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Keywords:

  • weed management;
  • seedbank;
  • Setaria faberi ;
  • giant foxtail;
  • Abutilon theophrasti ;
  • diversified crop rotations;
  • soil microorganisms;
  • seed pathogens;
  • organic matter

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Diversified cropping systems can have high soil microbial biomass and thus strong potential to reduce the weed seedbank through seed decay. This study, conducted in Iowa, USA, evaluated the hypothesis that weed seed decay is higher in a diversified 4-year maize–soyabean–oat/lucerne–lucerne cropping system than in a conventional 2-year maize–soyabean rotation. Mesh bags filled with either Setaria faberi or Abutilon theophrasti seeds and soil were buried at two depths in the maize phase of the two cropping systems and sampled over a 3-year period. Setaria faberi seed decay was consistently greater at 2 cm than at 20 cm burial depth and was higher in the more diverse rotation than in the conventional rotation in 1 year. Abutilon theophrasti seeds decayed very little in comparison with seeds of S. faberi. Separate laboratory and field experiments confirmed differences in germination and seed decay among the seed lots evaluated each year. Fusarium, Pythium, Alternaria, Cladosporium and Trichoderma were the most abundant genera colonising seeds of both species. A glasshouse experiment determined a relationship between Pythium ultimum and S. faberi seed decay. Possible differences in seed susceptibility to decay indicate the need to evaluate weed seedbank dynamics in different cropping systems when evaluating overall population dynamics and formulating weed management strategies.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Crop rotations comprised of crops with different life cycles can have detrimental effects on weed population growth (Liebman & Gallandt, 1997), with different planting and harvest dates among crops preventing or reducing either plant establishment or seed production by weeds (Bastiaans et al., 2008). In diversified cropping systems, the use of forage legumes as green manure and livestock manure to provide organic sources of nutrients and organic matter can reduce weed emergence, by affecting small seeded weeds through the release of allelochemicals or by providing substrates for other organisms that inhibit seedling growth (Liebman & Davis, 2000; Mohler et al., 2012).

Previous work by Liebman et al. (2008) at a long-term field experiment established in Iowa, USA, assessed the effect of simple and more diverse cropping systems on weed seed densities in soil. Over a 4-year period, decline of an experimentally supplemented seedbank of Setaria faberi Herrm. (giant foxtail) was greater in a 2-year maize (Zea mays L.) – soyabean [Glycine max (L.) Merr.] rotation than in a 4-year maize–soyabean–small grain + lucerne (Medicago sativa L.)–lucerne rotation. Abutilon theophrasti Medik. (velvetleaf) seed densities in this experiment declined significantly in the 2- and 4-year systems. Rodents and insects were found to prey upon seeds of both S. faberi and A. theophrasti at the experimental site (Heggenstaller et al., 2006; Williams et al., 2009), but weed seed losses due to decay remain poorly understood.

Weed seed decay rates can vary substantially among crops and crop management systems (Chee-Sanford et al., 2006; Davis et al., 2006). Beneficial effects of diversified cropping systems on soil physical and chemical characteristics, such as increased organic matter content, greater aggregate stability, higher water retention in drought conditions and slower nutrient release (Buyer & Kaufman, 1996; Chee-Sanford et al., 2006), can impact the soil microbial population distribution and community structure (De Cauwer et al., 2011), potentially influencing the colonisation and decay of weed seeds by soil microorganisms. Several studies have addressed the potential of agricultural systems that are less dependent on external, non-renewable resources to reduce the weed seedbank through enhanced weed seed decay or reduced seedling recruitment (Liebman & Davis, 2000; Ullrich et al., 2011). Contrasting results, however, were obtained. Davis et al. (2006) found higher S. faberi and A. theophrasti decay in soil from a conventionally managed system than in soil from a cropping system with low external inputs, and suggested that soil organic amendments had an inhibitory effect upon weed seed decay. Ullrich et al. (2011) did not find a consistent system effect on weed seed decay when they compared conventional cropping systems to organic systems receiving greater amounts of organic amendments and exhibiting higher soil microbial biomass. De Cauwer et al. (2011), on the other hand, determined that ambient seedbank density was lowest in plots amended with compost with a low C:N ratio, whereas Kremer and Li (2003) associated higher proportions of weed-inhibiting bacteria with cropping systems with soils containing high levels of organic matter.

The present study was conducted to test the hypothesis that a more diverse 4-year crop rotation system would promote higher weed seed decay, by enhancing the development of a more diverse soil microbial community that facilitates greater seed colonisation by fungi and Oomycetes, as compared with a simpler 2-year crop rotation system. The experiment evaluated seed decay of S. faberi and A. theophrasti, two important weeds in maize and soyabean in the US Midwest (Forcella et al., 1992; Buhler & Hartzler, 2001) with different seed coat thickness. This is an important factor that may influence the ability of soil microorganisms to decompose seeds (Davis et al., 2008). The seed coat of A. theophrasti, formed by a cutinised palisade layer, is an important barrier for pathogen, gas and water penetration (Davis et al., 2008) and is the main reason for the formation of persistent seedbanks of this species. Mature seeds of S. faberi, in contrast, are capable of freely imbibing water and dissolved gases (Dekker, 2003).

