Field and greenhouse evaluations of soil suppressiveness to Heterodera glycines in the Midwest corn-soybean production systems

This research was conducted at two field sites in Waseca County in Southern Minnesota for four years. Site 1 was located at the University of Minnesota Southern Research and Outreach Center (44°04′21″ N 93°31′21″ W) in Waseca, Minnesota, which had been planted to soybean continuously for 37 years and no-tillage had been practiced for the past 11 years before the experiment was established in 2009. Site 2 was in a commercial field (43°52′38″ N 93°43′14″ W) in Minnesota Lake, Minnesota, which had been in soybean monoculture for more than 20 years by 2005 when the field was planted to corn for four years prior to this experiment. The soil at Site 1 was a Nicollet clay loam (fine loamy, mixed, mesic Aquic Hapludoll), and the soil at Site 2 was Webster clay loam (fine loamy, mixed, mesic Endoaquoll). The soils in both fields were demonstrated to be suppressive to SCN (Chen, 2007a; Bao et al., 2011).

The experiment was a split-plot design with no-tillage and conventional tillage as main plots, and the crop sequence-biocide treatments as subplots with four replicates (Fig. A1). The crop sequence-biocide treatments were carried out from 2009 to 2012 (Table A1): (i) corn (C)/soybean (S) (susceptible to SCN) annual rotation (C-S-C-S) without biocide, (ii) rotation of corn/SCN-resistant soybean (R)/corn/SCN-susceptible soybean (C-R-C-S) without biocide, (iii) monoculture of SCN-susceptible soybean (S-S-S-S) without biocide, (iv) S-S-S-S with bactericide streptomycin treatment, (v) S-S-S-S with captan (N-trichloromethylthio-4-cyclohexene-1, 2-dicarboximide) fungicide treatment, and (vi) S-S-S-S with the general biocide formaldehyde. At Site 2, the formaldehyde treatment was omitted considering its high toxicity and proximity of the site to a residential house. The main plot was 16.2 m long and 13.71 wide, each subplot was 7.6 m long and 4.57 m wide which included six rows of crops (Fig. A1). All treatments were repeated annually from 2009 to 2012 at both sites.

For the formaldehyde treatment, 6.8 liter of 38% formaldehyde (Formalin) in 220 liters water was applied by irrigation in the four central rows (3000 L formalin per ha) 3 wk before planting. Appropriate safety instructions were followed when applying formalin. For the streptomycin and captan treatments, 18 grams of streptomycin sulfate (Sigma S 5601) (7.75 kg a.i./ha) and 27 grams a.i. of captan (80% wettable powder) (11.6 kg a.i./ha) each in 220 liters of water were applied by a pump from a tank into the surface of four central rows 1 wk before planting and every 2 wk after planting for two months (five times per year).

The conventional tillage treatment was fall chisel plowing after harvesting, and field cultivation followed by a finishing implement prior to planting. Fertilizer application was based on soil fertility test recommendations of the University of Minnesota Soil Test Laboratory. Fertilizer nitrogen in the form of urea with Agrotain nitrogen stabilizer was applied to corn plots only at the rate of 180 kg/ha in 2009, and 225 kg/ha in 2011. No other fertilizers were applied in soybean and corn during the 4yr. Corn and soybean were planted between late May and early June, and harvested between early October and mid-November depending on the soil and weather conditions each year. The SCN-resistant soybean cultivar was Latham EX547 RR N (PI 88788 source of resistance), SCN-susceptible soybean cultivar was Pioneer brand 92B13, and corn cultivar was DeKalb 46–61. All of the soybean and corn cultivars were resistant to glyphosate (2-phosphonomethylamino acetic acid), and glyphosate was used for both pre-emergence and post-emergence weed control. No insecticide or additional fungicide was used.

A soil sample consisting of 20 soil cores (2-cm diameter, 20 cm deep) was collected from each subplot in a systematic pattern across the two central rows at planting to assess initial (Pi), midseason (Pm) approximately 2 months after planting), and final population (Pf) at harvest. The soil was passed through a 5-mm aperture sieve and mixed thoroughly. The soil samples were stored in a cool room (4 °C) before being processed. Cysts were extracted from a subsample of 100 cm³ of soil with a semiautomatic elutriator (Byrd et al., 1976) and separated from soil particles and debris with centrifugation in a 63% (w/v) sucrose solution (Chen and Liu, 2005). Eggs were released from the cysts mechanically (Faghihi and Ferris, 2000) and collected in a 50-mL tube. The number of eggs was counted in a 0.5 to 2.0 mL aliquot, depending on the egg population density, and the total number of eggs in 100 cm³ of soil was derived. Soybean yields were measured from a 4.57-m length of the two central rows with a small plot combine. The soybean yield was standardized at 13% moisture.

To validate soil suppressiveness of each field treatment, soil samples from each plot were collected at midseason each year from 2009 to 2012 for a greenhouse assay. However, the results of 2011 were not reported because of insufficient SCN infection of soybean in the greenhouse. Approximately 4.5 kg soil was taken from 10 locations in each plot systematically to depth of 15 to 20 cm with a shovel. Each of the soil samples was passed through 5-mm aperture sieve, mixed thoroughly, and divided into three subsamples. Each subsample received one of three treatments: (i) 100% autoclaved field soil, (ii) 10% autoclaved field soil + 90% untreated field soil, and (iii) 100% field soil. The soil was autoclaved for 1 hr at 121°C.

