Mustard cover crop

The mustard cover crop in this study was a proprietary mixture of ‘Caliente Rojo’ brown mustard and ‘Nemat’ arugula (Eruca sativa) (High Performance Seeds, Inc., Moses Lake, WA, USA). ‘Caliente Rojo’ brown mustard is marketed to contain high concentrations of glucosinolates. ‘Nemat’ arugula is a trap crop for certain nematodes (Meloidogyne sp.). It suppresses population growth of certain nematodes by not allowing nematodes to reproduce (Melakeberhan et al. 2006). Although ‘Nemat’ arugula was included in the proprietary mixture, nematode responses to mustard cover crops were not within the scope of this study.

Sites and treatments

The study was conducted at four sites. Three sites were commercial fields managed by farmer cooperators. The fourth site was a New Mexico State University research farm. The commercial fields were near Columbus, NM, USA (lat. 31.797°N, long. 107.857°W); Deming, NM, USA (lat. 32.225°N, long. 107.775°W); and Las Uvas, NM, USA (lat. 32.605°N, long. 107.350°W). The university research farm was the Leyendecker Plant Science Research Center [hereafter, Leyendecker (lat. 32.202°N, long. 106.743°W)] near Las Cruces, NM, USA. The nearest two study sites were separated by 51 km. At Columbus, the study site featured sandy loam soil (Jal series, fine-loamy, carbonatic, thermic Typic Haplocalcids). At Las Uvas, the study site featured silty clay loam soil (Verhalen series, fine, smectitic, thermic Typic Haplotorrerts), and at Deming, the study site featured loam soil (Gila series, loamy, mixed, superactive, calcareous, thermic Typic Torrifluvents). At Leyendecker, the study field featured clay loam soil (Glendale series, fine-silty, mixed, superactive, calcareous, thermic Typic Torrifluvents).

The mustard cover crop was seeded at 7 lb/acre, as recommended by the seed company (High Performance Seeds, Inc. 2022). At Columbus, the mustard cover crop was seeded into raised beds using a mechanical planter, whereas at Deming, Las Uvas, and Leyendecker, the mustard cover crop was seeded on flat ground using a mechanical grain drill. Raised beds at Columbus were listed rows spaced 38 inches apart and with top ridges smoothed. For each raised bed, there was one row of mustard cover crop. After seeding and throughout the cover crop growing season, fields were irrigated three to four times as needed. At Columbus, Deming, and Las Uvas, fields were irrigated with a subsurface drip. At Leyendecker, the field was flood-irrigated.

At each site, the mustard cover crop was compared against bare ground. These treatments (mustard cover crop, bare ground) were arranged in parallel plots and replicated three times at Columbus, Deming, and Leyendecker. At Las Uvas, parallel plots of mustard cover crop and bare ground were replicated twice. Treatment plot dimensions differed among study sites. At Deming, treatment plots were 394 ft long and 40 ft wide. At Leyendecker, plots were 131 ft long and 13 ft wide, and at Columbus plots were 1148 ft long and 59 ft wide. At Las Uvas, treatment plots were 951 ft long and 66 ft wide. Within each mustard cover crop plot, six equally spaced sampling locations were established along the longitudinal axis. Each sampling location was paired with a sampling location in the adjacent bare-ground plot.

To end mustard cover crops, aboveground biomass was mowed and incorporated into the soil by tillage. Tillage programs for termination included furrow plowing (Columbus only), disking, and rotary tilling. Termination concluded with the creation of raised beds for direct-seeded chile pepper. Within 48 h of cover crop termination, study sites were irrigated using a subsurface drip at Columbus, Deming, and Las Uvas, and flood-furrow irrigation at Leyendecker. Table 1 provides information on cover crop seeding and termination dates.

Table 1.Mustard cover crop seeding and termination dates for the four sites in this study.

