Winter Production of Asian Leafy Greens in High Tunnels Using Biodegradable Mulches

: Use of season extension tools such as high tunnels and diverse vegetable crops have been crucial in improving competitiveness of vegetable growers in Mississippi who operate on small- to medium-sized farms. Chinese cabbage, also known as pak choy or bok choy, has become increasingly popular due to numerous cultivar choices, fast maturity, high productivity, tolerance for frost, and its potential use for winter production in high tunnels in a subtropical climate. Five Chinese cabbage cultivars including ‘Asian Delight’, ‘Black Summer’, ‘Red Pac’, ‘Rosie’, and ‘Tokyo Bekana’ were evaluated for plant growth, yield, and mineral nutrient concentrations when grown with three types of biodegradable plastic mulches (BDMs) and one polyethylene (PE, or plastic) mulch in a high tunnel in two experiments from 30 October 2019 to 18 March 2020. The ﬁve tested cultivars varied in plant height, widths, leaf SPAD, fresh and dry plant weights, marketable yield, and macro- and micro-nutrient concentrations. ‘Tokyo Bekana’ produced the highest marketable yield and fresh and dry plant weights in both experiments. The three BDMs resulted in similar marketable yield and mineral nutrients in tested cultivars and similar temperatures of leaf, mulch, and substrate compared to the PE mulch. The high tunnel provides a viable way for the winter production of selected Chinese cabbage cultivars in a subtropical climate with possible different yields between production cycles due to varying microenvironment in those months.


Introduction
Vegetables are an important part of specialty crop production and agriculture in Mississippi [1,2]. Vegetable growers in the state generally operate on small-to medium-sized farms and market their produce locally through farmer's markets, community supported agriculture (CSA), local restaurants, and direct on-farm sale [3,4]. As growers seek to diversify their production and increase competitiveness at local markets, Asian leafy greens have become increasingly popular cool-season crops with their wide adaptability, high productivity, and relatively short maturity, along with many cultivar choices [5]. Asian leafy greens are suitable for fall or spring planting in the southern states of the United States (US). Non-heading Chinese cabbages (Brassica rapa var. chinensis), also known as pak choy or bok choy, are of the most grown Asian leafy greens. They perform the best with daily average temperatures of 13-21 • C, tolerate frost, and have the potential to be produced during the mild winter in southern US with some protection from season extension tools [5,6]. Selection of suitable cultivars for cold tolerance and productivity is crucial for winter production [7].
High tunnels, or hoop houses, are constructed with metal frames covered with polyethylene films, with no automatic cooling, heating, or lighting systems [8,9]. High tunnels raise air and soil temperatures, lower frost risks, and provide shield from rain, resulting in advanced harvests, increased yield, improved crop quality, and decreased disease pressure [4,10,11]. With elevated temperatures compared to outdoors, high tunnels can extend the growing season by 1 to 4 weeks in the spring and 2 to 8 weeks in the fall [7].

Plant Cultivation and Experimental Design
Seedlings (40-42 days old) were hardened off approximately one week prior to planting and transplanted into the high tunnel located at the R. R. Foil Plant Science Research Center of Mississippi State University (lat. 33.45 • N, long. 88.79 • W; USDA hardiness zone 8a) on 30 October2019 and 11 February 2020 for the first and second experiment, respectively. The high tunnel measured 29.0 m long and 9.1 m wide, oriented north to south, and was placed in full sun. The high tunnel had metal frames covered with 0.15 mm (6 mil) clear polyethylene film and had side curtains and two doors on end walls opening to 1.5 m and 3 m high, respectively (Tubular Structure, Lucedale, MS, USA). Within the experiment duration, side curtains and end doors of the high tunnel were closed when the air temperature was below 4.4 • C, and remained open otherwise with temperatures above 4.4 • C.
Due to the heavy clay soil in the high tunnel not being suitable for growing vegetables, five raised beds were constructed with composted pine bark that was introduced into the high tunnel, serving as five replications (blocks). Each raised bed measured 27.4 m long, 80 cm wide at the base, 45 cm wide at the top, 15 cm high, and was spaced 1.2 m centerto-center. Before mulch was laid onto the bed, granular lime at a rate of 2.96 kg·m −3 (Soil Doctor Pelletized Lawn Lime; Oldcastle, Atlanta, GA) and a controlled release fertilizer 15N-3.9P-10K (Osmocote ® 15-9-12 plus, 3-4 months; ICL Specialty Fertilizers, Summerville, SC, USA) at a rate of 4.75 kg·m −3 were incorporated into the raised beds. One drip tape (15.9 mm in diameter, 0.91 L per hour; Netafim, Tel Aviv-Yafo, Israel) was laid onto the center of each raised bed with 30 cm emitter spacing.
Each raised bed was then divided into four equal sections approximately 6.9 m in length. Each section was randomly covered with one of the four mulch types. Three biodegradable plastic mulches (BDMs) and one black polyethylene (PE, also referred as plastic) film were tested in this study and are summarized in Table 1. Twelve plants from each cultivar were transplanted into the raised beds in staggered double rows 25 cm between plants and 25 cm between rows. This experiment was set up in a split-plot design with factorial arrangement of treatments and five replications. Mulch type served as the main plot factor and was randomly distributed within a block. Within each main plot, Asian green cultivar served as the sub-plot factor and was randomly distributed within each main plot. There were twelve single-plant subsamples in each sub plot.