An isolate of SCN HG Type 2.5.7 cultured on SCN-susceptible soybean in pots with autoclaved soil in the greenhouse was used as inoculum. The eggs of this population were extracted from the soil utilizing a similar method as described above, and then hatched in 4 mM ZnCl₂ hatching solution (Chen et al., 2000a, 2000b). J2 that hatched within the second and fifth day were collected and rinsed thoroughly as inoculum.

Each subsample of soil was placed in a 15-cm-diameter pot. Seven soybean ‘Freeborn’ (PI 88788 source of resistance) seeds, that had been treated with 0.5% NaOCl for 3 min, were sown in each pot. Freeborn, which was susceptible to the population of HG 2.5.7 with a female index of 65% but resistant to the SCN populations (HG type 0) from the field plots, was used to minimize the effect of initial populations in the untreated soil. Pots were arranged in completely randomized blocks and maintained in the growth room with an average temperature of 28°C (range 20–30°C). After 1 wk, the plants were thinned to provide four plants, and 5,000 SCN J2 were added in six holes, 3 cm deep, around the soybean plants in each pot. The soil was supplied with a P-N-K fertilizer (0.08 g P₂O₅ + 0.0.08 g N + 0.04 g K₂O/pot) after 4 wk of planting to minimize the effects of autoclaving on soil fertility.

Plant heights and total dry shoot weights per pot were measured at the termination of the experiments (60 d after inoculation). After cutting soybean shoots at the soil surface, the soil ball was broken and thoroughly mixed. Nematode egg population densities were determined with the procedures described previously (Chen and Liu, 2005).

The general linear model (GLM) procedure in Statistical Analysis System (SAS) Version 9.2 (SAS Institute, Cary, NC) was used to perform the split-plot analysis of variance (ANOVA). Dependent variables were evaluated for normality and transformed as necessary before performing the ANOVA. Significant differences were reported at P < 0.05 unless otherwise stated. In the field experiments, egg population density was transformed by x ^0.2 to x ^0.5 and yield data were not transformed. In contrast, egg population density was transformed by log(x), and shoot dry weight data were not transformed in the greenhouse experiment.

There were no interactions between the tillage and crop-biocide treatments except for 3 of the 12 sampling times (Table 1). Significant interactions were observed at harvest 2010 and midseason 2012 at Site 1 and at midseason 2010 at Site 2 (Tables 1 and 2). There were sporadic differences between tillage for individual crop sequences and no consistency between sites or among years. For example, tillage increased SCN population density in streptomycin treated plots in fall 2010, and C-R-C-S with no biocide treatments in the midseason 2012 at Site 1. Tillage also increased egg population density in streptomycin treated plots in the midseason 2010 at Site 2. In contrast, conventional tillage decreased SCN population density in the S-S-S-S, captan treatment in the midseason 2012 at Site 1, and C-R-C-S, no biocide treatment in the midseason 2010 samples.

When data were analyzed for individual tillage treatments, the SCN population density was always greatest in the formaldehyde treatment at Site 1 (Table 2). Captan increased the SCN population density in the no-tillage plots at Site 1 only in the midseason of 2012 (Table 2). Streptomycin did not affect SCN egg population density at these three sampling occasions (Table 2). The C-R-C-S treatment reduced egg densities consistently. The SCN egg population density was progressively less in susceptible soybean grown in monoculture with conventional tillage, monoculture with no-tillage, and rotated with corn with conventional tillage (Table 2). In contrast, SCN egg population density was less in the no-till than conventional tillage in the C-S-C-S rotation at midseason 2012 at Site 1 (Table 2). The one year of corn in 2009 reduced SCN population density at the midseason 2010 susceptible soybean in both tillage treatments at Site 2 (Table 2).

Conventional tillage increased soybean yield 3.89% in 2010 at Site 1 (Fig. 1A) and decreased soybean yield 7.38% in 2009 at Site 2 (Fig. 1C). No tillage effect on soybean yield was detected in any other year (Fig. 1A,C). At Site 1, biocide-crop sequence affected soybean yield only in 2010, resistant soybean plots had greater yield than susceptible soybean, but the plots with formaldehyde treatment had similar yield as resistant soybean (Fig. 1B). In the same year (2010), yield was less in monocultures of susceptible soybean treated with streptomycin than in susceptible soybean following corn or resistant soybean following corn (Fig. 1D). In 2012, the soybean yield was greater in plots with corn-soybean rotation than soybean monoculture, regardless of biocide treatments (Fig. 1D).

Figure 1:

Soil from both sites treated with autoclave-heating dramatically reduced the level of nematode suppression as it increased SCN egg population densities in the soil two months after planting (Fig. 2A,B). The smallest egg population density was generally in the 100% untreated field soil. Adding 10% of the untreated field soil into autoclaved soil also dramatically reduced SCN egg population density as compared with the autoclaved soil. The treatment of 10% field soil mixed with 90% autoclaved soil had greater egg population densities in 2009 and 2010, but slightly reduced egg population densities in 2012, as compared with the 100% field soil (Fig. 2A,B). No crop rotation, tillage, or biocide effects on the egg population density were detected in the greenhouse assays. Neither was there a difference in plant dry weights in 2009 and 2010, but untreated field soil had greater dry weight than the other two treatments containing 10% or no untreated soil in 2012 (Fig. 2C,D).

Figure 2: