1. Introduction
Nitrogen (N) management on organic farms can be difficult due to the nature of certified organic fertilizers. Organic farmers use compost, manure, legume cover crops, dried organic products such as feathers and blood meal, or liquid fertilizers such as fish emulsion to increase crop productivity. Additionally, there are many specialty products on the market containing plant growth regulators or phytohormones, intended to impact plant growth characteristics to increase yield and/or quality. The use of these fertilizers is often imperfect; liquid fertilizers and meals are energy intensive to produce and ship, and the yield impacts of specialty products are often inconclusive [
1,
2,
3]. Nitrogen mineralization rates of manures and meals are difficult to predict or control [
4,
5]. To improve the predictability of N inputs, organic farmers often turn to fertilizers such as liquid fish fertilizer to supply their crops with supplemental N mid-season. Purchasing and transporting fertilizers such as fish fertilizer can be costly and can have a large carbon (C) footprint, contrary to the aims of organic agriculture. By growing cyanobacterial fertilizer (cyano-fertilizer) organically on-farm, organic farmers can harness the N-fixing ability of these prokaryotes and potentially decrease the cost and C footprint of purchasing and transporting traditional organic fertilizers [
6,
7,
8]. While cyano-fertilizer has a lower N concentration than fish fertilizers, fish fertilizers are usually diluted prior to application, and both can be applied multiple times throughout the growing season through drip irrigation systems.
Phytohormones are marketed by manufacturers to stimulate plant growth when applied externally. Organic farmers can purchase products such as liquid seaweed extract to apply phytohormones to crop foliage or as a soil soak. Seaweed products are best known for their auxin and cytokinin contents. Studies have found that cyanobacteria can produce an elaborate array of secondary plant compounds, including auxins, cytokinins, and abscisic acid [
9,
10]. These compounds can affect the nutritional value and water use efficiency of plants [
11,
12]. Conclusions regarding the impacts of added phytohormones vary, and often the dosage and the location of application have different effects. In a study evaluating the response of tomatoes, carrots, and potatoes to a cytokinin-containing product, only the tomatoes produced a greater yield in response to the added cytokinin [
1]. Seaweed concentrate was reported to increase tomato seedling growth when used as a soil soak, while tomato yield was increased through foliar application [
13]. In another study, seaweed extract was applied to Russet Burbank potatoes for seven years with no increase in yield, but the Lemhi Russet variety showed a yield response in three of the five years [
14]. In our study, fish emulsions and cyano-fertilizer were applied to the soil with irrigation, and seaweed concentrates were applied foliarly to better understand the impacts of each on the carrots.
Carrot (
Daucus carota var. sativus) is a member of the Umbelliferae family [
15]. Carrots have a moderate N requirement and perform well with 100 to 135 kg available N ha
−1 [
15]. The timing of N application is of equal importance, as carrots must be “spoon-fed” N to prevent growth spurts and cracking [
15]. Carrots can take up 72–250 kg N ha
−1 depending on the soil conditions [
15]. In a dry year, net N removal for carrots was 72–81 kg ha
−1, which is lower than previously cited values of 150 kg ha
−1 in Finland and 178 kg ha
−1 in Michigan, USA [
16]. In the same study, carrot yields did not respond to an N rate of 110 kg ha
−1, and carrots usually had sufficient soil N without fertilizer application [
16]. Conversely, other researchers reported that the yield and quality parameters of carrots were maximized at 140–160 kg N ha
−1 depending on the planting date [
17].
In this study, carrots were grown in Fort Collins, CO, USA, in the 2014 and 2015 growing seasons to evaluate the impact of liquid organic fertilizers and foliar seaweed extracts on yield and quality. The specific objectives were to (1) evaluate cyano-fertilizer compared to hydrolyzed and non-hydrolyzed liquid fish fertilizers in providing adequate N to optimize the yield and quality of carrots without leaving excessive residual inorganic N in the soil post harvest, (2) characterize the impact of foliar liquid seaweed on carrot characteristics, and (3) evaluate the potential impact of phytohormones in seaweed, fish fertilizer, and cyano-fertilizer on carrots.
2. Materials and Methods
2.1. Site Description and Planting
Field experiments were conducted during the 2014 and 2015 growing seasons on certified organic land at the Colorado State University (CSU) Horticultural Research Center (4300 E County Road 50, 80524) in Fort Collins, CO, USA. The soil in this location is a fine, smectitic, mesic Aridic Argiustoll of the Nunn series [
18]. The soil pH was 8.1, and the organic matter content was 2.7% in the top 30 cm. The average maximum temperature during the growing season (1 May–30 September) was 26 °C in both 2014 and 2015, and the average minimum temperature was 18 °C in 2014 and 17 °C in 2015. There were 2.1 cm and 6.1 cm of rain during the growing season in 2014 and 2015, respectively. The plot location within the field was moved from year-to-year to avoid residual treatment effects. Pre-season inorganic soil N analyses (0–60 cm) were performed by Ward Laboratories Inc. in Kearney, NE, USA, in 2014 and by the CSU Soil, Water, Plant Testing Laboratory (Fort Collins, CO, USA) in 2015. The soils were extracted with 2 M KCl, and nitrate (NO
3−-N) and ammonium (NH
4+-N) were measured by automated colorimetry. The sample results were averaged to obtain the pre-season soil inorganic N value used in determining N application rates (
Table 1). The target N rate to meet the carrot crop’s N needs was 135 kg N ha
−1, including both the soil inorganic N and N fertilizer applied.