Microenvironment in the High Tunnel
Environmental conditions including air temperature, relative humidity (RH), and photosynthetically active radiation (PAR) were recorded in the high tunnel. A data logger (HOBO USB Micro Station H21-USB; Onset Computer Corp., Bourne, MA, USA) was installed in the center of the high tunnel. A temperature and RH sensor (HOBO S-THB-M002; Onset Computer Corp.) and a quantum sensor (HOBO S-LIA-M003; Onset Computer Corp.) were connected to the data logger to monitor air temperature, RH, and PAR at one-hour intervals. Daily light integral (DLI) was calculated by multiplying average daily PAR with 0.0864 as described by Torres and Lopez [41]. Growing degree days were calculated daily by [(Daily maximum temperature + daily minimum temperature)/2base temperature]. Cumulative GDDs between certain time periods were estimated by summing daily GDDs. The base temperature used for Asian greens was 4 • C [7]. The microenvironment in the high tunnel is presented in Figures S1-S3.

Data Collection and Plant Harvest
Before harvest, two plants from each sub-plot were randomly selected to measure plant height, widths, and relative leaf chlorophyll content, measured as Soil-Plant Analysis Development (SPAD) readings, on 4 December 2019 and 16 March 2020 in the first and second experiment, respectively. Plant width was calculated as the average of width 1 (widest points apart) and width 2 (width at perpendicular direction to width 1). Leaf SPAD was measured on three fully expanded leaves of each selected plant using a chlorophyll meter (SPAD 502 Plus; Konica Minolta, Inc., Osaka, Japan). Three readings measured from the selected leaves, one reading per leaf, were averaged to represent relative chlorophyll content of a given plant.
Temperatures on leaf surface, mulch surface, substrate temperature and moisture were also measured with two readings from each cultivar (or sub plot). Leaf surface temperature was measured on two plants in each plot, using a handheld infrared dual-laser thermometer (Southwire, Carrollton, CA, USA). Two mulch temperature readings were also collected in each sub-plot using the same thermometer. Substrate temperature at 10 cm depth was measured using a digital thermometer probe (Fisherbrand TM Traceble TM ; Pittsburgh, PA, USA). Substrate moisture at 6 cm depth was measured using a soil moisture sensor (ML2x; Delta-T Devices, Cambridge, UK) toward the center of each plot with two readings from each sub-lot. The moisture sensor was connected to a soil sensor reader (HH2; Delta-T Devices) for instant moisture readings.
After plant growth and temperature data were collected, all plants were harvested at crown level to measure fresh yield on 5 December 2019 and 18 March 2020 for the first and second experiment, respectively. The twelve plants in each plot were separated into marketable yield and culled for unmarketable yield based on plant size and leaf quality. The number of plant in each category was also recorded. Besides total yield, two marketable plants were measured for individual fresh plant weight, and oven dried at 60 • C until constant weight. The dry weight of each plant was then measured.

