N2-fixation and weed competition: breaking the connection between crops and weeds

The field experiments were conducted at the Center for Environmental Farming Systems in Goldsboro, NC. From past experiments, soils are known to be sandy, have very low nitrogen status with organic matter levels below 0.5%. The fields were fertilized with all nutrients except nitrogen. Fields were drilled to soybean with high (N93-1264) and low (cv. Young) N-transfer genotypes (see Fig.1) in separate areas. The genotypes are quite similar in terms of growth rate, height, branching, and reproductive characteristics.
A series of circular plots were established in the soybean fields. Soybean occurring inside the plot were removed. Scarified sicklepod or prickly sida seeds were planted into separate, replicated plots. The weeds were planted in three concentric rings plus a center grouping, which will provide a range of separation distances from the crop (see adjacent figure). Treatments were constructed to test weed response as a function of weed: (1) density, (2) distance from soybean (0.25 to 1.00 m from plot edge), (3) N transfer into a weed patch, and (4) low versus high N-transfer soybean. The treatments were : 1) the complete set of rings filled with weed plants, 2) weeds in the outer ring removed, 3) weeds in the outer and inner ring removed, 4) weeds in the outer two rings removed, and 5) all weeds removed except in the center. Interspecific and intraspecific interference for plants at different positions in the patch were quantified. The basic model describing interference among neighboring plants is written:
P(W)=Y – XWNA(N)
where P(W) is the performance (e.g., fecundity, total or reproductive biomass) of the weed plants, Y is the performance of individuals with neighbors removed, XWN- is the slope of the regression of performance, and A(N) is the performance (e.g. biomass) of neighbors. The model can be enhanced by substituting a spatially explicit term for A(N) to account for the differences in proximity to neighboring plants.
Non-destructive measurements of plants in the patches were made throughout the growing season, until maturity. Observations included height, stem and canopy diameter, leaf number, SPAD meter readings [Spectrum Technology’s “chlorophyll meter” (Plainfield, IL)], etc. for individual plants within a ring. Periodically, individual leaves from separate weed plants in a ring, and from randomly selected soybean plants in the field, were sampled to quantify N2-fixation, N transfer, and N uptake from the soil over time. During maturation, senescent fruits on the weeds will be collected and catalogued to maintain records for individual plants in different positions within the plot.
The amounts of N originating from soil N and from the N2-fixing soybean will be determined using the 15N natural abundance technique. The 15N abundance in soybean and weed tissues indicate the relative contributions of N from the soil and N2-fixation by the soybean plants using the equation:
Amount of fixed N = ( 1 - atom % 15N excess of weed with soybean ) x weed total N
atom % 15N excess of weed alone

We know from the previous experiments that atom % 15N abundance is sufficiently different in nodulating N2-fixing plants and non-nodulating plants to clearly distinguish contributions from the N2-fixation process. Individual leaves from many plants in a ring will be sampled during the growing season, and seeds will be harvested at maturity from plants in a ring, and the tissues combined to provide the large sample size necessary to optimize the accuracy of the estimates. Separate patches established without surrounding soybean provided the baseline 15N profiles for soil N. Total N and 15N enrichments were determined using a Finnegan MAT DELTAplus Isotope Ratio Mass Spectrometer.
To verify that the mycorrhizal network was in place for all experiments, soil cores were taken periodically during the growing season. Modified standard methods were used to stain roots for the detection of mycorrhizal colonization and to assess colonized root length using the grid-line intersect method.
Two weed mixtures of one mycorrhizal host (sicklepod) and one non-mycorrhizal host (Palmer amaranth) species were established in plots (patches) that have been drilled to high (N93-1264) or low (cv.‘Young’) N transfer soybean genotypes. Patches developed at two different density levels, either 5 or 10 plants m-2, in 2 m2 microplots to give total weed numbers of 10 or 20 plants per patch. Proportions of the two species were varied in a replacement series design. Patches with Palmer amaranth alone indicated the degree of competition from the soybean genotypes without the involvement of transferred N. Based upon previous observations, we anticipate that the competitive effects of these two soybean genotypes will be similar. The weed species assemblages will be non-destructively monitored throughout the season, and plants will be harvested late in reproduction.
The experiment will also include sets of plots within the soybean fields that receive split rate N fertilizer additions for a total application rate of ~140 kg ha-1. That will, potentially, remove the beneficial effects of N transfer from the N2-fixing crop into a low N environment, allowing examination of inter-species interactions with only the detrimental competitive effect of soybean, as well as assessment of the extent that inorganic N interferes with the N transfer process. Additional plots will be fumigated or treated with fungicide to allow assessment of inter-species competition with suppressed mycorrhizal populations and, presumably, N-transfer. Also, weed mixture plots will be established without surrounding soybean to allow evaluation of the mycorrhizal and non-mycorrhizal weed interactions on low N soils without influence from soybean, N2-fixation, or N-transfer.
The replacement series method is indicated for experiments examining the degree of interference that may occur between two species. In this type of experiment, total density of the assemblage is held constant while the proportion of competing species is varied. The resulting biomass is then expressed as a fraction of the yield of each respective species when grown alone.
To determine how limitations in N impact weed competitiveness and reproductive output, experiments examined nitrogen fertilization effects on sicklepod growing in an old-field successional area with a Tarboro sandy loam soil (Typic Udipsamment, pH of 5.5, 0.3% humic matter, 5-22 ppm inorganic nitrogen). The experiments were conducted in 2000 and 2001 at the Center for Environmental Farming Systems in Goldsboro, North Carolina, USA. A randomized complete block design was used, with four nitrogen treatments and five replicates. Individual plots had an area of 1 m2 and were surrounded by a 1 m buffer strip within blocks and a 2 m buffer strip between blocks.
Sicklepod seeds were obtained from Azlin Seed Company (Leland, MS, USA), who had grown the seed-yielding parental plants at an application rate of 67 kg N ha-1. Nitrogen treatments were established by applying ammonium nitrate fertilizer (NH4NO3) at four levels: 0, 112, 224, and 448 kg N ha-1. In each plot, sicklepod seeds and ammonium nitrate fertilizer were broadcast concurrently (on June 9, 2000 and June 16, 2001). At time of emergence (five days, on average), plots were thinned to four seedlings of uniform size per plot, with one in each 0.25 m2 section of the plot.
During the summer growing season, weed control was maintained by hand within plots and by mowing within the buffer areas. Fruits (legumes) were harvested at maturity, but prior to dehiscence. One plant per plot was selected for fruit collection. Fruit removal continued until all mature legumes had been collected. Harvests were completed on December 5 in 2000 and November 20 in 2001. Following final fruit collection, plants were harvested, oven dried at 60 C for 48 h and then weighed.
After threshing seeds from fruits by hand, a mesh sieve was used to separate seeds into two seed-size classes: small (<2.4 mm) and large (>2.4 mm). Seeds of each category were counted and weighed on a per plant basis. One-way analysis of variance was performed on untransformed data to test for the effects of nitrogen fertilizer treatment on measures of growth and reproduction. Nitrogen concentrations in the seed were determined using an Elemental Analyzer (Model Flash EA 1112, ThermoQuest, Rodano, Milano, Italy). Results and discussion below often focus on nitrogen content, which is calculated from nitrogen concentration x seed mass.
Seed Mass Distributions
A total of 700 seeds per nitrogen treatment were selected randomly and weighed to the nearest 0.01 mg using an electronic micro-balance. The distributions of seed masses were compared using two-sided pairwise Kolmogorov-Smirnov non-parametric tests. Skewness (g1) and kurtosis (g2) were determined. Skewness values indicate the direction and degree of the tail of the distribution curve, if different from a normal distribution, while kurtosis values represent the peakedness.