The hypothesis that S. faberi and A. theophrasti seed decay would be higher when seeds are buried in the soil at 2 cm than at 20 cm was also tested. Burial of S. faberi and A. theophrasti seeds increases both the level of viability and dormancy, as well as the longevity of the seeds, possibly because of decreased oxygen at greater depths (Dekker, 2003; Davis & Renner, 2007). The identity of the most predominant fungi and Oomycetes colonising the seeds and potentially causing seed decay was also investigated. These microorganisms are known to be important seed decay causal agents for weeds (Wagner & Mitschunas, 2008) and crops (Agarwal & Sinclair, 1988). Finally, a glasshouse experiment and a laboratory experiment were carried out to test the effect of specific pathogens on seed decay and seed lot physiological differences.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Description of site, crop management and seeds

The study was carried out from 2008 to 2010 at Iowa State University's Marsden Farm, in Boone County, Iowa, USA (42°01′N, 93°47′W; 333 m a.s.l.). The experiment in which the study was conducted was initiated in 2002 to evaluate crop productivity, weed productivity and density, energy use efficiency, and economic performance characteristics of a 2-year maize–soyabean rotation and a 4-year maize–soyabean–lucerne+oat–lucerne rotation (Liebman et al., 2008; Davis et al., 2012). Soils at the experiment site are Clarion loam (fine-loamy, Typic Hapludolls), Nicollet loam (fine-loamy, Aquic Hapludolls) and Webster silty clay loam (fine-loamy, Typic Endoaquolls). The mean concentrations of macronutrients and organic matter and the mean soil pH are shown in Table 1.

Table 1. Mean concentration of macronutrients, organic matter (OM) and mean soil pH determined from soil samples taken to a depth of 20 cm at the experiment site
RotationYearP (mg kg−1)K (mg kg−1)OM (g kg−1)pH
  1. Soil samples were taken on 22 May 2008, 1 May 2009, and 25 April 2010.

2-year200829.1102.148.56.4
200928.4136.854.57.0
201047.9183.349.67.1
4-year200836.197.345.37.3
200922.9128.473.67.6
201020.5114.943.56.9

A randomised complete block design with four replicates was used, and each crop phase of each rotation system was grown every year in a separate plot, for a total of nine plots per block. Plot size was 18 × 85 m. In 2008, the maize and soyabean plots were split in halves to plant (i) a genetically engineered (GE) maize hybrid followed by a GE soyabean variety and (ii) a non-GE maize hybrid followed by a non-GE soyabean variety in each plot (Gómez et al., 2013). Seed decay of S. faberi and A. theophrasti was studied over a 3-year period in the maize plots of the 2- and 4-year cropping systems planted with the GE hybrid. The GE maize hybrid contained transgenes to control both Ostrinia nubilalis Hübner (European corn borer) and Diabrotica spp. (maize rootworms). Weed seeds were buried in these particular plots because there was no soil disturbance after the maize was planted and therefore the risk of disturbing the seeds was minimal. Weed management was performed in these plots by applying a mixture of the pre-emergence herbicides S-metolachlor [acetamide, 2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)-(S)] and isoxaflutole [5-cyclopropyl-4-(2-methylsulfonyl-4-trifl uoromethylbenzoyl) isoxazole]. Information on crop identity, planting and harvest dates, seed density, seed mass and inter-row spacing is provided by Gómez et al. (2013).

Crop management practices varied among rotation systems. Synthetic fertilisers were applied in the 2-year rotation, whereas composted cattle manure and reduced rates of synthetic fertilisers were applied in the 4-year rotation, based on soil tests from each crop rotation (Gómez et al., 2013). No synthetic nitrogen was applied to maize plots in the 4-year rotation in 2009 and 2010. Composted cattle manure was applied to lucerne plots during the fall preceding maize in the 4-year rotation at a rate of 16.2 Mg ha−1 (fresh weight basis). Details of the farming practices carried out in the 2- and 4-year crop rotations, as well as crop productivity and economic returns from 2008 to 2010 are provided by Gómez et al. (2013).

Setaria faberi and A. theophrasti seeds evaluated in 2008, 2009 and 2010 were collected from a field located 13 km east of the experimental site in October 2006, 2008 and 2009, respectively. It was not possible to harvest and test seeds produced at the Marsden Farm site due to the paucity of plants of S. faberi and A. theophrasti surviving control practices and reaching reproductive maturity. The seeds collected were stored in a cold room at a temperature of 5°C and 40% relative humidity until 1 day before burial. Germination of each seed lot was evaluated by placing six batches of 50 seeds each in Petri plates with moist filter paper for 14 days. The plates were placed in a growth chamber in cycles of 30/20°C for 15.5 and 8.5 h, respectively. Germinated seeds were counted and discarded on a daily basis; seeds that did not germinate after 14 days were tested for their viability with a solution of tetrazolium (2,3,5 triphenyl tetrazolium chloride) at 1% m/v. Another sample of seeds from each seed lot was tested for seedborne fungi and Oomycetes using two different growth media: four batches of 15 seeds were placed in Petri plates containing potato dextrose agar (PDA, 39 g L−1 of water) plus the antibiotics streptomycin sulphate (33 mg L−1) and neomycin sulphate (40 mg L−1), while four other batches of 15 seeds were placed in Petri plates with maize meal agar (CMA, 17 g L−1 of water), quintozene (130 mg L−1) and the antibiotics vancomycin (300 mg L−1) and pimaricin (5 mg L−1). The PDA medium was selected to isolate several fungi and Oomycetes that colonise seeds, whereas the CMA medium was chosen as a Pythium-selective media.

