Reniform Nematode Management Using Winter Crop Rotation and Residue Incorporation Methods in Greenhouse Experiments

The objectives were investigated in a repeated pot experiment conducted in a polycarbonate greenhouse at the University of Florida Entomology and Nematology Department in Gainesville, FL. Two trials, Trial 1 and Trial 2, were performed in a completely randomized design, with six replicates that utilized winter crops and method of crop residue incorporation as the only factors. A total of 10 treatments (Fig. 1) were evaluated: three winter crops (carinata, oat, or hairy vetch) grown in rotation with 1) no shoot OM incorporated (1–3), 2) fresh shoot OM incorporated (4–6), or 3) dry shoot OM incorporated (7–9); and a fallow control (10). This experiment simulated crop rotation without shoot incorporation (harvested), incorporating fresh residue, and incorporating dried residue. For each treatment, fresh roots were reincorporated because this mimics field production practices. Crops evaluated were the major variety of carinata available at the time of experiment establishment, ‘Avanza 641’ (Nuseed Co., Sacramento, CA), and unknown varieties of hairy vetch and oat.

Figure 1:

The RN population for this research came from a naturally infested field in Tift County, Georgia, and was maintained in cotton plants in greenhouse conditions. The inoculum was obtained based on Hussey and Barker (1973), as explained in Sandoval and Grabau (2023). Soil used in this experiment was autoclaved at 121 °C for 90 minutes in an Amsco Lab (OH, USA) 250 LV autoclave. The soil was a Chipley-Foxworth-Albany complex (91% sand, 6.8% silt, and 2.4% clay with 1.7% OM) from the University of Florida North Florida Research and Education Center–Suwannee Valley near Live Oak, FL.

The experiment had three stages: 1) establishment, 2) winter crop residue application, and 3) evaluation (Fig. 1). During the first stage, winter crops were planted in clay pots (15 cm top diameter) containing 1000 cm³ of autoclaved soil that was mixed in a plastic bag with 3500 RN (eggs and juveniles). Nematodes were inoculated into each pot on the same day as inoculum extraction. In the second stage, at winter crop termination, the entire plant was removed from the pot. For all treatments, the roots were cut into pieces smaller than 0.5 cm and were reincorporated into the same pot with the original soil from the first stage. The plant shoot was discarded for treatments without OM incorporation. For the dry OM treatments, after harvesting the winter plants, the shoot was placed in paper bags in an oven (SPX, Blue M Electric Co, Blue Island, IL) for three days at 60°C (140° F). For the dry OM and fresh OM treatments, the dry or fresh shoots of the winter crops were ground into small particles in a domestic coffee grinder. Subsequently, the OM was mixed in a plastic bag with the soil from the same pot as the first stage of the experiment. Pots were placed in the greenhouse and after 12 days, cotton seeds (‘Deltapine 1646B2XF’, Bayer CropScience, St. Louis, MO) were planted directly into the soil and left to grow until harvest, two months later. Information about trial schedules is provided in Table 1.

The final plant densities of each crop were based on recommended planting density for field production as adapted to 15-cm-diameter pots as measured at the top of the pot (area= 176.7 cm²). Therefore, the final density for the first stage was six oat, two carinata, two canola, or one hairy vetch per pot (Seepaul et al., 2019; Harker et al., 2015; Forsberg and Reeves, 1995; Bamford and Entz, 2016). Winter crops were initially seeded at double the recommended density, but one week after planting, pots were thinned to the final density specified for each crop. Cotton in the second stage of the experiment was also seeded at double the recommended planted density but 5 days after planting was thinned to the recommended density of 2 plants per pot.

The soil temperature was measured using a HOBO MX TidbiT 400 (Onset Computer Corporation, MA, USA). The average temperature while the winter crops were growing and incorporated was 19.6°C (max=36.8°C, min=6.3°C) for Trial 1, and 21.4°C (max=48.8°C, min=8.0°C) for Trial 2. The temperature when the cotton plants were growing was 23.4°C (max=46.0°C, min=8.2°C) for Trial 1, and 25.6°C (max=46.0°C, min=11.0°C) for Trial 2. No supplemental light or fertilizer was used, and plants from all treatments were watered daily by hand.