Offspring Growth
A time-course study was used to evaluate the effects of seed size and nitrogen treatment on growth of seedlings using a two-way factorial design. Seeds from the small and large seed-size classes (described above) were germinated in paper rolls in a germination chamber for 72 h in the dark. The paper rolls were placed vertically into 4-L beakers and kept moist by capillary action from a 0.1 mM CaSO4 solution. Upon emergence of the radicle, seedlings from each seed size class were transferred into eight 50-L continuous flow, solution culture systems. The continuous-flow solution culture systems were located in a controlled-environment growth room in the Southeastern Plant Environment Laboratory at North Carolina State University, Raleigh, N.C. A combination of cool-white fluorescent and incandescent lamps provided a photosynthetic photon flux (PPF) of 700 ± 50 _mol m-2 sec-1 at wavelengths of 400 to 700 nm and a photomorphogenic radiation (PR) of 12 W m-2 between wavelengths of 700 to 850 nm. The seedlings were grown in a modified Hoagland’s solution containing low nitrogen (0.3 mM KNO3) or high nitrogen (1.0 mM KNO3). Solution pH was automatically monitored and maintained at 5.8 ± 0.2, and temperature was maintained at 30 ± 0.5 ºC. Seedlings were harvested every five days until day 40 (after transplant). Shoot and root tissues were separated at harvest, oven dried at 60 ºC for 48 h, and then weighed. Linear regression analysis of total biomass over time was performed on square-root transformed data. The square-root transformation was selected because it increased homogeneity of variance across harvest dates and gave a more linear biomass response. Using the General Linear Model Procedure (SAS, 2000), multiple contrast procedures were performed on the transformed data to test for equality of slopes of linear regressions (representing relative growth rate) for all combinations of nitrogen and seed factors.
Replacement Series
The effects of seed mass on seedling competitiveness were examined in a replacement series experiment. The two-factor experiments included densities of 10 or 20 plants pot-1 exposed to a low or high nitrogen treatment with four replications per treatment. Small seeds from the low-N treatment and large seeds from the high-N treatment in the field study were used. The small:large seed-size class ratios for the 10 plants pot-1 density were 10:0, 7:3, 5:5, 3:7, and 0:10. The ratios for the 20 plants pot-1 density were 20:0, 15:5, 10:10, 5:15, and 0:20.
Seeds were germinated in paper rolls in a germination chamber for 72 h in the dark, as before. Upon radicle emergence, seedlings of each seed-size class were placed in 19 L pots (with drain holes) containing river bottom sand, and placed in controlled environment chambers. Seedlings from each seed size were labeled with small colored stakes. The chambers provided a 12-h photoperiod plus a 2-h night interruption to suppress flowering. A combination of metal halide, high-pressure sodium and incandescent lamps supplied a PPF of 900-1100 µmol m-2 s-1 for the 12-h photoperiod. Only incandescent lamps were used for the night interruption.
Pots were watered once daily with one of two nutrient solutions. The nutrient solutions were a modified Hoagland’s solution (Downs and Thomas 1991) with or without 1.0 mM KNO3 and were applied to saturation (approximately 2 L). In addition, pots in the low nutrient treatment were saturated with the nutrient solution containing 1.0 mM KNO3 once per week. All treatments were harvested 50 days after transplanting. Shoots were removed and placed in a drying oven at 60 ºC for 48 h. Relative per plant yield (dry biomass) was calculated for plants originating from each seed size class:

Calculated parameters:
Relative yield of plants from small seeds (s) RYs = Ysl / Yss
Relative yield of plants from large seeds (l) RYl = Yls / Yll

Where: Ysl = mean per-pot shoot biomass of seedlings from small seeds when grown with seedlings from large seeds; Yls = mean per-pot shoot biomass of seedlings from large seeds when grown with seedlings from small seeds; Yss = mean per-pot shoot biomass of seedlings from small seeds in monoculture; Yll = mean per-pot shoot biomass of seedlings from large seeds in monoculture. Actual RY was compared to expected relative yield (EY, a theoretical value calculated assuming plants were uniformly competitive in mixture and in monoculture) for each ratio of small: large seed size proportions using paired t-tests.