Seed burial

The experiment was laid out as a split-plot design with four blocks. The main plot treatment was the crop rotation system (2 and 4 year). Subplots were arranged in a completely randomised design in both rotation systems to evaluate the effects of weed species identity (S. faberi and A. theophrasti), at two burial depths (2 and 20 cm), at 11 extraction times.

Eleven sets of 32 nylon mesh bags (10 × 19 cm, pore size of 0.08 cm) were prepared each year. Sixteen of the bags in each set were filled with 30 seeds per bag of S. faberi mixed with 458 cm3 of soil collected from either the 2- or the 4-year rotation, whereas the other 16 bags were filled with A. theophrasti seeds and soil following the same procedure. Mixing soil with seeds in the bags was intended to reduce seed-to-seed contamination by soil fungi and Oomycetes, which can happen when high densities of seeds are buried within mesh bags. Soil from the first 2 cm and from 18 to 20 cm was collected the day before the bag filling in each of the plots where the bags were going to be buried and separately used plot by plot. Once filled, the bags were stored in a cold room at 4°C until they were taken to the field, 12 h later. The seed density within each bag was equivalent to 6550 seeds m−2 at 0–10 cm depth. Previous field studies have reported S. faberi seed densities ranging from 100 to 25 500 and A. theophrasti seed densities of 100–7000 seeds m−2 (Forcella et al., 1992; Lindquist et al., 1995).

Bags were placed into the soil by marking a 4.1 × 1.9 m grid on the ground containing 45 cells, each 0.45 × 0.38 m (the longer side oriented parallel to maize rows). Bags were randomly assigned to grid cells and buried at either 2 or 20 cm in-between the crop rows. Bag burial areas were then covered with continuous sheets of non-coloured plastic for 1 h to protect the seeds while pre-emergence herbicides were applied to the rest of the plot area. Seed burial occurred on 21 May 2008, 1 May 2009 and 21 April 2010, 1 day after the maize was planted.

Seed recovery and classification

Every 2 weeks for the first 14 weeks after burial and every 4 weeks for the subsequent 12 weeks, one bag per weed species, burial depth and crop rotation was recovered from each of the four replicates. The initial 2-week interval between extractions was intended to make a more precise differentiation between decayed and germinated seeds; some non-infected seeds that germinate but do not reach the surface could be mistaken for decayed seeds once the vegetative parts that emerged from them decompose in the soil (Davis & Renner, 2007). One set of bags remained in the soil overwinter and was extracted on 23 April 2009, 9 April 2010 and 10 April 2011. To allow tillage operations in the plots in preparation for the next crop season, however, this last group of bags was temporarily removed from the soil in the fall of each year, placed in individual plastic bags, stored inside a dark plastic bag in a cold room at 4°C and buried again in the same microplot 3 or 4 days later. In summary, the time the bags remained buried varied from 2 weeks to up to 1 year.

All the bags recovered from the soil were placed individually inside mesh tubes and washed in an elutriator for 120 min. The bags were then placed on top of laboratory benches and air-dried overnight using two 60 W fans. The seeds were recovered manually using nested sieves and classified as germinated, dormant or decayed. The following criteria were followed to classify S. faberi seeds: (i) those seeds that exhibited root or shoot growth, separation between the palea and the lemma larger than 0.1 cm, or aperture of the placental pore, were classified as germinated; (ii) seeds that appeared intact but collapsed under a pressure of c. 0.832 kg cm−2 (measured with the Wagner FDX Algometer) performed with forceps, a method known as the crush test (Borza et al., 2007), were classified as decayed; and (iii) seeds that looked intact and did not collapse under pressure in the crush test were considered dormant. Dormant seeds recovered at 6 and 10 weeks after burial, and those that had overwintered, were tested for viability with tetrazolium, as described above. Classification of decayed and dormant A. theophrasti seeds was performed similarly to that for S. faberi seeds, whereas intact A. theophrasti seeds that showed a lateral seed coat opening, signs of root or shoot emergence, or seed shells showing signs of a similar lateral aperture were considered germinated.

Identification of fungi and Oomycetes colonising the seeds

All the recovered seeds were placed on the two growth media described above. Half of the seeds recovered from each bag were placed on Petri plates containing PDA plus antibiotics and the other half were plated in CMA plus antibiotics. These two media were selected to isolate Fusarium, Alternaria, Cladosporium and Pythium, which have been associated with S. faberi and A. theophrasti seeds in previous studies and can cause seed decay (Kirkpatrick & Bazzaz, 1979; Davis & Renner, 2007). Seed colonisation by Trichoderma was also recorded, because some strains within this genus are used as biocontrol agents against some pathogenic Pythium species (Naseby et al., 2000).The Petri plates were then placed in a growth chamber for 7 days at a temperature of 25°C, with constant light. Seeds were evaluated at 2, 4 and 7 days after planting, and the cumulative proportion of seeds colonised by each microorganism was determined by the end of the evaluation period. Fungi and Oomycetes were visually identified by analysing their mycelia and spores under the microscope and by observing their mycelia colour and growth patterns on the growth media.

