulate litter decomposition,
 which can result in a net carbon loss from grasslands. In other
 ecosystems, the net result on decomposition would depend on the relative
 cover of vegetation, aboveground litter, and bare soil. This study
 highlights that glyphosate application should be considered when
 evaluating sustainable management to preserve and enhance soil carbon
 storage in grasslands.
description: Study site and application of glyphosate The
 study site was a humid mesophytic grassland in the Flooding Pampa, a vast
 region of around 9 million hectares in the province of Buenos Aires,
 Argentina. The mean annual temperature is around 15°C and the mean annual
 rainfall is 885 mm (Soriano and Paruelo 1992). The landscape has a
 treeless physiognomy and an extremely flat topography with periodic
 flooding during autumn–spring in lowland, except in ridge areas with
 well-drained sandy soils (Burkart et al. 1998). The field experiment was
 carried out in a commercial livestock farm (35° 01´S, 57° 50´ W). The
 plant community is dominated by C<sub>3</sub> and
 C<sub>4</sub> grass species (see details in Druille et al.
 2015). Soil is classified as a typical Natracuol (US Soil Taxonomy),
 characterized by having a nonsaline acid A1 horizon, and a highly alkaline
 saline B2 horizon (Lavado and Taboada 1988). Glyphosate
 was not applied at the study site before, even though glyphosate
 application in the late summer is a common practice in the region with a 3
 l/ha dose (Rodriguez and Jacobo 2010). We applied this dose (1440 g of
 acid equivalent/ha) of a commercial glyphosate formulation Glacoxan® in
 field and greenhouse experiments with a 20 l backpack sprayer with a
 constant pressure of 3 bars. Pathways of
 single-glyphosate application To evaluate pathways of
 single application of glyphosate effects through living plants (1), leaf
 litter (2), and soil (3), we set up a litter decomposition experiment in a
 greenhouse. For these pathways, we used plant material that is naturally
 found in the field at the end of the summer when glyphosate is applied in
 the Flooding Pampa. At that time of the year,
 <em>Paspalum</em> <em>dilatatum</em>, the native
 dominant perennial C<sub>4</sub> grass, can be found as a
 living plant and as plant litter. In turn, <em>Lolium</em>
 <em>multiflorum</em>, which is an introduced annual winter C3
 forage grass, is only found dead as plant litter. Considering that in this
 grassland the vast majority of <em>L. multiflorum</em> plants
 are endophyte-infected with <em>Epichloë</em>
 <em>occultans</em> (Gundel et al. 2009), in this experiment we
 used <em>L. multiflorum</em> plants associated with the
 endophyte. Together, for the living plant pathway we used <em>P.
 dilatatum </em>(1) and for the leaf litter pathway (2) we used
 litter produced by <em>P. dilatatum</em> and <em>L.
 multiflorum</em> with endophyte plants.
 <em>Paspalum dilatatum</em> and <em>Lolium
 multiflorum</em> plants were grown in 1 m x 1 m monoculture plots in
 the experimental campus of the School of Agronomy at the University of
 Buenos Aires. <em>L. multiflorum</em> plants grew from seeds
 with naturally high level (82%) of endophyte association (E+), and from
 seeds without endophyte (E-) obtained experimentally following Omacini et
 al. (2004). We collected fresh senesced plant litter of both species and,
 in the lab, sorted leaf litter from other plant organs. Then, in the
 <em>P. dilatatum</em> plots, we removed all dead plant
 material to applied glyphosate on living plants. After 15 days, we
 collected <em>P. dilatatum</em> plants killed by glyphosate
 and separated the leaf litter from other organs. We determined the total
 carbon (%C) and nitrogen (%N) content of all types of litter by Dumas
 combustion with a TruSpec elemental analyzer (LECO, St. Joseph, MI, USA)
 at the University of Buenos Aires. We prepared
 litterbags containing leaf litter from <em>P. dilatatum</em>
 plants killed by glyphosate (Plant Gx) and from naturally senesced
 <em>P. dilatatum</em> and <em>L. multiflorum</em>
 E+ plants. Litterbags were made of fiberglass mesh, which is the most
 common used material for litter decomposition studies (Harmon et al. 1999,
 Bradford et al. 2002), and that we have successfully used before (Omacini
 et al. 2004, Vivanco and Austin 2006, 2019).  We used 0.5 g of each litter
 type in 11 cm x 9 cm litterbags with a 3 mm opening on the upper face and
 a 2 mm opening on the lower face. We prepared plastic containers with 1.2
 kg of soil from the study site, which had not received prior glyphosate
 treatment. Half of the litterbags containing naturally senesced leaves
 were sprayed with glyphosate (Litter G+) and the other half was sprayed
 with water (Litter G-). Half of the soil containers were sprayed with
 glyphosate (Soil G+) and the other half was sprayed with water (Soil G-).
 We assigned litterbags (Plant Gx, Litter G+, Litter G-) to soil containers
 (Soil G+, Soil G-) in a factorial design and kept them moistened with
 regular watering (n=5). We assessed litter
 decomposition as litter mass loss over time. We collected litterbags after
 140 and 270 days of incubation. Litterbags were dried for 48 h at 65°C;
 soil and debris were removed from litter and were oven-dried again for
 determination of dry mass. We estimated the decomposition constant k using
 a single exponential decay model by regressing the log of the fraction of
 mass remaining against time. The decomposition constant integrates the
 dynamics of litter mass loss over time and it is a useful parameter to
 compare between litter types and treatments (Wieder and Lang 1982). We
 used ln (M<sub>t</sub>/M<sub>o</sub>) = –kt, where
 M<sub>o</sub> is the initial dry mass,
 M<sup>t</sup> is the dry mass at time t, and k is the
 decomposition constant (Swift et al. 1979). Linear regressions were
 performed by setting the intercept to zero. In the few cases when samples
 did not fit a significant regression, values were considered outliers and
 were replaced by the mean of the treatment, following the missing value
 procedure (Steel and Torrie 1980, Vivanco and Austin 2008).
 Pathways of repeated annual application of glyphosate in natural
 grasslands We evaluated pathways of repeated annual
 application of glyphosate through legacies in ecosystem properties (4) and
 through the enhancement of endophytic grass (5) (Fig. 1) on decomposition
 of leaf litter and roots in a field experiment in the Flooding Pampa. In
 this field experiment, we previously studied the impacts of glyphosate
 application on beneficial soil microorganisms (Druille et al. 2013, 2015,
 2016). We established 10 plots (1.5m x 1.5 m) in an area of similar
 floristic composition and randomly assigned them to control (Ecosystem G-)
 or glyphosate application (Ecosystem G+) treatments. Every April (late
 summer in the southern hemisphere) for three consecutive years, we applied
 3 l / ha of water to Ecosystem G- plots and 3 l / ha (1440 g acid
 equivalent / ha) of commercial glyphosate formulation (Glacoxan®) to Ecosystem G+ plots.
 We applied these treatments using a 20 l backpack sprayer with a constant
 pressure of 3 bars. Cattle grazing was avoided during the experiment by
 keeping an electric wire around the experimental area. To avoid biomass
 accumulation and the consequent aging of grasslands, we made a harvest of
 plant biomass using a lawn mower set to leave 10 cm stubble every year
 before application of the treatment. To evaluate
 pathways of repeated annual application of glyphosate, we used litter
 produced by plants of <em>L. multiflorum</em> with (E+) and
 without (E-) endophyte that was accumulated above and below ground (leaf
 and root litter). We prepared 14 cm x 14 cm litterbags made of 2 mm
 fiberglass mesh. We placed leaf litterbags on the ground and root
 litterbags buried 5 cm belowground. Considering that the place where the
 litter was deposited (above and belowground) can interact with the type of
 litter (leaf and root litter), we placed a common substrate (stem litter)
 litterbags on the ground and buried at 5 cm to assess the effects of the
 above and belowground environment. The experiment started 15 days after
 the third year of application of glyphosate (n = 4) and we collected
 litterbags at 30, 140 and 260 days. We assessed ash-free dry mass (500°C
 oven for 4 h) to estimate the decomposition constant k as described in
 Section 2.3. Together, this experiment evaluated the relative importance
 of pathways 4 and 5 and provides information about the effect of an aerial
 symbiosis on root decomposition of the host, which has not been evaluated
 previously. We assessed above and belowground ecosystem
 properties in Ecosystem G- and Ecosystem G+ plots. We measured plant cover
 in December (when the last litterbag pickup occurred) in 10 plots of each
 level of glyphosate application. For estimation of plant cover, we used
 the line intercept method proposed by Canfield (1941). We determined
 potential water evaporation at ground level by measuring the water loss of
 wet filter papers. We used preweighted oven-dried filter papers and wet
 them in the field to full water-holding capacity. Filter papers were
 weighed immediately before and after incubation on the ground for 1 hour
 at midday in May to calculate water loss. We measured two filter papers
 per plot in 5 replicates for each level of glyphosate
 application. We determined soil gravimetric water
 content from 10 cm depth soil cores taken in August and December (second
 and third litterbag harvest dates, respectively). We also determined soil
 organic matter content and soil potential respiration from soil cores
 taken in May, approximately one year after the decomposition experiment
 was installed in the field. Soil organic matter content was determined by
 total combustion in an oven at 500°C for 4 hours. We determined soil
 potential respiration by incubating a 15-g sample at 25°C, in a 200-ml
 vial with gastight septum caps. The soil was pre-incubated at water field
 capacity for 48 h without seedlings or any plants.
 CO<sub>2</sub> production was measured 2, 4 and 7 days after a
 24-h incubation period with an infrared gas analyzer (PP Systems EGM-4,
 Amesbury, Massachusetts, USA). We used five replicates per level of
 glyphosate application for soil measurements.