Subsamples of 100 cm³ soil for nematode extraction were taken after winter crops and cotton were harvested. Nematodes were also extracted from the entire cotton root system at harvest and cotton root weight was measured to calculate RN density on roots. Nematode extractions from roots were done using the same method for the inoculum preparation as previously explained, while the extraction from soil was done by the sucrose centrifugation method (Jenkins, 1964). Nematode counting and identification were done using a 400X inverted Primovert microscope (Carl Zeiss Inc, Thornwood, NY). Reniform nematode soil abundances (vermiform nematodes) at winter crop and cotton termination, RN population densities (eggs and vermiform nematodes) per gram of roots, and reproduction factor (RF, RF= final population/initial population, final population= RN eggs and vermiform in soil and roots) (Seinhorst, 1967) are reported.

Plant growth assessments were made at termination of winter crops and cotton. For winter crops, the total fresh weight of crop residues to be incorporated for each respective treatment was measured. Only root weight was measured for rotation with no OM incorporation treatments, but root and shoot fresh weights were measured for rotation with fresh OM or dry OM treatments. Additionally, the total OM rate applied per pot (%w/w) was calculated for each treatment ((∑OM matter applied*100)/1500 g soil). For the dry OM treatments, the ∑OM matter applied = dry shoot weight+ fresh root weight. For the fresh treatments: ∑OM= fresh shoot weight + fresh root weight. For the no OM treatments, ∑OM= fresh root weight. For this experiment, 1500 g soil was equal to 1000 cm³ soil. The variables measured for cotton plants were height, root fresh weight, shoot fresh and dry weight, Soil Plant Analysis Development (SPAD), and canopy cover. SPAD values were taken on the third fully expanded leaf from the apex using a SPAD-502 chlorophyll meter (Konica Minolta Sensing Inc., Osaka, Japan). To assess the canopy cover, each pot was placed in the center of a 1 m² white cardboard, and photos were taken at 1 m height, parallel to the top of the pot. Pictures were cut to only show 1 m² using the computer imaging program GIMP (GNU Image Manipulation Program) 2.10.20 (The GIMP Team, Berkley, CA) and were evaluated using Canopeo (Patrignani and Ochsner, 2015).

Data analysis was done in RStudio version 2021.09.0 (The R Foundation for Statistical Computing, Vienna, Austria). To ensure ANOVA model assumptions were met, normality was assessed by the Shapiro-Wilk test, and homogeneity of variances by Levene's test. For nematode and plant parameters, trials were analyzed separately because of trial-by-treatment interactions (ANOVA, P ≤ 0.05). Plant parameters that did not meet the statistical assumptions were square root or ln(x+1) transformed to meet them. The % w/w OM applied per pot for the rotation treatments with fresh or dry OM incorporation and the plant parameters were analyzed by one-way ANOVA. For the winter crops and cotton, the plant parameters with significant treatment effects (ANOVA, P ≤ 0.05) were analyzed by Tukey's honesty significant test (HSD). Nematode variables were analyzed by Kruskal-Wallis non-parametric test because variables did not meet ANOVA model assumptions. For nematode variables, means were separated by multiple comparisons with Bonferroni's method at a 5% significance level, if the Kruskal-Wallis's test was significant (P < 0.05). Nematode soil populations at winter crop termination were analyzed by crop only because crop-OM incorporation treatment combinations had not been implemented yet. Data is displayed as boxplots in the figures. The black center line represents the median (50^th percentile), the lower limit of boxes symbolizes the 25^th percentile, the upper limit of boxes the 75^th percentile, whiskers indicate the 5^th and 95^th percentiles, and outliers denote values beyond the upper and lower whiskers and are indicated as black dots.

Winter crops are significantly affected by RN abundance in the soil at winter crop termination. The abundance of RN in 100 cm³ soil was higher in carinata and hairy vetch than in fallow and oat in Trial 1 (Fig. 2A). In Trial 2, consistent with Trial 1, hairy vetch had greater RN abundance than fallow and oat, but in this trial, carinata had intermediate values (Fig. 2B).