After analysing results from 2008, a secondary field experiment was carried out in 2009 to determine whether fungi and Oomycetes were internal or external colonisers of the seeds. Following the procedure described above, one set of 32 bags was filled with S. faberi and A. theophrasti seeds mixed with soil, and then buried at 2 cm next to the microplots of the main study. The bags were exhumed 26 weeks after burial and the seeds recovered by washing the soil in the elutriator. Dormant seeds were surface sterilised by submersion in a solution of 0.1% v/v of sodium hypochlorite for 2 min followed by a 5 min rinse with deionised water on a strainer. The seeds were then placed either in PDA plus streptomycin sulphate and neomycin sulphate or CMA plus vancomycin and pimaricin. They were then placed in a growth chamber, and colonising fungi and Oomycetes were visually identified following the procedure previously described.

Glasshouse study

Decayed seeds of S. faberi recovered in 2008 and 2009 from the main experiment were mostly colonised by the pathogens Fusarium spp. and Pythium spp. A glasshouse experiment was then set out in 2010 to answer the questions (i) are Fusarium and Pythium species causing S. faberi seed decay? and (ii) is the soil from the studied crop rotations enhancing or suppressing the decay process? Soil from plots corresponding to the 2- and 4-year rotations to 20-cm depth was collected, and half of the soil from each rotation was pasteurised by microwaving 4 kg of soil at a time for 8 min in full power on a 900 W microwave oven, to eliminate pathogenic soilborne fungi and Oomycetes, but not other soil microorganisms (Ferriss, 1984). Each treated soil, pasteurised and non-pasteurised, was separated into three fractions. One fraction was inoculated with Pythium ultimum Trow, another fraction was inoculated with Fusarium sporotrichoides Sherb. and the third fraction was used as a control with no microbial inoculation. These pathogens were selected for this experiment due to their high incidence on the plated seeds. Pythium ultimum inoculum was obtained from S. faberi seeds that remained buried in the soil for 8 weeks and were placed on CMA plus antibiotics after being recovered. Fusarium sporotrichoides inoculum was obtained following the same procedure, except that PDA plus antibiotics was used instead of CMA. Inoculum was prepared by transferring the microorganisms to 9-cm-diameter Petri plates that were placed in a growth chamber for 7 days at 25°C, with constant light, to allow them to fill the plate.

The soil needed to fill a 1 L pot was mixed with the inoculum finely sliced, in a ratio of one Petri plate with inoculum per pot (Zhang & Yang, 2000). The control treatment consisted of soil mixed with CMA only. Thirty S. faberi seeds were placed in each pot at c. 2 cm below the soil surface. The soil in each plot remained saturated with water at all times. The pots were placed on glasshouse benches in a completely randomised design with four replicates. After 5 weeks, the soil was washed and the seeds recovered and classified as germinated, dormant or decayed, following the procedure described above.

Seed lot differentiation

Setaria faberi seed decay was considerably higher in 2008 than in 2009. Seed decay might be influenced by the maternal environment because the seed coat is maternally derived (Schutte et al., 2008), and seed lots evaluated in 2008 and 2009 were harvested in different years and thus were of different ages. Therefore, a field and a laboratory experiment were carried out in 2010 to determine whether a seed lot effect on the S. faberi and A. theophrasti seeds could explain differences in seed decay and germination among years. One set of mesh bags was prepared to test in the field seed lots of S. faberi and A. theophrasti harvested in 2006, 2008 and 2009 that had been used in the main study from 2008 to 2010. The bags were buried at 2 and 20 cm in microplots within the maize plots of the 2- and 4-year crop rotations. The bags were filled and buried following the procedure previously described. All the bags were exhumed 26 weeks after burial and the seeds recovered and classified as germinated, dormant and decayed, following the criteria previously explained.

A saturated salt accelerated ageing (SSAA) test was also performed in 2010 to evaluate, in controlled conditions, the vigour of the three seed lots of S. faberi and A. theophrasti evaluated in the field trial from 2008 to 2010. The SSAA test provides a more sensitive index of small seed quality than the germination test, as well as a consistent ranking of seed lot performance (Bennett et al., 2004). A sample of 200 seeds of each seed lot was surface sterilised by submersion in a solution of 0.1% v/v of sodium hypochlorite for 2 min followed by a 5-min rinse with deionised water. The salt solution used in the SSAA test was prepared by dissolving 135 g of sodium chloride in 400 mL of water. This solution was stored for 3 days in an oven at 30°C before it was used. One SSAA box per seed lot was used, and 40 mL of the saturated salt was poured on the bottom of the box. The boxes were then placed in the ageing chamber for 72 h at 41°C. Once the ageing process was completed, two replicates of 100 aged seeds each per seed lot were transferred to plastic boxes with blotter paper. The boxes with the seeds were then placed in a growth chamber with a temperature set to oscillate between 14 and 26°C on a 16- and 8-h cycle respectively. The seeds were observed daily, and the germination percentage was recorded after 11 days.

