Terrestrial Non-Parasitic Nematode Assemblages associated With Glyphosate-tolerant and Conventional Soybean-Based Cropping Systems

During the 2011/12 growing season, rhizosphere soil was collected from soybean plants that were cultivated at eight local fields. Four of these fields were planted with glyphosate-tolerant and four with conventional soybean cultivars (Fig. 1), representing the two soybean ecosystems. Concurrently, soil samples were also collected from a third ecosystem, viz. natural vegetation (representing a reference system) either adjacent to, or within 50 to 100 m from the soybean fields sampled. From each of these three ecosystems, at each sampling locality 80 rhizosphere soil samples were collected, pooled and 20 sub-samples examined.

Glyphosate had been applied continuously for a minimum of 5 years prior to our study in the fields where glyphosate-tolerant soybean and/or maize cultivars were cultivated. However, in the fields planted with conventional soybean no glyphosate-tolerant cultivars were grown and no glyphosate applied for at least 5 years prior this study or never before. No crop cultivation has taken place for at least 10 years prior to this study in the areas where the natural vegetation was sampled.

During the 2012/13 growing season, the same fields sampled during the preceding season were sampled again as well as nine additional fields and adjacent natural vegetation (Fig. 1). Five of these additional fields were planted with glyphosate-tolerant and four with conventional soybean cultivars, with information about the soybean cultivar planted, crop history and soil properties for each field sampled are supplied (Table 1). Soil properties for each site were determined by the EcoAnalitica Laboratory of North-West University (NWU, Potchefstroom) using internationally-accredited protocols (Walkey and Black, 1947; Bouyoucos, 1962; Beretta et al., 2014). Mean rainfall and temperature data, obtained from the database of the Agricultural Research Council, Institute for Soil, Climate and Water, AgroClimatology for each site, from planting of the soybean crops until nematode sampling are also listed (Table 2). Rip and till was the soil cultivation practice used in all soybean fields sampled.

Nematodes were extracted from 200 g soil samples using the decanting and sieving method (Hooper et al., 2005), and counted and identified to genus level using a 1-ml Hawksley slide and light microscope (1,000 × magnification) (Doncaster et al., 1967). This process was repeated once for each sample and the mean of the two counts were used for data analyses. At least 30 individuals from each genus per sample were, after counting, fixed in a heated formaldehyde-propionic-acid-water (FPG) solution (100 ml of a 40% formalin solution, 10 ml propionic acid and 890 ml distilled water). The glass dish with the fixed nematodes were placed in an incubator at 40°C for 72 hr and the FPG solution stepwise replaced with glycerin (Marais et al., 2017). The fixed nematodes were hand-picked from the glycerin using a fine-tip needle and permanently mounted in glycerin on glass microscope slides according to the paraffin-ring protocol (Hooper, 1986). Genus identification of nematodes was done and verified by Dr Antoinette Swart, a nematode specialist-taxonomist of the Agricultural Research Council – Plant Health and Protection (Roodeplaat, South Africa).

Fig. 1

The experimental site consisted of a small field (0.028 ha plot) situated on the premises of the Agricultural Research Council’s Grain Crops Institute, Potchefstroom, South Africa. The study was conducted over two consecutive growing seasons (2013/14 and 2014/15) with soybean being cultivated during the first and maize during the second season. The soil of the plot contained 94% sand and 6% clay. The organic matter content ranged from 0.18% (2013/14 season) to 0.23% (2014/15 season), while soil pH (H2O) was 8 for the 2013/14 season and 7.8 for the 2014/15 season. The history about crops grown and herbicides applied on the experimental site, glyphosate application dosages and dates during the experimental period, nematode sampling dates and rainfall, and temperature data are supplied (Table 3).

The experimental plot was split into two halves (0.013 ha each), which were divided by a fallow, 2-m buffer strip. Before planting the plot for the first season with soybean, weeds that grew on the experimental plot were mechanically hoed and left on the experimental plot. This is the practice that local farmers use. On 18 November 2013, at the beginning of the 2013/14 growing season, seeds of the glyphosate-resistant soybean cultivar LS 6164R were planted after the soil was ripped and tilled using a tractor. The soybean seeds were planted (170 per row) in 5-m-long rows with intra-and inter-row spacings of 3 cm and 0.9 m, respectively. Each seed was coated with Bradyrhizobium japonicum race WB74 at the recommended dosage rate (Soygro Pty Ltd; www.soygro.co.za). The layout of the experiment was a split-plot design with 12 replicates. Each row represented a replicate.