Organic “Nectar” (F1) carrot (
Daucus carota var. sativus) seeds were purchased from Johnny’s Selected Seeds (Johnny’s Selected Seeds, Winslow, ME, USA). This variety is said to be uniform and flavorful and 17.7–20.3 cm in length, with medium-tall tops that hold up well to leaf blight. The carrots were double planted (sub-rows) at a seeding rate of 494,000 seeds ha
−1 in 3.05 m-length rows per plot, with 7.6 cm spacing between the plants and 76 cm centers between the rows (2.3 m
2 plot
−1); the planting and harvest dates are shown in
Table 2. Each sub-row was planted 7.6 cm away from the center of the drip tape. After emergence, the carrots were thinned to contain approximately 40 plants per 3.05 m sub-row, for a total of 80 carrots surrounding each drip tape. To minimize edge effects, the center 10 plants (5 per sub-row) were flagged for measurements as representatives of the row.
2.2. Experimental Design
The study was designed as a randomized complete block design with a 4 × 2 factorial scheme (4 soil treatments × 2 foliar seaweed treatments) with four replications (32 plots). The following treatments were compared: four soil treatments (one control and three fertilizer treatments) with and without foliar seaweed for a total of eight treatments. The three N fertilizers used in this experiment were: cyano-fertilizer, hydrolyzed fish fertilizer, and non-hydrolyzed fish fertilizer. The cyano-fertilizer (
Anabaena spp. cyanobacteria) was grown on-farm [
19] and had an average of 23.3 mg N/kg or <1% N by weight (Total Kjeldahl N). Neptune’s Harvest hydrolyzed fish fertilizer (2-4-1) and Alaska non-hydrolyzed fish fertilizer (5-1-1) were purchased from Neptune’s Harvest (Gloucester, MA, USA) and Fort Collins Nursery (Fort Collins, CO, USA), respectively. In fertilizer manufacturing, the term ‘hydrolyzed’ generally means that the whole fish is cold processed in water and is broken down using naturally occurring enzymes, whereas non-hydrolyzed typically means heat processed and evaporated to concentrate the nutrients.
The three fertilizers varied in N concentration and were applied at equal N rates (the application dates are shown in
Table 3). The N fertilizers were injected into a drip irrigation system (described below) and applied directly to the soil. The control group received no N fertilizer, and water supplied through the N treatments was calculated and supplied to the control rows the next day to equalize the water application. In 2014, Seacom PGR Organic Seaweed Concentrate (0-4-4) was purchased from Johnny’s Selected Seeds (Winslow, ME, USA), but in 2015, the Colorado Department of Agriculture would not accept this product under its organic certification; therefore, in 2015, Neptune’s Harvest Organic Seaweed Plant Food (0-0-1) purchased from Neptune’s Harvest (Gloucester, MA, USA) was utilized instead. Both seaweed products were chosen because the seaweed was cold processed to retain the integrity of biological molecules and because they contained no N, reducing interference with the effects of N fertilizers. The seaweed extract was applied foliarly using a backpack sprayer following the manufacturer’s recommendations (
Table 4).
2.3. Irrigation System and Fertilizer Application
A drip irrigation system was installed to supply water and the N fertilizers. The system utilized two 24.4 m headers running lengthwise, outlining the plot with drip tape rows in between. A ball valve was installed at each end of the drip tape where it joined with the headers for the purpose of selectively closing the rows to facilitate fertigation through the irrigation system. Large ball valves were installed at the ends of the headers so that low-pressure, clean water could be flushed through the lines between treatments to minimize cross-contamination. The drip tape used was 15 mil, with 20 cm spaced emitters and 4.1-L min−1 m−1 (John Deere, Moline, IL, USA). Irrigation was automated to run for 45 min 5 days a week. Irrigation water was not applied 2 days a week to prevent overwatering. On days that fertilizer was applied through the irrigation system, irrigation water was not applied. The applied irrigation water for the carrots totaled 82 cm and 61 cm in 2014 and 2015, respectively.