Data analysis

Proportions of germinated, dormant, decayed and viable seeds were arcsin (√x) transformed to meet analysis of variance requirements for normal distribution. Analyses of variance were then performed using the mixed procedure of SAS for analysis of split-plot experiments. Crop rotation system, weed species identity, burial depth and extraction time were considered fixed factors, and replication and year were considered random factors. Non-linear regression was used to analyse the 2008 seed decay in the two crop rotations and depths, using DataFit software (version 9.0; Oakdale Engineering, Oakdale, PA).The proportion of weed seeds colonised by each microorganism was transformed and analysed similarly to proportions of germinated, dormant and decayed seeds. The glasshouse experiment was analysed using the GLM procedure of SAS; treatment (microorganisms and control), soil type (pasteurised and non-pasteurised) and crop rotation were considered fixed factors, whereas replication was treated as a random factor.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Initial viability of S. faberi and A. theophrasti seed lots ranged from 96% to 100% and from 95% to 100% respectively. Germination of S. faberi seeds was 14%, 11% and 0%, whereas A. theophrasti germination was 11%, 20% and 5% for 2008, 2009 and 2010 respectively. Fusarium, Alternaria and Cladosporium were the main colonisers of the seeds before burial; Penicillium was found colonising S. faberi seeds only in 2008 (Appendix S1). Pythium was not detected colonising the seeds before burial.

Germination, decay and dormancy of recovered seeds

A significant year effect (P < 0.05) was detected for the response variables germinated, dormant and decayed. Because interactions between species and the other factors were significant (P < 0.05) within years, data were analysed by species. We focus hereafter on seed decay results, which were the focus of this study.

Setaria faberi

In 2008, seed decay was higher in the 4-year rotation than in the 2-year rotation (P = 0.009), and also higher at 2 cm than at 20 cm (P = 0.014; Table 2). In 2009, seed decay was again higher at 2 cm than at 20 cm (P = 0.002), although it was much lower than in 2008. No differences between rotations were detected. Seed decay varied among extraction times in 2008 and 2009. A Gompertz function fitted the data of seed decay in 2008, but not in 2009. Seed decay increased over time in 2008 until reaching a maximum value of 29.4% at 14 weeks in the 4-year rotation and 26.8% at 22 weeks in the 2-year rotation (Fig. 1). When compared between burial depths, seed decay was highest at 2 cm (31.8%) after 22 weeks since burial (Fig. 1). In 2009, only burial depth had an effect on seed decay over time (Table 2). In 2010, the number of decayed seeds was higher at 2 cm than at 20 cm (P = 0.027), although these values were several orders of magnitude lower than in the two previous years (Table 2).

Table 2. Proportion of germinated, decayed and dormant Setaria faberi and Abutilon theophrasti seeds recovered from 2008 to 2010 in the main experiment, averaged over 10 extraction times. Transformed [arcsin (√x)] means are shown in parentheses
Seed conditionYear2-year rotation4-year rotation anova a SEb
2 cm20 cm2 cm20 cmRotationDepthRotationaDepth
  1. a

    Analysis of variance of transformed data. Data are P-values.

  2. b

    Standard error of transformed [arcsin (√x)] means.

S. faberi
Germinated20080.26 (0.52)0.17 (0.41)0.32 (0.59)0.16 (0.40)0.33<0.010.080.05
20090.35 (0.63)0.11 (0.32)0.27 (0.53)0.11 (0.30)0.20<0.010.050.03
20100.04 (0.16)0.03 (0.12)0.06 (0.20)0.04 (0.15)0.220.040.750.02
Decayed20080.23 (0.45)0.20 (0.41)0.28 (0.52)0.22 (0.46)0.010.010.750.01
20090.02 (0.09)0.02 (0.06)0.03 (0.14)0.01 (0.05)0.31<0.010.110.02
20100.01 (0.04)0.01 (0.03)0.02 (0.08)0.01 (0.03)0.220.030.090.01
Dormant20080.51 (0.80)0.63 (0.93)0.40 (0.68)0.62 (0.91)0.07<0.010.020.02
20090.63 (0.92)0.87 (1.22)0.68 (0.98)0.88 (1.25)0.34<0.010.380.03
20100.95 (1.39)0.97 (1.42)0.92 (1.32)0.96 (1.40)0.100.000.230.02
A. theophrasti
Germinated20080.03 (0.11)0.04 (0.13)0.05 (0.19)0.03 (0.13)0.050.340.060.02
20090.22 (0.48)0.22 (0.47)0.32 (0.60)0.22 (0.48)0.090.000.000.02
20100.03 (0.12)0.02 (0.09)0.03 (0.13)0.02 (0.08)0.990.010.310.02
Decayed20080.01 (0.05)0.01 (0.04)0.01 (0.05)0.01 (0.03)0.390.130.890.01
20090.01 (0.04)0.01 (0.03)0.01 (0.02)0.00 (0.02)0.130.530.770.01
20100.00 (0.00)0.00 (0.01)0.00 (0.00)0.00 (0.00)0.850.800.170.00
Dormant20080.96 (1.42)0.96 (1.42)0.93 (1.36)0.96 (1.42)0.070.100.060.02
20090.77 (1.09)0.77 (1.08)0.67 (0.97)0.79 (1.11)0.220.000.000.02
20100.97 (1.45)0.98 (1.47)0.97 (1.43)0.98 (1.50)0.970.020.180.02
image

Figure 1. Gompertz function fitted to Setaria faberi seed decay among crop rotations (mean of decay at 2 and 20 cm) and burial depths (mean of decay at 2- and 4-year rotations) over the 2008 crop season. Seed decay in 2009 and 2010 was significantly lower and did not follow any specific pattern. Data points represent the mean and vertical bars represent SEM.

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Abutilon theophrasti Decay of A. theophrasti seeds was lower than 2% in 2008, 2009 and 2010 (Table 2). No significant differences were found between crop rotations and between burial depths (> 0.05). Abutilon theophrasti seeds remained mostly dormant in 2008 and 2010 (Table 2).