After germination, soybean seedlings were irrigated with ~25 mm water three times a week using a sprinkler irrigation system, except when it had rained sufficiently. When naturally occurring weeds were 10 to 20 cm tall, one half of the experimental plot was treated with glyphosate (active substance 360 g/l glyphosate present as 441 g/l of the potassium salt at a dosage rate of 2 l/ha) using a knapsack sprayer. Applications were done early in the mornings to avoid wind and possible drift of the product as specified by the owner company of the product used. The other half of the experimental plot was not treated with glyphosate or any other herbicide and represented the control. Weeds in the non-treated plot were removed using a hand hoe and left on the soil. This meant that the upper surface of the soil was disturbed during the hoeing action and organic material was left on the soil to decompose.

Before planting, as well as 10 to 20 days after each glyphosate application and also at 120 to 140 days after planting (i.e., at crop maturity), rhizosphere soil and the root systems of nine soybean plants from each replicate were collected, thoroughly mixed and one sub-sample per replicate used for nematode analyses.

During the winter of 2014, no crop was grown and both halves of the experimental plot were left fallow without any weed control being applied. However, before planting seeds of the glyphosate-resistant maize cultivar DKC 80–30 RR on 18 November 2014 of the follow-up growing season, glyphosate was applied on the same plot half where glyphosate had been applied during the previous season (where soybean was planted). Again, the other half of the experimental plot was not treated and the weeds hand hoed and left on the soil. Ten days later, the soil was ripped and tilled and seeds of commercially available maize cultivar DKC 80–30 RR planted. Twenty-five maize seeds were planted per row, each being 5-m long, with intra- and inter-row spacings of 20 cm and 0.9 m, respectively. Two glyphosate applications were done as described above for the preceding soybean crop.

Ten to 20 days after each glyphosate application and also at 120 to 140 days after planting (i.e., at crop maturity), rhizosphere soil and the root systems of three maize plants from each replicate were collected, thoroughly mixed and one sub-sample per replicate used for nematode analyses. The same protocols were used for soil and root sampling, nematode extraction, counting, and identification to genus level as described for the commercial field sampling study.

Thirty-two non-parasitic nematode genera were collectively identified from soils of the three ecosystems, with 65% identified from soils of glyphosate-tolerant soybean fields, 72% from conventional soybean fields, and 88% from natural vegetation sites (Table 4). The genera identified were represented by different feeding groups and functional guilds, and included bacterivores, fungivores, predators, and omnivores.

The predominant non-parasitic nematodes from glyphosate-tolerant soybean sites for the 2011/12 season were Aphelenchus, Acrobeles, and Acrobeloides (Table 5). Aphelenchus occurred in soils from all of glyphosate-tolerant soybean sites while Acrobeles and Acrobeloides occurred in only 50%. For conventional soybean, the predominant genera were Panagrolaimus, Acrobeloides, and Aphelenchus. Panagrolaimus occurred at 75% of the sites, with Acrobeloides and Aphelenchus occurring in 50%. In soils from natural vegetation sites, the predominant genera were Acrobeles, Aphelenchus, and Acrobeloides. Acrobeles occurred at all sites, while Aphelenchus and Acrobeloides were found at 75% of the sites.

For the 2012/13 season the predominant genera identified from soils of glyphosate-tolerant soybean sites were Aphelenchus, Acrobeles, and Eucephalobus (Table 5). Aphelenchus occurred at all sites and Acrobeles and Eucephalobus at 89% and 78%, respectively. For conventional soybean, the predominant genera were Aphelenchus, Eucephalobus, and Acrobeloides. Aphelenchus occurred at all sites, with Eucephalobus and Acrobeloides occurring at 44% and 67%, respectively. In soils from natural vegetation sites the predominant genera were Aphelenchus, Eucephalobus, and Panagrolaimus. Aphelenchus and Panagrolaimus occurred at all sites and Eucephalobus at 83%.

Although the abundance of the predominant genera (Acrobeles, Acrobeloides, Aphelenchus, Eucephalobus, and Panagrolaimus) varied substantially for the three ecosystems, it did not differ significantly between ecosystems according to t-Test analyses (Table 6).

Mixed Models analysis showed significant (P ≤ 0.05) interactions for fungivores, omnivores, and predators for Season*Locality and for predators for Season* Ecosystem*Locality (Table 7). Due to relative low F-ratios for this interaction for fungivores, and the absence or very low numbers for predators and omnivores (ranging between 2 and 7 for omnivores and 2 and 4 for predators (Table 4) further discussion of the data is abstained from.

Season significantly (P ≤ 0.05) affected the abundance of all four nematode trophic groups (bacteri-, fungi-, omnivores, and predators) (Table 7). The abundance of bacterivores (873 ± 426 vs. 120 ± 430 nematodes/200 g soil), fungivores (283 ± 150 vs. 88 ± 152 nematodes/200 g soil) and omnivores (1.6 ± 0.9 vs. 0.5 ± 0.9 nematodes/2.00 g soil) was significantly higher in Season 2 compared with Season 1. By contrast, predator abundance was significantly (P ≤ 0.05) higher in Season 1 (0.8 ± 0.15 nematodes/200 g soil) than Season 2 (0.38 ± 0.12 nematodes/200 g soil). However, due to either the absence or very low numbers for predators and omnivores discussion of the data for these two trophic groups is abstained from.

Ecosystem affected only predator abundance significantly (P ≤ 0.05), with significantly higher population densities in glyphosate-tolerant (1 ± 0.2 nematodes/200 g soil) compared with conventional soybean (0.3 ± 0.2 nematodes/200 g soil) and natural veld (0.2 ± 0.1 nematodes/200 g soil). However, the very low predator numbers recorded for all three ecosystems warrants no further discussion.

Locality significantly (P ≤ 0.05) affected omnivore and predator abundance but warrants no further discussion due to very low population densities recorded for these to trophic groups (Tables 5 and 7).

According to CCA analyses, no differences were apparent for the nematode assemblages present in soils from the three ecosystems when data for the sites were combined (data not shown). However, when the three ecosystems were plotted per site, distinct variations existed among the respective nematode communities for the three ecosystems with the cumulative explained variation (Axes 1 and 2) for the different locations for both seasons ranging from 22% to 82% (data not shown). An example is that of Edenville (Fig. 2) with a cumulative explained variation of 48.9%. For the other localities, similar differences between the nematode communities for the three ecosystems were observed (data not shown) although the nematode assemblages associated with each ecosystem differed among the localities.

According to faunal analysis, soils from the majority of the sites (54%) of the three ecosystems plotted in Quadrant D due to their Enrichment Index (EI) and Structural Index (SI) being <50% for both seasons (Fig. 3a). Such soils were dominated mainly by the presence of fungivores, especially Fu2. Forty-six percent of the sites plotted in Quadrant A due to their EI being >50% and SI being <50%. These soils were dominated by bacterivores, mainly belonging to Ba1 and Ba2. None of the sampling sites plotted in the Quadrants B and/or C.

The metabolic footprints (data pooled for sites from each ecosystem for each season), for the three ecosystems were small (Fig. 3b). The EI for the three ecosystems was intermediate (38%) to moderately high (68%) and the SI very low (<10%) for both seasons. Small differences were evident for both natural vegetation (plotted in Quadrant D for the two respective seasons) and glyphosate-tolerant (plotted in Quadrant A for the two respective seasons) ecosystems. However, for the conventional soybean ecosystem the difference for the two seasons were more pronounced, plotting in Quadrant A (2011/2012 growing season) and D (2012/2013 growing season). This phenomenon was probably due to a higher percentage of Fu2 being present in soils during the 2013 season.

Fig. 2

Fig. 3

All nematode genera identified from the experimental plot were present in soil samples taken before the study commenced. Their numbers were, however, low and ranged between two and seven per 200 g soil.

Fourteen non-parasitic nematode genera were identified from rhizosphere soil samples. In general, higher numbers of non-parasitic nematodes were recorded during the 2014/15 compared with the 2013/14 growing season (Table 8). Aphelenchus was most abundant and always occurred in higher numbers in glyphosate-treated plots. Aphelenchoides only occurred in the glyphosate-treated half of the plot while Tylenchus only occurred in non-treated halves of both crops. Acrobeloides, Cephalobus, Eucephalobus, and Panagrolaimus always occurred in higher numbers in the glyphosate-treated compared with the non-treated half of the plots.

Substantial differences were apparent for non-parasitic nematode assemblages present in soils of the soybean-maize cropping system for the glyphosate-treated (plotted below the red line in Fig. 4) compared with the non-treated plot halves (plotted above the red line in Fig. 4). Data for the non-treated soil of all sampling dates plotted in Quadrants A and B, with EI >45% due to domination by bacterivores (Ba2 in particular representing Acrobeles, Acrobeloides, and Eucephalobus). One sample from the non-treated maize plants plotted in Quadrant B with a high SI (86%) due to the presence of predators (Pr5) belonging to the genera Aporcelaimellus and Discolaimium. By contrast, all samples from the glyphosate-treated plot half, except for one, plotted in Quadrants C and D with a low EI (<35%). This was substantiated by the presence of fungivores, Fu2 in particular belonging to Aphelenchus, and Aphelenchoides while Fu4 was also present and represented by Tylencholaimus.

Fig. 4