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Data collection for objective 1

Two months after cover crop planting, mustard stand densities and percentages of ground occupied by mustard cover crops were determined with 0.25-m² rectangular quadrats at each sampling location (18 sampling locations at Columbus, Deming, and Leyendecker; 12 sampling locations at Las Uvas). Also at each sampling location and just before cover crop termination, aboveground biomass of weeds and cover crop were harvested from 0.25-m² quadrats. Weed biomass and cover crop biomass were bagged separately and oven-dried at 65 °C for 72 h.

Data collection for objective 2

Seeds of Palmer amaranth were collected from Leyendecker in Aug 2018. Seeds were obtained by hand-clipping seed-bearing inflorescences. These inflorescences were dried under room conditions for 14 to 20 d. Dried inflorescences were hand-threshed, and sequential combinations of sieving and forced-air separation were used to separate seeds from chaff. Seeds were then stored in an airtight container at 4 °C. Before use in our study, seeds were assayed for viability using a 1.0% aqueous solution of tetrazolium [2,3,5-triphenyl chloride (Association of Official Seed Analysts 2000)]. Tetrazolium assay results indicated that 96% of the Palmer amaranth seeds were viable at the onset of our study.

Packets (2 × 3 cm) containing 50 Palmer amaranth seeds were made using nylon netting (No-See-Um Netting; Equinox Ltd., Williamsport, PA, USA). The netting was sealed using a heat sealer. Packets were buried at each sampling location after the cover crop was ended, but before the irrigation that occurred after cover crop termination. Packets were buried at a depth of 2 inches. One day before chile pepper seeding, which was 20 to 24 d after packet burial, packets were recovered from the field and brought to the laboratory. At the laboratory, packets were opened and seeds were placed on moistened filter papers in petri plates. Petri plates were placed under a stereoscope and individual seeds were pressed gently using forceps. Seeds that did not collapse under gentle pressure were considered viable and are hereafter referred to as persistent. Responses to gentle pressure have been used for assessments of weed seed viability (Borza et al. 2007; Khan et al. 2022), including assessments of seed viability for Palmer amaranth (Korres et al. 2018). To our knowledge, previous reports for pressure assessments of seed viability did not include information on the amounts of force applied to individual seeds. For information on the amount of force required to rupture weed seedcoats, see Davis et al. (2016).

Persistent seeds were subjected to germination assays conducted in petri plates that were placed in a chamber set to 35/25 °C (day/night) with a 14-h photoperiod. At 2-d intervals for 14 d, germinated seeds were counted and removed. At the conclusion of the 14-d germination assay, seeds that did not germinate were assayed for viability using a 1.0% aqueous solution of tetrazolium. Results from viability assays were used to adjust quantities of persistent seeds determined with forceps and gentle pressure. Specifically, the number of nonviable seeds within each packet was subtracted from the number of persistent seeds determined with forceps and gentle pressure.

After termination of cover crops, but before irrigation, soil from each sampling location in cover crop and bare-ground plots was collected from the top 7 cm using a hand shovel. These soil samples were used in a study conducted in a greenhouse at Leyendecker. Throughout the study, the greenhouse was set to maintain an air temperature of 24 ± 4 °C. Soil from each sampling location in cover crop and bare-ground plots was dispensed into cylindrical plastic pots (diameter, 9 inches; depth, 8.5 inches), which produced pots with and pots without mustard biomass in the soil. After filling, pots were irrigated to field capacity. With no further irrigation, soils were incubated for 4 weeks under greenhouse conditions. After the 4-week incubation period, ‘NM 6–4’ chile pepper plants at the two-leaf stage were transplanted into pots (one plant per pot). Chile pepper plants were watered with a sprinkler canister daily and fertilized once at the 10-leaf stage with 5N–4.4P–8.3K fertilizer. Plants were monitored for symptoms of Verticillium wilt every 7 d. Verticillium wilt is caused by a soil-borne fungus, Verticillium dahliae. It is prevalent in chile pepper fields across southern New Mexico (Sanogo and Carpenter 2006) and was observed at study sites before our study. Symptoms of Verticillium wilt include wilting, even when plants receive adequate watering (Fig. 1), followed by foliar chlorosis and premature senescence (Goldberg 2010).

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The greenhouse study was ended 60 d after transplanting, which is when chile pepper plants started bearing fruit. Data collected at termination included plant height, fresh and dry biomass of shoots, and dry biomass of roots. Before collecting data on roots, plants were placed on a 10-mm mesh screen and roots were washed carefully with water. Root and shoot dry weights were determined after biomass was dried in an oven for 72 h at 65 °C.

Data analysis

All statistical analyses were performed using R (version 4.1.0; R Foundation for Statistical Computing, Vienna, Austria). For objective 1, site effects on mustard stand densities and ground coverage at 2 months after seeding were determined with analysis of variance (ANOVA) followed by tests for least significant difference. ANOVA was also used to assess site effects on mustard cover crop biomass at termination. For presentation purposes, data on mustard cover crop biomass were converted to pounds per acre because this unit of yield is commonly used in management guides for cover crops (Clark 2007). To determine site effects on weed biomass at cover crop termination, data were analyzed with a Kruskal-Wallis test followed by post hoc Conover tests for nonparametric data using the R library PMCMRplus. For objective 2, quantities of persistent Palmer amaranth seeds were converted to percentages of seeds buried. Quantities of germinated seeds were converted to percentages of persistent seeds. Percentage data for seed persistence and germination were analyzed with generalized linear mixed models fitted with a binomial distribution using the R library lme4. Models were developed separately for each site. In these models, treatment (mustard cover crop, bare ground) was the fixed effect; the sampling location within a replication was the random effect. For the greenhouse study, data on chile pepper plant size were sorted by study site and analyzed separately with paired t tests. These tests compared plants grown in soil with mustard biomass against plants grown in soil without mustard biomass.

Objective 1: Determine whether a mustard cover crop seeded in fall produces enough biomass for biofumigation in early spring

At 2 months after cover crop planting, mustard stand densities and ground coverage differed among study sites (Table 2). Mustard stand densities were greatest at Leyendecker (241 plants/m²) and lowest at Las Uvas (32 plants/m²). Mustard stand densities at Columbus (85 plants/m²) and Deming (106 plants/m²) were less than the mustard stand density at Leyendecker but greater than the mustard stand density at Las Uvas. The percentage of ground covered by the mustard cover crop at 2 months after cover crop seeding was greatest at Deming (85%) and Leyendecker (89%), and lowest at Las Uvas (5%). The percentage of ground covered by the mustard cover crop at Columbus (50%) was less than the percentage of ground covered at Deming and Leyendecker, but greater than the percentage of ground covered at Las Uvas. Low stand density and low ground coverage for the mustard cover crop at Las Uvas was associated with frost shortly after seeding. Moderate stand density and moderate ground coverage for the mustard cover crop at Columbus corresponded with late planting; however, the Columbus site did not receive frost shortly after cover crop seeding.

Table 2.Stand densities and groundcover for a cover crop mixture of ‘Caliente Rojo’ brown mustard and ‘Nemat’ arugula at 2 moths after cover crop seeding.

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At termination, mustard biomass differed among sites (Table 3). The mustard cover crop at Leyendecker produced the maximum amount of biomass in this study (598 g⋅m^–2); the mustard cover crop at Las Uvas produced the least amount of biomass (125 g⋅m^–2). For three sites (Columbus, Deming, and Leyendecker), the range of mustard cover crop aboveground biomass at termination was comparable to previous reports of mustard cover crop biomass production in regions outside of New Mexico. For example, brown mustard and white mustard (Sinapis alba) seeded at 8.9 lb/acre in fall and spring in the U.S. Great Lakes region had ∼1605 to 4730 lb/acre of dry aboveground biomass at termination (Björkman et al. 2015). Brown mustard seeded at 7.1 lb/acre in summer in France had ∼1965 to 3390 lb/acre of dry aboveground biomass at termination (Motisi et al. 2009). However, a mixture of brown mustard and white mustard seeded at 19.6 lb/acre in fall in California had ∼9800 lb/acre of dry aboveground biomass at termination in early spring (Brennan and Smith 2005), suggesting that the cover crop seeding and production procedures in our study did not sustain maximum levels of mustard cover crop biomass.