Viability of dormant seeds

Viability of dormant seeds recovered at 6, 10 and 14 weeks after burial was analysed similarly to germination, decay and dormancy. The triple interaction between year, species and depth was significant (P = 0.0073); therefore, the data were analysed by year and species. All A. theophrasti seeds classified as dormant were viable every year; likewise, all S. faberi seeds classified as dormant in 2009 and 2010 were viable. In 2008, viability of S. faberi seeds recovered from 2 cm was lower than viability of seeds recovered from 20 cm (P = 0.0134), 81% vs. 91% respectively. No significant differences were found between crop rotations or extraction times.

Overwintering seeds

Setaria faberi seeds that remained buried in the soil over the 2008–2009 winter continued to decompose. Over 42% of the seeds recovered from 2 cm and over 27% of the seeds recovered from 20 cm were decayed (Table 3). However, no significant differences (> 0.05) were determined between rotations or burial depths. Decay of seeds of S. faberi overwintering in 2009–2010 and 2010–2011, on the other hand, was lower than 4% (Table 3).

Table 3. Proportion of germinated, decayed and dormant Setaria faberi and Abutilon theophrasti seeds that remained overwinter in the main experiment. Seeds were buried in spring in sets of mesh bags at 2- and 20-cm depth in two cropping systems and recovered the following spring
Seed conditionYear2-year rotation4-year rotationSource of variabilityaSEb
2 cm20 cm2 cm20 cmRotationDepthRotationaDepth
  1. a

    Analysis of variance of transformed data. Data are P-values.

  2. b

    Standard errors.

S. faberi
Germinated20080.200.170.370.150.270.030.120.06
20090.430.120.280.110.390.000.240.06
20100.050.010.070.020.460.090.790.08
Decayed20080.420.270.450.340.460.060.770.06
20090.030.020.040.010.730.280.730.06
20100.000.020.020.010.900.240.100.06
Dormant20080.380.570.180.510.020.010.220.05
20090.550.860.680.880.410.000.330.07
20100.950.970.910.970.660.170.460.09
A. theophrasti
Germinated20080.040.040.090.040.070.680.430.04
20090.110.200.250.240.110.270.180.05
20100.020.010.030.030.370.650.930.08
Decayed20080.000.000.020.000.180.130.130.00
20090.000.000.010.000.390.360.360.00
20100.000.000.000.000.00
Dormant20080.960.960.900.960.060.610.390.05
20090.890.800.740.760.090.340.170.05
20100.980.990.970.970.370..64740.930.08

Abutilon theophrasti seeds remained mostly dormant in 2008–2009 and 2010–2011 winters, similar to the 2008 and 2010 growing seasons. Seed decay was <2% during the three winters (Table 3). No significant differences were determined between crop rotations and burial depths for decayed seeds in any of the 3 years. Viability of dormant A. theophrasti seeds was 99%, 99% and 100% in 2008–2009, 2009–2010 and 2010–2011 winters respectively.

Fungi and Oomycetes colonising S. faberi and A. theophrasti seeds

Seed colonisation by fungi and Oomycetes was analysed by year and species due to the significant interactions (P < 0.05) between these two factors. Pythium, Fusarium, Alternaria, Trichoderma and Cladosporium were the predominant genera from both S. faberi and A. theophrasti seeds.

Setaria faberi Overall, there was higher colonisation of S. faberi seeds by Pythium, Fusarium and Trichoderma in 2008 than in 2009 or 2010 (Appendix S2). In 2008, seed colonisation by Pythium was slightly higher in the 2-year rotation than in the 4-year rotation (P < 0.0001), whereas in 2009 seed colonisation was highest at 2 cm regardless of the crop rotation (P = 0.013). Pythium incidence on recovered seeds varied among extraction times in each of the 3 years (P < 0.001; Fig. 2). There were significant differences in seed colonisation by Fusarium among extraction times (P < 0.0001), but not between crop rotations or burial depths (Fig. 2). Trichoderma seed colonisation was highest at 2 cm in the 2-year rotation and at 20 cm in the 4-year rotation (P < 0.05; Fig. 2); higher colonisation in the 2-year rotation than in the 4-year rotation was also detected at certain extraction times (P < 0.05). Seed colonisation by Alternaria varied among extraction times every year (P < 0.0001; Fig. 2) and was higher at 20 cm than at 2 cm, although this difference was significant only in 2010 (P = 0.024). Cladosporium seed colonisation also fluctuated among extraction times (P < 0.0001; Fig. 2).

image

Figure 2. Mean Setaria faberi seed colonisation by fungi and Oomycetes over crop seasons 2008–2010 in the main experiment. Seeds were buried in sets of mesh bags at two depths (2 and 20 cm) in two cropping systems (2- and 4-year rotations) and recovered over a period of 22 weeks.

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Abutilon theophrasti Similar to S. faberi seed colonisation, A. theophrasti seed colonisation by Pythium, Fusarium and Trichoderma was highest in 2008 (Fig. 3). Pythium colonisation was higher in the 2-year rotation than in the 4-year rotation at certain extraction times in 2008, 2009 and 2010 (P < 0.05). Seed colonisation by Fusarium and Alternaria varied among extraction times (P < 0.05), but not between burial depth or crop rotation, whereas Trichoderma colonisation was highest at 2 cm in the 2-year rotation over the 3 years (P < 0.05). Colonisation of A. theophrasti seeds by Cladosporium was higher in the 2-year rotation than in the 4-year rotation at certain extraction times in 2008 (P = 0.003); in 2009, it was highest at 20 cm in the 2-year rotation and in 2010 it fluctuated among extraction times (P < 0.0001).

image

Figure 3. Mean Abutilon theophrasti seed colonisation by fungi and Oomycetes over crop seasons 2008–2010 in the main experiment. Seeds were buried in sets of mesh bags at two depths (2 and 20 cm) in two cropping systems (2- and 4-year rotations) and recovered over a period of 22 weeks.

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In the field experiment designed to identify fungi and Oomycetes colonising S. faberi seeds internally, it was found that over 50% and 20% of the seeds were colonised by Fusarium and Alternaria respectively (Appendix S3). No significant differences were detected between crop rotations. Trichoderma growth was observed in <9% of the seeds recovered from the 2-year rotation.

Glasshouse study

The proportion of S. faberi decayed seeds was higher in soil inoculated with P. ultimum (0.14) than in soil inoculated with F. sporotrichoides (0.08) or the control (0.09; P = 0.02; SE = 0.015). No significant differences were detected between crop rotation or soil treatment (pasteurised or non-pasteurised).

Seed lot differentiation

Setaria faberi Seed decay of S. faberi seeds was highest in the seed lot harvested in 2006 (Appendix S4). Within this seed lot, seed decay was higher in the 4-year rotation than in the 2-year rotation (P = 0.005). A similar result was obtained in our overall field experiment, when this particular seed lot was evaluated in 2008 (Table 2).

Abutilon theophrasti Abutilon theophrasti seed decay was negligible for any of the seed lots (Appendix S4).When the seeds were aged in the laboratory, significant differences (P < 0.001) in germination among S. faberi and A. theophrasti seed lots were determined. Similar to what was observed in the field, seed germination was highest in the 2008 seed lot, lowest in the 2009 seed lot and intermediate in the 2006 seed lot (Appendix S5). These findings support the hypothesis that inherent seed lot differences could potentially affect the germination, decay and dormancy of the seeds once they are buried in the field.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

It has been suggested that weed seed decomposition by microbes plays an important role in reducing the persistence of the soil weed seedbank (Chee-Sanford et al., 2006; Wagner & Mitschunas, 2008). However, the great number of factors that influence weed seed decay make this phenomenon so variable over time, location, weed species and cropping systems that determining a consistent effect of a particular cropping system on weed seed decay is extremely difficult. It is established in seed and plant pathology that the key elements that need to be considered when analysing a disease are the environment, the host and the pathogen, that is, the disease triangle (Agrios, 1995). Weed seed decay should be studied with similar criteria.

Applying the concept of the disease triangle, this study found that environmental conditions might have affected S. faberi seed decay when we compared burial depths, cropping systems with contrasting soil management practices and crop seasons. Setaria faberi seeds buried at a shallow depth presented higher decomposition during 1 year, possibly because favourable environmental conditions of light, moisture and temperature for seed colonisation and germination. Although important differences in total soil organic matter content between the cropping systems were not determined, except in 2009, the component that changes more rapidly and thus has the greatest impact on aggregate stability, affecting the seed environment, is the light fraction of the organic matter (Shepherd et al., 2002). This study also detected differences in host (seed) susceptibility to microbial-related decay, both among S. faberi seed lots and between weed species. Weed seed germination, dormancy and decay in the soil are influenced by genetic traits, the maternal environment in which the seed develops and the environment that the seed encounters once it enters the soil seedbank (Wolf et al., 1998; Schutte et al., 2008). Considering that S. faberi and A. theophrasti seeds evaluated in this study from 2008 to 2010 were harvested in the same field but in different years, it is possible that intrinsic seed lot differences related to the maternal environment might be the cause of the observed differences in germination and decay, as suggested by the SSAA test and the parallel seed lots field experiment carried out in 2010. Setaria faberi seeds have a great deal of plasticity in phenotypic expression and even genetically identical seeds might differ in their dormancy characteristics (Dekker, 2003), making them more or less susceptible to microbial decay. Differences in seed decay among years could also be attributed to physiological and chemical factors. Although we did not determine the concentration of seed exudates in the spermosphere of S. faberi or A. theophrasti seeds, it is known that the presence and quantity of specific exudate components released during seed germination are directly correlated with disease incidence, particularly for diseases caused by Pythium and Fusarium species (Begonia & Kremer, 1994; Nelson, 2004). Seed age, seed coat integrity and environmental variables such as temperature may influence the concentration of certain organic molecules in the spermosphere (Nelson, 2004). Thus, it is plausible that physiological differences among seed lots and environmental fluctuations among years could have affected the microbially mediated seed decay process.

Finally, a possible relationship between the pathogen P. ultimum and S. faberi seed decay was determined by following Koch's postulates: we found P. ultimum growing in buried S. faberi seeds, we isolated the pathogen in pure media and we determined, in our glasshouse experiment, that P. ultimum caused higher S. faberi seed decay than the control when inoculated in pasteurised soil. Incidence of P. ultimum on S. faberi weed seeds was highest in 2008, concurring with higher seed decay, than in 2009 and 2010. Despite the high proportion of S. faberi seeds colonised internally by Fusarium species, determined in our parallel field experiment, the effect of F. sporotrichoides on seed decay was not as important as the effect of P. ultimum. It is important to note that P. ultimum might not be the primary agent causing seed decay, but a pathogen that would colonise the seed once another microorganism triggers the decay process.