Mineral Nutrient Analyses
Each dry plant was ground to pass through a 1 mm sieve with a grinder (Wiley mini mill, Thomas Scientific, Swedesboro, NJ, USA) for mineral nutrient analyses. Combustion analysis was used for the determination of total nitrogen (N) concentration with 0.25 g of dry tissue using an elemental analyzer (vario MAX cube; Elementar Americas Inc., Long Island, NY, USA). A dry tissue sample of 0.5 g was digested with 1 mL of 6 M hydrochloric acid (HCl) and 50 mL of 0.05 M HCl to obtain the concentrations of phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), copper (Cu), iron (Fe), manganese (Mn), zinc (Zn), and boron (B) using inductively coupled plasma optical emission spectrometry (SPECTROBLUE; SPECTRO Analytical Instruments, Kleve, Germany). Plant samples were tested at the Mississippi State University Extension Service Soil Testing Laboratory. Concentrations of macronutrients (mg·g −1 ) and micronutrients (µg·g −1 ) in Asian green plants are presented on a dry weight basis.

Statistical Analyses
Two-way analysis of variance (ANOVA) was performed on all tested variables. All data were analyzed using the PROC GLMMIX procedure of SAS (version 9.4; SAS Institute, Cary, NC, USA). Where indicated by ANOVA, means were separated using Tukey's Honest Significant Difference (HSD) test at p < 0.05. Data from the two experiments were compared as repeated measures, where the experiment date was used as a factor to analyze its effect.

Results
The experiment date affected all measured dependent variables in this study, showing varying trends between the November and February experiments (data not shown). Therefore, data from the two experiments are presented separately.

Plant Height, Widths, and Leaf SPAD
The Asian green cultivars varied in plant height, width, and relative leaf chlorophyll content, measured as SPAD readings in both experiments. Mulch type did not affect any of the three variables in either experiment ( Table 2).

Fresh and Dry Plant Weights
Fresh and dry plant weights varied among cultivars in both experiments (Table 3). Mulch type affected fresh and dry plant weights in November 2019 (Table 4).      (Table 4).

Marketable and Unmarketable Yields
Marketable yield in both experiments and unmarketable yield in November 2019 varied among cultivars. Mulch type did not affect marketable or unmarketable yield in either experiment ( Table 3).
Trends of marketable yield among cultivars were similar in both experiments, where 'Tokyo Bekana' produced the highest marketable yields of 5510 kg·ha −1 in November and 14,915 kg·ha −1 in February, higher than any other cultivars. Lower, 'Asian Delight' and 'Black Summer' produced similar intermediate yields of 3596 kg·ha −1 and 3834 kg·ha −1 in November, and 10,837 kg·ha −1 and 11,959 kg·ha −1 in February, respectively. The two red-leaf cultivars 'Red Pac' and 'Rosie' produced the lowest yields of 1434 kg·ha −1 and 1883 kg·ha −1 in November, and of 4500 kg·ha −1 and 5442 kg·ha −1 in February, respectively. Unmarketable yield ranged from 76.6 kg·ha −1 to 769.9 kg·ha −1 in November, equivalent to 1.45% to 16.7% of total yield. 'Black summer' produced the highest unmarketable yields in November, with the main reason observed to be malformed leaves. There was no difference in unmarketable yield among cultivars in the February experiment due to large variations, ranging from 18.4 kg·ha −1 to 159.3 kg·ha −1 equivalent of 0.17% to 2.53% of total yield.  (Table 5).

Macronutrients
Concentrations of macronutrients including N, P, K, Ca, and S in the November experiment and N, P, K, Mg, and S in the February experiment varied among cultivars ( Table 6). The five tested cultivars had similar Mg concentrations in November and similar Ca concentrations in February. Mulch type did not affect macronutrient concentrations in tested Asian green cultivars.  Calcium concentration was generally similar among cultivars ranging from 15.6 mg·g −1 in 'Black Summer' to 20.4 mg·g −1 in 'Tokyo Bekana' in November, except that 'Tokyo Bekana' had higher Ca concentration than 'Black Summer'. There was no difference in Ca concentration among cultivars, or mulch type in the February experiment.
Magnesium concentration was similar among cultivars ranging from 3.35 mg·g −1 to 3.64 mg·g −1 in November. In February, Mg concentration ranged from 4.04 mg·g −1 in 'Rosie' to 4.84 mg·g −1 in 'Asian Delight', with cultivars generally having similar Mg concentrations except that 'Asian Delight' had higher Mg concentration than 'Rosie'.
In November, 'Black Summer' had higher S concentration of 7.18 mg·g −1 than the other four cultivars with similar S concentrations of 5.67 mg·g −1 to 6.18 mg·g −1 . In February, S concentration ranged from 5.97 mg·g −1 in 'Asian Delight' to 7.3 mg·g −1 in 'Black Summer', with cultivars generally having similar S concentrations except for 'Black Summer' having a higher S concentration than 'Asian Delight'.