Table 3.Dry aboveground biomass for a cover crop and weeds co-occurring in quadrats at cover crop termination.

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Weed biomass at cover crop termination was lower at Deming (0.1 g⋅m^–2) and Leyendecker (0 g⋅m^–2) than Columbus (4.2 g⋅m^–2) and Las Uvas (5.0 g⋅m^–2; Table 3). Deming and Leyendecker also featured relatively high levels of cover crop ground coverage at 2 months after cover crop seeding (Table 2), which suggests early-season measurements of cover crop ground coverage foreshadow cover crop suppression of co-occurring weeds at termination. At sites where the mustard cover crop was well established (Columbus, Deming, Leyendecker), weed biomass at cover crop termination was less than 1% of mustard biomass. These results were generally consistent with previous studies that reported mustard cover crops were more competitive than weeds (Björkman et al. 2015; Brennan and Smith 2005).

In a previous study in Washington state, white mustard produced 3747 to 4014 lb/acre of aboveground dry biomass by the time of termination (Al-Khatib et al. 1997). After incorporation into the soil, the white mustard biomass reduced weed density by 17% at 30 d after planting pea. In the same study, rapeseed (Brassica napus) produced 5620 to 7315 lb/acre of dry biomass, and after incorporation into the soil, the rapeseed biomass reduced weed density by 34% in pea (Al-Khatib et al. 1997). In potato, biofumigation with 3658 to 5175 lb/acre of dry rapeseed biomass reduced weed densities by 73% to 85% (Al-Khatib et al. 1997). These results from a previous study (Al-Khatib et al. 1997) suggest that mustard cover crops require 3658 to 7315 lb/acre of dry aboveground biomass at termination to suppress pests in subsequent cash crops. According to this literature-based estimate for biomass requirements, and considering the results of our study, a fall-seeded mustard cover crop in southern New Mexico produces enough biomass for biofumigation in early spring, provided the mustard cover crop does not experience frost shortly after seeding.

Objective 2: Determine whether biomass from an overwinter mustard cover crop inhibits a weed, but does not affect chile pepper plants adversely

At sites where the mustard cover crop was well established (Columbus, Deming, and Leyendecker), incorporated mustard biomass reduced the number of viable Palmer amaranth seeds in the soil (Fig. 2A). Palmer amaranth seeds that persisted in soil with mustard biomass had lower rates of germination than seeds retrieved from the soil without mustard biomass (Fig. 2B), which suggests that mustard cover crop biomass induced secondary dormancy of Palmer amaranth seeds. These results are consistent with previous studies that indicated 1) compounds derived from glucosinolates strongly suppressed Palmer amaranth seedling emergence (Norsworthy and Meehan 2005) and 2) germination of redroot pigweed was completely inhibited by pesticidal compounds analogous to compounds derived from decaying mustard biomass (Teasdale and Taylorson 1986).

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Under greenhouse conditions, chile pepper plants grown in soil with mustard biomass were larger than chile pepper plants grown in soil without mustard biomass (Table 4). Mustard-induced increases in chile pepper plant size were especially prominent in roots, as root dry weights were up to 160% greater in soil with mustard biomass compared with soil without mustard biomass. Mechanisms by which mustard cover crop biomass could have enhanced chile pepper plant growth include increased soil nitrogen (Brennan et al. 2013; Weinert et al. 2002), increased soil organic matter (Agarwal et al. 2022a), reduced soil pH (Rudolph et al. 2015), and suppression of soil-borne pathogens (Larkin and Griffin 2007), including V. dahliae (Subbarao and Hubbard 1996). Suppression of soil-borne pathogens may have been an important causal factor for enhanced plant growth in our study because chile pepper plants in soil with mustard biomass did not exhibit symptoms of Verticillium wilt, whereas Verticillium wilt symptoms were prevalent in chile pepper plants grown in soil without mustard biomass (Table 4).

Table 4.Shoot fresh weight, shoot dry weight, root dry weight, and Verticillium wilt incidence for chile pepper plants grown in different soils under greenhouse conditions.

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The general absence of phytotoxicity from mustard biomass in our study was consistent with previous studies that indicated radish (Raphanus sativus) biomass did not inhibit the emergence of lettuce [Lactuca sativa (Lawley et al. 2012)], and white mustard biomass promoted the growth of onion and celery [Apium graveoens (Wang et al. 2010)]. However, greenhouse results in our study were somewhat inconsistent with results from a field study by Rudolph et al. (2015). Rudolph et al. (2015) determined that brown mustard cover crops seeded in September and ended in November or December did not influence vegetative biomass of chile pepper that was seeded the following April or May, although two brown mustard cultivars (Caliente 199 and Pacific Gold) increased fruit yield for the second of two harvests in one of two experimental runs. Inconsistent results for mustard effects on chile pepper between the results of the study of Rudolph et al. (2015) and our study may reflect inherent environmental differences that complicate comparisons between field and greenhouse studies. Also, inconsistent results between Rudolph et al. (2015) and our study may have been caused by differences in termination times for the fall-seeded mustard cover crops. In our study, fall-seeded mustard cover crops were ended in February or March, whereas in Rudolph et al. (2015), fall-seeded mustard cover crops were ended in November or December. Thus, the inconsistent results between Rudolph et al. (2015) and our study suggest that promotion of chile pepper growth with a biofumigant cover crop is more likely to occur when the cover crop is ended 20 to 24 d before chile pepper seeding, rather than 115 to 134 d before chile pepper seeding.

The promotional effects of mustard cover crop biomass on chile pepper plant size, combined with the inhibitory effects of mustard cover crop biomass on Palmer amaranth seed persistence and seed germination, suggest that biofumigation possibly provides at least two benefits to chile pepper production: 1) enhanced crop growth and 2) suppression of specific crop pests. This two-part hypothesis needs to be confirmed with further field studies. In addition to the cover crop mixture of ‘Caliente Rojo’ brown mustard and ‘Nemat’ arugula, cultivars and species in the mustard family that might be used for biofumigation in southern New Mexico include ‘Caliente 199’ brown mustard. A previous study in New Mexico indicated that an overwinter cover crop of ‘Caliente 199’ brown mustard improved soil quality by increasing the diameter of stable soil aggregates (Antosh et al. 2020). Another previous study in New Mexico determined that ‘Caliente 199’ brown mustard generally had greater concentrations of glucosinolates than ‘Caliente 61’ brown mustard and ‘Arcadia’ broccoli (Brassica oleracea var. italica), with glucosinolate concentrations in ‘Caliente 199’ brown mustard significantly greater in 1 of 2 years (Rudolph et al. 2015). Future studies should compare mustard cultivars, including Caliente Rojo and Caliente 199 brown mustards, for their potential as biofumigant cover crops supporting chile pepper production in New Mexico.

Possible benefits from biofumigation must be balanced against potential hazards from the mustard cover crop. Notably, brown mustard cover crops could increase population densities of southern root-knot nematode [Meloidogyne incognita (Rudolph et al. 2015)]—a chile pepper pest that reduces fruit yield. Also, mustard family plants in the southwestern United States are hosts to Beet curly top virus and its vector, the beet leafhopper (Circulifer tenellus) (Creamer et al. 2003, 2005). If beet leafhoppers on a mustard cover crop persist in the local environment after the cover crop is ended, these leafhoppers may transmit the virus to cause curly top disease in chile pepper. Direct evidence of Beet curly top virus transfer from mustard cover crops to chile pepper via beet leafhopper is lacking, but further research is needed.