Abutilon theophrasti seeds, conversely, remained viable or germinated during the season, even when a high proportion of seeds was colonised by Pythium and Fusarium species. High persistence of A. theophrasti seed in the seedbank was also reported in studies by Buhler and Hartzler (2001), whereas other studies suggest that once the integrity of A. theophrasti seed coat is compromised, microbially mediated seed decay occurs readily (Kremer & Spencer, 1989; Davis & Renner, 2007).

With regard to the effect on seed decay of environment–host–pathogen interactions, the present study did not consistently demonstrate that a more diverse 4-year crop rotation system would promote higher weed seed decay, as compared with a simpler 2-year crop rotation system. Although a significant difference in S. faberi seed decay between rotations was detected in 2008, the fact that seed decay was low in the following 2 years indicated that a clear effect of cropping system did not occur. When the seed lot evaluated in 2008 was studied again in 2010 in a seed lot differentiation experiment, however, the highest seed decay was observed, again, in the 4-year rotation. As mentioned above, the effect of cropping systems on seed decay might be related to multiple biotic and abiotic interactions occurring at a specific site and time. It is also important to consider that seedbank depletion is often observed after a larger period of time than the interval evaluated in this study.

Setaria faberi and A. theophrasti population dynamics must be considered when designing weed management strategies in our circumstances. This study found that S. faberi seed decay can be as important to seedbank depletion as seed germination, which suggests that any effort made towards enhancing microbial decomposition of seeds would reduce significantly the burden of control placed on post-emergence weed management tactics. Similar decay rates for S. faberi were detected by Davis et al. (2006) in controlled conditions, but field experiments by Buhler and Hartzler (2001) and Schutte et al. (2008) reported seed decay rates of up to two times higher, depending on seed lot, burial location and year. These previous studies, however, did not account for mortality attributable to fatal germination. This study also found that certain S. faberi seed lots can exhibit very low annual decay rates. Abutilon theophrasti seed decay has been found to range from 16% to 60% (Buhler & Hartzler, 2001; Davis et al., 2006; Schutte et al., 2008), whereas the present study found that it was not an important seedbank depletion factor. These differences among studies show a high ecological variability in agricultural fields and indicate the necessity of evaluating weed population dynamics in different cropping systems to make weed management programmes as effective as possible.

The variability of S. faberi and A. theophrasti seed decay results in this study suggests that other factors, which were not measured, might be also involved in the seed decay process. Those factors may include: the effect of soil bacteria on the seed coat and embryo (Chee-Sanford et al., 2006), the presence of antimicrobial compounds on seeds that prevent microbial colonisation (Davis et al., 2008), the C:N ratio in the soil (De Cauwer et al., 2011), the existence of ‘safe-sites’ in the soil that prevent the decay of certain seeds (Conn & Werdin-Pfisterer, 2010), spatial heterogeneity and patchiness in microbial population distributions (Chee-Sanford, 2007), the effect of the competition for light, water and N by the crop on nutrient composition of the weed seed (Cardina & Sparrow, 1997) and seed damage by insects and vertebrates (Kremer & Spencer, 1989; Schutte et al., 2008). It is important, therefore, that future research on seed decay takes a broad view of the biological interactions that weed seeds, and the weed plant itself, encounter during weed life cycles.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Funding for this study was provided by the USDA National Research Initiative (Project 2006-35320-16548) and the Leopold Center for Sustainable Agriculture (Projects 2007-E09 and E-2010-02). We thank Mark Gleason and Susana Goggi for their assistance with the design of some of the experiments and for their comments on the manuscript. We also thank David Sundberg, Adriana Chacón, Madeline Tomka, Brady North, Nick Siepker, Ranae Dietzel, Mike Cruse, Meghann Jarchow and Sarah Hirsch for their help with field and laboratory activities, and Mercedes Díaz for her assistance with morphological identification of Fusarium species.

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  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
wre12052-sup-0001-AppendixS1.xlsapplication/msexcel34KAppendix S1 Proportion of S. faberi and A. theophrasti seeds colonised by fungi by year before burial.
wre12052-sup-0002-AppendixS2.xlsapplication/msexcel34KAppendix S2 Proportion of recovered S. faberi and A. theophrasti seeds, from the 3-year experiment, colonised by fungi and Oomycetes by burial depth.
wre12052-sup-0003-AppendixS3.xlsapplication/msexcel43KAppendix S3 Proportion of S. faberi surface sterilised seeds colonised by fungi by rotation. Seeds were exhumed from soil after 26 weeks and surface sterilised before being placed in growth media.
wre12052-sup-0004-AppendixS4.xlsapplication/msexcel28KAppendix S4 Proportion of germinated, decayed and dormant S. faberi and A. theophrasti seeds recovered in 2010 from the seed lots evaluation experiment. Results are presented by depth within rotation by year.
wre12052-sup-0005-AppendixS5.xlsapplication/msexcel36KAppendix S5 Proportion of S. faberi and A. theophrasti seeds that germinated in the saturated salt accelerated ageing test performed to determine intrinsic differences among seed lots.

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