Micronutrients
Concentrations of micronutrients including Cu, Fe, Mn, Zn, and B in both experiments varied among cultivars. Mulch type did not affect micronutrient concentrations in tested Asian green cultivars (Table 7).

Discussion
The microenvironment varied between the two experiments in terms of air temperatures, RH, and DLI ( Figures S1-S3). Repeated measure showed higher soil temperature in raised beds in February than November in each cultivar plot (data not shown). Using 4 • C as the base temperature, the cumulative growing degree days (GDDs) in the November experiment was 245 GDDs, compared to 352 GDDs in the February experiment. The daily light integral ranged from 2.71 mol·m −2 ·d −1 on 14 November to 25.1 mol·m −2 ·d −1 on 1 November 2019 in the first experiment, and from 3.66 mol·m −2 ·d −1 on 4 March to 30.9 mol·m −2 ·d −1 on 6 March 2020 in second experiment. There were nine and five days with minimum air temperature below 0 • C in the November and February experiment, with the lowest minimum temperature of −3.21 • C and −3.57 • C, respectively ( Figure S1). The Chinese cabbage cultivars are known to tolerate frost [6]. We did not observe visual damage of plants in either experiment. The high tunnel provides a viable way for winter production of Asian greens in Southeastern US.
As a result of varying microenvironment between the two experiments, the marketable yield of each cultivar in the February experiment was significantly higher than that in the November experiment (data not shown), averaging 2.71 (in 'Tokyo Bekana') to 3.14 (in 'Red Pac') times the marketable yield of those in the November experiment. Fresh plant weight was also significantly higher in the February experiment (data not shown), averaging 1.66 to 1.97 times of those in November. Such differences in yield were found when all Asian green cultivars were harvested at 36 days after planting (DAP) in both experiments. Fresh plant weights of tested Asian green cultivars were higher or similar to reported ranges when leafy green cultivars were produced in Northwest US during winter months, with a much longer growth period, harvested at 71 DAP [7]. Growers could expect different yields between production cycles during cool seasons and could possibly increase the growth period to increase yields during cooler months. On the other hand, growing Asian green cultivars in warmer months can result in faster harvest for satisfactory yield.
The growing density in the current study was 18,182 plants per acre (equivalent to 44,944 plants per hectare) in the high tunnel. This is in general agreement with the reported planting density of 14,520 plants per acre with leafy greens including mustard (Brassica juncea), kale (Brassica oleracea acephala group), and collard (Brassica oleracea acephala group) varieties with a slightly higher spacing of 38 cm [42], compared to the 25 cm spacing used in the current study. With similar density among cultivars, yield was mainly a function of fresh plant weight. Yields of red leaf cultivars 'Red Pac' and 'Rosie' can potentially be increased by increasing planting density due to their vertical growth habits and small crown widths.
Leafy green vegetables including bok choy cultivars are known to be a rich source of mineral nutrients. Macro-and micro-nutrient concentrations in tested bok choy cultivars were generally in agreement with reported ranges [43]. Besides basic nutrition, bok choys contain high levels of fiber and abundant bioactive compounds including many B-vitamins and carotenoids-antioxidants that play roles in blocking early stages of cancer [44]. Concentrations of phytochemicals affected by cultural practices, including the high tunnel and biodegradable mulches, merits further investigation.
The three tested BDMs resulted in similar fresh and dry plant weights and marketable yields compared to the plastic mulch. However, BioTelo increased fresh and dry plant weights compared to Organix A.G. in the November experiment. Concentrations of all tested macro-and micro-nutrients varied among cultivars, but were not affected by mulch type. Therefore, plastic much and BDMs resulted in similar fresh yield and quality of tested Asian green cultivars in a high tunnel in the current study. This is in agreement with multiple reports in the production of cucumber (Cucumis sativus), sweet corn, tomato, strawberry (Fragaria × ananassa), and pepper in open field or high tunnel systems [21,[37][38][39]45].