N fertilizers were applied directly through the drip irrigation system utilizing the row valves to control application to the appropriate rows. The cyano-fertilizer was grown in a production raceway on-farm and applied at full strength using a sump pump placed in the raceway [
19]. Fish fertilizers were diluted into livestock watering tanks to match the cyano-fertilizer N concentration for each application and applied with a sump pump. The N content of the cyano-fertilizer was measured on the day of fertigation using a DR3900 Benchtop Spectrophotometer (Hach, Loveland, CO, USA) to measure total Kjeldahl nitrogen.
Based on the 2014 pre-season soil sampling, the fertilizer N requirement for carrots was 85 kg N ha
−1 to reach a total of 135 kg N ha
−1. Due to weather challenges, only 68 kg N ha
−1 were applied to the carrots before harvest (
Table 3). In 2015, contamination of raceways with a predatory microbe negatively impacted cyano-fertilizer production. No cyano-fertilizer was applied after 23 July 2015. Therefore, cyano-fertilizer was applied at a rate of 28 kg N ha
−1, less than the recommended rate of 81 kg N ha
−1 (
Table 3). All 2015 data reflect the reduced application rates for cyano-fertilizer, although the fish emulsions were applied at the full recommended rates (
Table 3).
2.4. Harvest Sampling and Analyses
On the date of harvest, the center 10 carrots from each row were harvested, and the tops were cut off at the crown. The carrot roots were examined for deformities. The number of individual carrots displaying branched roots, root knobs, cracks or splits, and underdeveloped root lengths were counted for each row (
Figure 1). The carrot root length and circumference at the crown were measured with a tape measure. Circumference was converted to diameter for statistical analysis. After harvest, each plot was soil sampled (0–45 cm) under the drip tape using a Giddings soil sampling rig (Giddings Machine Company, Windsor, CO, USA), and the samples were air-dried, ground, sieved through a 2 mm mesh, extracted using a 1:10 soil to solution ratio in 2 M KCl, and analyzed for NO
3−-N and NH
4+-N. In 2014, the extracts were analyzed with the auto-analyzer (Alpkem, Gorenjska, Slovenia) at the CSU EcoCore Analytical Services Laboratory in Fort Collins, CO, USA. In 2015, the extracts were analyzed by the CSU Soil, Water, and Plant Testing Laboratory in Fort Collins, CO, USA, with a Lachat (Lachat Instruments, Milwaukee, WI, USA) auto-analyzer.
Phytohormone analyses were conducted at the Proteomics and Metabolomics Facility, CSU. The fertilizer samples were adjusted to pH 7.0 with 1 N NaOH and extracted three times with water-saturated n-butanol followed by vacuum drying [
20]. The extracts obtained were filtered through membrane filters (pore size 0.45 μm). Supernatants were harvested by centrifugation at 5000×
g for 20 min at 4 °C, homogenized in liquid nitrogen using a cold mortar and pestle at 4 °C, and extracted using 80% methanol containing 10 mg L
−1 butylated hydroxytoluene at 4 °C. The samples were methylated with diazomethane and dissolved in heptane, and analysis performed with a gas chromatograph—mass spectrometer (GC-MS) [
21]. The amounts of phytohormone applied over the growing season were determined by multiplying the measured concentrations by the amount of fertilizer applied (
Table 5).
2.5. Statistical Analysis
All statistics were performed using Statistical Analysis Software 9.4 (SAS Institute, Inc., Cary, NC, USA). The PROC Mixed statement was used, and the experimental design was run as a 4 × 2 factorial scheme. The treatment and foliar seaweed applications were the fixed effects, and blocks, or replicates, were treated as a random variable. There were several factors that made it difficult to compare the 2014 and 2015 seasons; there was much more rain in 2015, and the foliar seaweed product was changed between seasons. For these reasons, the years were analyzed separately. The slice statement was used to analyze the effects of foliar seaweed extract. An adjusted F-test of fixed effects was performed using the REML method. The least-square means were estimated with the LSMEANS statement and compared with the PDIFF statement. p-values < 0.05 were considered significant in all cases.
5. Conclusions
Cyano-fertilizer treatment resulted in the same or higher carrot yields and carrot length than the fish fertilizers in both years. The cyano-fertilizer was also equal to the fish fertilizers in % root knobs, % underdeveloped roots, and post-harvest soil NO3−-N concentration. Therefore, cyano-fertilizer is a viable alternative to liquid fish fertilizers.
Foliar seaweed applications had no significant impact on carrot yield in either year. However, in one of the two study years, foliar seaweed application decreased root knobs when applied with non-hydrolyzed fish fertilizer and decreased underdeveloped roots in the no fertilizer control.
Although the cyano-fertilizer, both fish fertilizers, and one of the seaweed products contained auxin and salicylic acid, none contained measurable cytokinin concentrations. Salicylic acid can improve resistance to root-knot nematodes, and this may possibly have been related to the knob formation on the roots. However, this requires further evaluation prior to drawing a conclusion regarding salicylic acid’s impact on carrot root knobs.