Figure 2:

Crops significantly affected RN abundance in the soil at cotton termination, but the incorporation type did not have a consistent effect. In general, oat and fallow managed final RN soil abundances better than carinata and hairy vetch. In Trial 1, oat no OM and dry OM had lower soil RN abundance relative to fallow (Fig. 3A). No OM from carinata, and no OM and dry OM from hairy vetch had greater RN abundances than fallow, and all other treatments were similar to fallow. In Trial 1, RN soil abundance did not vary by incorporation type in oat, but carinata and hairy vetch did have greater RN soil abundance for no OM than fresh OM and intermediate abundance for dry OM (Fig. 3A). In Trial 2, all of the treatments had similar or greater soil RN abundance relative to fallow (Fig. 3B). The oat treatments, for all incorporation types, were not different from fallow. However, in Trial 2, all carinata and hairy vetch treatments increased RN abundance relative to fallow (Fig. 3B). In Trial 2, there were no differences in the final RN soil abundance among OM incorporation types within each crop (Fig. 3B).

Figure 3:

In general, RN root abundances varied by crop but did not consistently vary by incorporation treatment (Fig. 4). Consistently in both trials, fresh OM from oat was the most effective treatment to manage RN root abundances and no OM and dry OM from hairy vetch were the least effective. In Trial 1, RN root abundances were generally lower in the oat treatments, greater in the no OM and dry OM from hairy vetch, and intermediate in the other treatments (Fig. 4A). The no OM and dry OM from hairy vetch had greater RN root abundance than fallow, and all other treatments were statistically similar to fallow (Fig. 4A). In Trial 2, the fresh OM from oat and fallow had generally lower RN abundances in the roots, while the no OM and dry OM from carinata and hairy vetch had the greatest abundances, and all the other treatments had intermediate values (Fig. 4B). In this trial, none of the oat treatments were different from fallow but all the carinata and hairy vetch treatments had greater RN abundances than fallow (Fig. 4B). In both trials, there were no differences in RN root abundances among OM incorporation types within each crop (Fig. 4).

Figure 4:

Crops significantly impacted the RN reproduction factor (RF), but incorporation treatment did not consistently affect RF (Fig. 5). Oat and fallow were generally good whereas hairy vetch no OM was generally poor for managing RN based on RF, with other treatments intermediate or variable. In Trial 1, the fresh OM and dry OM from oat were the only treatments that reduced RF relative to fallow (Fig. 5A). In Trial 1, RF did not vary by incorporation treatment within oat, but within carinata and hairy vetch, RF was greater with no OM than fresh OM and intermediate for dry OM (Fig. 5B). In Trial 2, RF was less for fallow than most treatments and the oat treatments had RF that, statistically, was similarly as low as fallow. In this trial, none of the treatments reduced RF compared to fallow, and any of the treatments from carinata and hairy vetch had greater RF than fallow (Fig. 5B). In this trial, the incorporation type, within crop, did not have any influence on the RF (Fig. 5B).

Figure 5:

In Trial 1, the fresh weight of residues was greater for oat than hairy vetch or carinata independently of the incorporation treatment but in Trial 2 there was more variation among treatments (Table 2). In Trial 2, oat without OM had greater fresh residue weight than any dry OM treatment or fresh hairy vetch (Table 2). The final OM rate applied per pot varied by treatment (Table 2). In Trial 1, OM rate was greater for oat than any other crop, regardless of incorporation type (Table 2). In Trial 2, the OM rate applied per pot was greater for fresh oat and carinata than most other treatments (Table 2).

In Trial 1, all plant parameters, except the shoot dry weight, were affected by treatments (Table 3). In Trial 2, the fresh shoot weight was the only plant parameter affected by treatments (Table 3). Therefore, in general, there were no consistent treatment effects on plant growth for the two trials (Table 3). In Trial 1, the canopy cover, height, and shoot fresh weight were each greater for carinata no OM and fresh OM than for the oat fresh OM. Cotton height and shoot fresh weight were also greater following carinata no OM and fresh OM than oat dry OM (Table 3). The root fresh weight did not have consistent results between trials (Table 3). In Trial 1, the root fresh weight of the cotton plants was greater following all oat treatments compared to the dry and fresh OM from hairy vetch and the fallow treatment, with all the other treatments intermediate (Table 3). In Trial 2, carinata fresh OM and hairy vetch no OM had greater root fresh weight than carinata dry OM and fallow; all other treatments had intermediate values (Table 3). In Trial 1, SPAD was greater following fallow than carinata no OM and hairy vetch treatments (Table 3). Additionally, oat no OM had a greater SPAD value than the dry or no OM treatment from hairy vetch. There were no differences in SPAD among the other treatments (Table 3).