In terms of the microenvironment, plastic mulch resulted in higher substrate moisture, of 19.0%, than BioTelo, of 16.8%, but similar substrate moisture to Organix A.G. or Weed-GuardPlus, of 17.5% and 17.6%, respectively, in the November experiment. This could result from the higher permeability and low thickness of BDMs, as discussed by Ghimire et al. [39]. Even with P-values lower than 0.05 in substrate temperature measured on December 3 2019 and leaf temperature measured on March 16 2020, multiple comparison showed no difference among mulch types. Similar soil temperatures were also found in raised beds covered with BDMs compared to standard plastic mulch [35]. When using BDMs in the production of leafy greens, irrigation program may need to be adjusted to compensate for possible increased moisture loss through biodegradable mulches.
Strengths of BDMs are reported to be lower than PE mulch [46]. Degradation of BDMs was reported to provide poor weed control for persistent weed species in pepper production [21]. We did not observe any tearing problems during laying or in production, or weed growth penetrating the BDMs, even when they were thinner (15 to 17.8 µm) than the plastic film (32 µm). The Organix A.G. mulch was reported to split shortly after layering and resulted in poor weed control when used to produce sweet corn in a Mediterraneantype climate with average air temperatures of 16.1 • C [39]. The different results are likely due to the short production cycle of leafy greens (36 days) in the current study and that the high tunnel reduces wind, rainfall, and solar radiation, all of which accelerate deterioration of BDMs. This agrees with Miles et al. [38] reporting higher values of rips, tears, and percent of visually observed deterioration in open fields than high tunnels.
One advantage of using biodegradable mulch to compensate for the high initial purchase cost is saving of labor and mechanics in mulch removal, because they are designed to be tilled into the soil or composted at the end of the growing season [47]. Some BDMs were reported to deteriorate by 65% to 100% 397 days after soil incorporation, whereas other types showed little deterioration over the same time [33]. However, high deterioration of BDMs measured as percent soil exposure does not necessarily suggest degradation. Soil burial tests showed a high percentage of the BDM area remained after 18 month [46]. A number of factors, biotic or abiotic, including water, oxygen, temperature, mulch thickness, and microbial community size and composition, impact the breakdown of BDMs [26,30,48,49].
The objectives in our study were to examine crop yield and quality of Asian green cultivars using BDMs versus PE mulch, without investigating deterioration after soil incorporation. Use of a high tunnel for the production of vegetable crops, especially for small growers in the state of MS, relies on fast turnaround of high tunnel growing space and maximum occupancy of various crops throughout the year. It remains unclear whether the timeframe of mulch biodegradation will interfere with planting of another crop shortly after one is harvested, considering that deterioration and degradation of biodegradable mulches are likely slower in a high tunnel production system compared to an outdoor environment, as described above. Best management practices in a high tunnel production system using BDMs require further investigation.

Conclusions
The five tested Chinese cabbage cultivars varied in plant height, widths, leaf SPAD, fresh and dry plant weights, marketable yields, and macro-and micro-nutrient concentrations, with 'Tokyo Bekana' producing the highest marketable yield and fresh and dry plant weights in both experiments. The three BDMs resulted in similar marketable yields and mineral nutrients in tested cultivars and similar microenvironment including mulch, substrate, and leaf temperatures compared to the plastic mulch without tearing problems in the short production cycle of Asian greens. The high tunnel provides a viable way for winter production of selected Chinese cabbage cultivars in a subtropical climate with marketable yield being significantly higher during warmer months with higher GDDs.  Mention of a trademark, proprietary product, or vendor, does not constitute a guarantee or warranty of the product by Mississippi State University or the USDA and does not imply its approval to the exclusion of other products or vendors that also may be suitable.