Optimization of Substrate and Nutrient Solution Strength for Lettuce and Chinese Cabbage Seedling Production in the Semi-Arid Environment of Central Myanmar

: The growing population of tropical countries has led to a new awareness of the importance of vegetables as a source of essential foods and nutrients. The success of vegetable cultivation depends to a large extent on high-quality seedlings. This work aimed at evaluating the effects of different substrates and different nutrient solution concentrations on the development of lettuce and Chinese cabbage seedlings in a semi-arid tropical area. Three independent experiments were conducted at the Soil and Water Research Station at Yezin Agriculture University, Myanmar (Myanmar, 19.83 ◦ N; 96.27 ◦ E). In all experiments a randomized block design was implemented with four treatments and three repetitions. In the ﬁrst experiment the adaptability of lettuce seedling to two substrates (namely a Hulls Manure mix composed by 50% of mature cattle manure and 50% of carbonized rice husk and a soil based substrate constituted by 70% local soil, 20% burned rice husk, and 10% fresh cattle manure) and two nutrient solutions with different electrical conductivities (ECs) (W 0.1 , stored rainwater with EC = 0.13 dS m − 1 and NS 1.2 , nutrient solution with EC = 1.20 dS m − 1 ) were tested. In the second and third experiments, two species (lettuce and Chinese cabbage) were assessed for their response to nutrient solution concentrations. In both crops, 4 fertigation treatments (W 0.1 ; NS 0.6 ; NS 1.2 ; and NS 1.8 ) were supplied, by modulating the concentration of a compound mineral fertilizer (15:15:15) in the following ranges: W 0.1 : 0 g L − 1 , electrical conductivity (EC) 0.13 dS m − 1 , NS 0.6 : 0.3 g L − 1 , EC of 0.60 dS m − 1 ; NS 1.2 : 0.6 g L − 1 , 1.2 dS m − 1 EC, and NS 1.8 : 0.9 g L − 1 , 1.8 dS m − 1 EC. Adopting different substrates and applying different nutrient solutions signiﬁcantly affected growth (fresh weight and leaf morphology) and some physiological parameters (stomatal conductance, leaf temperature, and leaf chlorophyll content) of lettuce and Chinese cabbage seedling. From the ﬁrst experiment, the combination of the soil based substrate and NS 1.2 treatments allowed us to improve the seedlings’ growth. In the second experiment, highest growth of lettuce and Chinese cabbage seedlings was associated with NS 1.2 and NS 1.8 , respectively. The presented results allow for the optimization of both growing media and nutrient solution management when lettuce and Chinese cabbage seedling are produced in the Central Dry Zone of Myanmar.


Introduction
Climate change and the world population increase are leading to a new awareness on the importance of vegetable crops as a source of food. This is particularly relevant, since vegetables can supply essential nutrients (e.g., vitamins and minerals) that are otherwise not available from other foods [1] Moreover, the current sanitary emergency due to the COVID19 pandemic will likely reinforce the importance of getting knowledge and grates figures from different crop features and management strategies to elaborate specific recommendations on the optimal management of seedling production.

Location
Three experiments were conducted in open field conditions at the Soil and Water Research Station of Yezin Agriculture University located in the University Campus, CDZ-Myanmar, 16 km from the Capital Naypyidaw (19 • 83 North and 96 • 27 East, 122 m a.s.l.) (Figure 1a,b). According to Köppen's classification, the local climate is the Aw type, which is tropical rainy with dry summer and rainy season concentrated between June and October.
Horticulturae 2021, 7, x FOR PEER REVIEW 3 of 15 etable seedling production methodologies can ensure better quality seedlings, as compared to current production techniques. The study integrates figures from different crop features and management strategies to elaborate specific recommendations on the optimal management of seedling production.

Location
Three experiments were conducted in open field conditions at the Soil and Water Research Station of Yezin Agriculture University located in the University Campus, CDZ-Myanmar, 16 km from the Capital Naypyidaw (19°83′ North and 96°27′ East, 122 m a.s.l.) (Figure 1a,b). According to Köppen's classification, the local climate is the Aw type, which is tropical rainy with dry summer and rainy season concentrated between June and October.

Plant Material and Crop Management
The first two experiments were performed on lettuce (Lactuca sativa L.) cv. Green wave (Evergreen seeds, Sunnyvale, CA, USA), while the third one was carried out on Chinese cabbage (Brassica juncea L.) cv. Pavito (East West Seeds, Nanning, China). Both cultivars are commonly sold in the local market of the main cities of CDZ-Myanmar such as Sagaing, Magway, and Mandalay. Sowing took place on 9 January 2020 (first experiment) and Horticulturae 2021, 7, 64 4 of 15 27 February 2020 (second and third experiment). Crops were sown manually in 105 cells plastic seedling trays. The sizes of the seedling tray were the following: 45 cm length; 28 cm width and 5.5 cm dept. Plant density was 833 plants m −2 . Seedling trays were placed on a simple wooden/bamboo frame covered with a 70% shading net (Figure 1c) to reduce sunlight intensity while preserving air circulation from the open sides. For five days after sowing, irrigation was carried out only with clear water three times per day (at 7 a.m., 11 a.m., and 3 p.m.). When 80% of seedlings presented fully expanded cotyledons, the germination process was considered completed, and treatments started. Plant fertilization was managed to supply 1 L per tray of nutrient solution twice a day (7 a.m. and 3 p.m.) while only water was supplied once a day at 11 a.m. using a 1-L watering can.

Treatments and Experimental Design
Experiment 1: A total of four treatments were considered, obtained by the factorial combination of two substrates (soil based substrate, SBS, and Hulls Manure mix, HM-mix) and two nutrient solution concentrations (water, W 0.1 , and water enriched with fertilizer, NS 1.2 ).
SBS (soil based substrate) refers to the local common substrate generally used to grow vegetables seedlings, composed by 70% of the local soil, 20% of burned rice husk, and 10% of fresh cattle manure. In this case, the rice husks are burned inside an iron tank up to turning husks into ashes. HM-mix (Hulls Manure mix) refers to a substrate formerly suggested for seedling production in the tropics [20], composed by 50% of carbonized rice husk and 50% of well sifted mature cattle manure. Physical and chemical substrates characterization was performed at the laboratory of the Yezin Agriculture University of Naypyidaw (Myanmar). Electrical conductivity (EC) and pH were determined using the conductivity meter DS-51 and pH meter F-51 (HORIBA, Kyoto, Japan). Organic matter (OM), cation exchange capacity (CEC), and water holding capacity (WHC) were determined using the Tyurin's method, leaching method, and Keen-Razcowski measurement method, respectively [21][22][23]. Available N, P, and K were also analyzed using the Alkaline permanganate method, Olsen's P-Malachite method, and ammonium acetate extraction [24,25].
The experimental design was a completely randomized block design with four treatments and three replicates of each essay ( Figure 2).

Rice Husk Carbonization Process
The HM-mix is composed by cattle manure enriched with carbonized rice husk. The carbonization of the rice husk is performed by using a metal chimney (height of 1.5 m and diameter of 0.15 m, Figure 3a,b) that features a square perforated metal burner (28 cm height) at its base, where combustion takes place. The carbonization process starts with lighting a wood fire, which is quickly covered with the chimney. At this point, the rice husk is poured directly over the burner, creating a cone in contact with the chimney itself ( Figure 3b) and undergoing a pyrolysis process. The carbonization process continued for around 3 h, during which rice husk was turned several times to ensure homogeneous carbonization. When full carbonization was reached, the chimney was removed and water was poured over the carbonized rice husk to stop the combustion (Figure 3c). The obtained carbonized rice husk (biochar) was then ready for use ( Figure 3d).

Rice Husk Carbonization Process
The HM-mix is composed by cattle manure enriched with carbonized rice husk. The carbonization of the rice husk is performed by using a metal chimney (height of 1.5 m and diameter of 0.15 m, Figure 3a,b) that features a square perforated metal burner (28 cm height) at its base, where combustion takes place. The carbonization process starts with lighting a wood fire, which is quickly covered with the chimney. At this point, the rice husk is poured directly over the burner, creating a cone in contact with the chimney itself ( Figure 3b) and undergoing a pyrolysis process. The carbonization process continued for around 3 h, during which rice husk was turned several times to ensure homogeneous carbonization. When full carbonization was reached, the chimney was removed and water was poured over the carbonized rice husk to stop the combustion (Figure 3c). The obtained carbonized rice husk (biochar) was then ready for use ( Figure 3d). Horticulturae 2021, 7, x FOR PEER REVIEW 6 of 15

Measurements
At 21 days after sowing, morphological (experiment 1: width and length of all plant leaves; experiment 2 and 3: seedling fresh weight, leaf number, width, and length of all plant leaves) and physiological (experiment 1: leaf chlorophyll content and temperature; experiment 2 and 3: stomatal conductance, leaf chlorophyll content, and leaf temperature) parameters were taken. Leaf temperature was assessed using an infrared thermometer model FLUKE 61 (Fluke Corporation, Everett, WA, USA). Stomatal conductance was measured using a handheld photosynthesis measurement system model CI-340 (Camas, WA, USA). Leaf chlorophyll content was estimated using SPAD 502 (Minolta, Osaka, Japan). Morphological measurements were obtained from 6 plants per treatment per each of the 3 replicates (n = 18). Physiological measurements were made on the upper surface of the canopy on three leaves per each sampled plant, on 6 plants per treatment in each of the three replications (n = 18).

Measurements
At 21 days after sowing, morphological (experiment 1: width and length of all plant leaves; experiment 2 and 3: seedling fresh weight, leaf number, width, and length of all plant leaves) and physiological (experiment 1: leaf chlorophyll content and temperature; experiment 2 and 3: stomatal conductance, leaf chlorophyll content, and leaf temperature) parameters were taken. Leaf temperature was assessed using an infrared thermometer model FLUKE 61 (Fluke Corporation, Everett, WA, USA). Stomatal conductance was measured using a handheld photosynthesis measurement system model CI-340 (Camas, WA, USA). Leaf chlorophyll content was estimated using SPAD 502 (Minolta, Osaka, Japan). Morphological measurements were obtained from 6 plants per treatment per each of the 3 replicates (n = 18). Physiological measurements were made on the upper surface of the canopy on three leaves per each sampled plant, on 6 plants per treatment in each of the three replications (n = 18).

Statistical Analysis
Data from experiment 1 were analyzed using two-way ANOVA (substrate × nutrient solution), while data from experiment 2 and 3 were analyzed using one-way ANOVA. Means were separated using the Tukey HSD (honestly significant difference) test [26] at p ≤ 0.05. Before the analysis, all data were checked for normality and homogeneity of the variance. Averages and standard errors (SE) were calculated. Statistical analysis was carried out using R statistical software (version 3.3.2).

Climate during the Experiments
The first experiment was carried out from 9 January to 29 January 2020. During cultivation, maximum air temperatures ranged between 28.

Experiment 1 3.2.1. Substrate
Results of physical and chemical substrates characterization are reported in Table 1. Both substrates have a subacid pH and a quite high EC, comparable to a moderately saline soil. Among the macronutrient analyzed, the available nitrogen content was higher in SBS than in the HM-mix. Contrarily, the HM-mix had higher phosphorous and potassium, organic matter content, and water holding capacity. The observed higher cation exchange capacity (CEC) of the HM-mixed substrate is also an indicator of a larger nutrient reserve available for the plant. Moreover, during irrigation/fertilization, water stagnation, and runoff on the surface trays with the SBS substrate occurred, highlighting its low permeability, low infiltration capacity, and poor drainage compared to the HM-mixed substrate.

Seedling Growth
For all parameters analyzed, no-significant interactions (p = 0.26 for leaf length; p = 0.20 for leaf width; p = 0.45 for leaf temperature; and p = 0.77 for leaf chlorophyll content) between the substrate and nutrient solution were observed, therefore the effect of the two factors was discussed separately. The adopted substrate only affected leaf length and width (Table 2), whereas the nutrient solution affected all parameters analyzed ( Table 3). As compared with SBS, HM-mix use resulted in increased leaf length and width by 14% and 17%, respectively (Table 2). Leaf length and width and leaf chlorophyll content were the highest in seedlings grown with nutrient solution NS 1.2 (18.8 SPAD value), with a 152%, 17%, and 39% increase from seedling watered with clear water (W 0.1 ), respectively (Table 3). On the other hand, the highest leaf temperature was observed in seedling watered with W 0.1 (26.2 • C).

Experiment 2 Seedling Growth
Productive and morphological parameters: Seedling weight was affected by nutrient solution, reaching the highest value in seedlings grown in NS 1.2 (Figure 4a). The nutrient solution also influenced the number of leaves (Table 4), although significant differences were detected only between W 0.1 and the other nutrient solution treatments. Leaf length was the highest in NS 1.2 and NS 1.8 treatments and decreased when the nutrient solution's mineral fertilizer concentration was reduced (Table 4). Finally, leaf width was not affected by nutrient solution treatments (Table 4).  Productive and morphological parameters: Seedling weight was affected by nutrient solution, reaching the highest value in seedlings grown in NS1.2 (Figure 4a). The nutrient solution also influenced the number of leaves (Table 4), although significant differences were detected only between W0.1 and the other nutrient solution treatments. Leaf length was the highest in NS1.2 and NS1.8 treatments and decreased when the nutrient solution's mineral fertilizer concentration was reduced (Table 4). Finally, leaf width was not affected by nutrient solution treatments (Table 4). Physiological parameter: Leaf chlorophyll content was the highest in NS1.8 and diminished with decreasing nutrient solution concentrations (Figure 4b). No significant difference was observed between NS1.2 and NS0.6, while the lowest value was obtained in W0.1 treatment (Figure 4b). Nutrient solution treatments affected stomatal conductance, which resulted to be the lowest in seedlings grown in W0.1, while no significant differences were detected among the other nutrient solution treatments (average value of 315 mmol m −2 s −1 ) (Figure 4c). Leaf temperature was also affected by the nutrient solution concentration, with the highest values in W0.1 (33.4 °C) and NS0.6 (31.1 °C), although, in this second case, without statistically significant differences as compared to NS1.2 and NS1.8 (Figure 4d). Significance codes: ***, significant differences at p ≤ 0.001, "ns" = not significant. Different letters indicate significant differences with the Tukey HSD test at p ≤ 0.05.

Experiment 3
Seedling Growth Productive and morphological parameters: When NS1.8 was supplied, Chinese cabbage seedlings weight was the highest, and progressively decreased with the reduction of nutrient solution concentration (Figure 5a). Additionally, leaf morphological parameters (leaf number, width, and length) were affected by nutrient solution concentration, show- Physiological parameter: Leaf chlorophyll content was the highest in NS 1.8 and diminished with decreasing nutrient solution concentrations (Figure 4b). No significant difference was observed between NS 1.2 and NS 0.6 , while the lowest value was obtained in W 0.1 treatment (Figure 4b). Nutrient solution treatments affected stomatal conductance, which resulted to be the lowest in seedlings grown in W 0.1 , while no significant differences were detected among the other nutrient solution treatments (average value of 315 mmol m −2 s −1 ) (Figure 4c). Leaf temperature was also affected by the nutrient solution concentration, with the highest values in W 0.1 (33.4 • C) and NS 0.6 (31.1 • C), although, in this second case, without statistically significant differences as compared to NS 1.2 and NS 1.8 (Figure 4d).

Experiment 3 Seedling Growth
Productive and morphological parameters: When NS 1.8 was supplied, Chinese cabbage seedlings weight was the highest, and progressively decreased with the reduction of nutrient solution concentration (Figure 5a). Additionally, leaf morphological parameters (leaf number, width, and length) were affected by nutrient solution concentration, showing higher values in plants grown with NS 1.8 and NS 1.2 (Table 5).

Discussion
Adopting different substrates and nutrient solutions significantly affected growth (fresh weight and leaf morphology) and physiological parameters (stomatal conductance, leaf temperature, and chlorophyll content values) of lettuce and Chinese cabbage seedlings grown in CDZ-Myanmar. Seedlings' quality affects their growth and yield after transplanting, given that good-quality seedlings exhibit morphological characteristics such as thick stems, thick leaves, dark green leaves, and large white roots [27]. In the first experiment, lettuce seedlings with the longest leaf length and width were associated with  (Figure 5b). The response in both stomatal conductance and leaf temperature was consistent with lettuce's observations, with lowest values in seedlings grown in W 0.1 and no statistically significant differences among other nutrient solution treatments (average value of 481 mmol m −2 s −1 ) (Figure 5c). Leaf temperature was the greatest in W 0.1 , while no statistically significant differences were detected among the other treatments (Figure 5d).

Discussion
Adopting different substrates and nutrient solutions significantly affected growth (fresh weight and leaf morphology) and physiological parameters (stomatal conductance, leaf temperature, and chlorophyll content values) of lettuce and Chinese cabbage seedlings grown in CDZ-Myanmar. Seedlings' quality affects their growth and yield after transplanting, given that good-quality seedlings exhibit morphological characteristics such as thick stems, thick leaves, dark green leaves, and large white roots [27]. In the first experiment, lettuce seedlings with the longest leaf length and width were associated with the HM-mix substrate and NS 1.2 nutrient solution treatments. Physiological parameters (leaf temperature and chlorophyll content values) were affected only by the nutrient solution ( Table 3). The combination of HM-mix and NS 1.2 allowed obtaining the most appropriate seedlings for transplanting. Although both substrates have similar EC and pH, the former quite high while the latter adequate for lettuce growth, the obtained results differ probably due to other HM-mix substrate's intrinsic physical and chemical features. According to the substrate analysis, SBS substrate showed larger nitrogen content as compared to the HM-mix substrate, probably resulted by the inclusion of fresh cattle manure [28] that negatively affected seedling growth. In a previous study by Sapkota et al. [29], elevated nitrogen concentration in the substrate depressed root metabolism leading to shorter roots. The high content of carbonized rice husks (biochar) in the HM-mixed substrate may be responsible of its good agronomic performances, given that rice husk features elevate the content of silicon and potassium [30]. The carbonization process is needed to avoid that rice husk turns to ash, keeping the original shape and ensuring several benefits associated with its capacity for decreasing bulk density, enhancing water holding capacity, adding organic carbon, increasing available nutrients, and ultimately increasing crop yields [31,32]. Accordingly, carbonized rice husk use as a growing substrate should be promoted among farmers, especially in those areas where it is readily available as in Asian countries, where rice residues are estimated at 560 million tons for rice straw and 112 million tons for rice husks [30]. Therefore, the promotion of this biochar production should also be stimulated to increase vegetable local quality seedling production.
The low permeability and poor drainage showed by SBS substrate during the irrigation/fertilization time, probably determined continuous waterlogged conditions reducing oxygen in the substrate and seedling growth [33,34]. Main constraints generated by water stagnation in agriculture include detrimental effects on both soil/substrate chemical and physical properties when anaerobic conditions persist repetitively [34]. Furthermore, the application of adequate nutrient solution assures higher and prompt nutrient availability to the seedlings, and the root growing media may affect plants' response to salinity [35,36]. According to the results obtained in the first experiment, the combination of HM-mixed substrate and NS 1.2 nutrient solution proved to be a valid option to produce quality lettuce seedling in CDZ-Myanmar.
Within second and third experiments, the definition of the optimal nutrient solution concentration was targeted. Albeit limited literature has addressed to date the definition of optimal nutrient solution strength on lettuce and Chinese cabbage nursery production in tropical areas, comparison can be made against existing literature on commercial hydroponic production. Accordingly, it was observed that whenever nutrient solution salinity levels are elevated (above 2.0 dS m −1 ), lettuce fresh yield and plant growth might be reduced [35]. Although the adopted substrate featured high electrical conductivity (EC) ( Table 1), the present study points out that the optimal nutrient solution EC for lettuce seedlings production was 1.2 dS m −1 (NS 1.2 ), while for the cultivation of Chinese cabbage seedlings ranged between 1.2 and 1.8 dS m −1 (NS 1.2 ; NS 1.8 ) (Tables 4 and 5). The effect of the initial high EC of the substrate on production was probably reduced by washing practices of substrates performed in the first five days by watering trays with only clear water. Bustamante et al. [37], in an experiment on the effect of washing treatments on different composts used as nursery growing media for seedling pepper production, showed that EC clearly decreased (up to 40.7%) in all growing substrates subjected to washing treatments. From an economic point of view, maximum recommendable fertilizer addition would be in the range of 0.8 g of fertilizer L −1 . Considering the cost for 1000 L nutrient solution preparation, it is possible to estimate a 33% economic saving that the farmer could obtain in preparing NS 1.2 as compared to NS 1.8 without compromising crop growth in lettuce. It is also clear that the use of clear water or nutrient solutions with low electrical conductivity for seedling production, such as in the case of W 0.1 and NS 0.6 treatments, does not guarantee the achievement of satisfactory seedlings size, resulting in lower leaf dimensions probably due to limited nutrients availability (Tables 4 and 5). For instance, in lettuce, leaf size was formerly affected by nutrient solution composition and water availability [29,38,39]. Moreover, seedlings grown under W 0.1 and NS 0.6 showed lower values of leaf chlorophyll content as compared to NS 1.2 and NS 1.8 . According to Trani et al. [19], lettuce seedlings, at the time of transplanting, should have intense green leaves of approximately 5 cm in length, a condition that was not met, in the hereby presented study, in W 0.1 . It emerges how obtaining lettuce seedlings of elevated quality is highly dependent on whether their nutritional requirements are met [40]. Besides, W 0.1 treatment, both in lettuce and Chinese cabbage, showed the lowest values for stomatal conductance, the highest foliar temperature, and the lowest chlorophyll content values compared to the other treatments (Figure 4). It is evident that seedlings under W 0.1 treatment had to deal with abiotic stressors, most probably nutritional deficits. Plants' stomatal conductance is closely related to plants' nutritional status, with a confirmed linear relationship with leaf nitrogen concentration for broad categories of vegetables [41]. The application of clear water (W 0.1 ) clearly did not meet the nutritional requirements of the seedlings, and fertigation yet at the nursery stage would be recommended to obtain seedlings suitable for transplant and marketing. On the other hand, it resulted in being also important not to provide too elevate fertilizer concentration, which would ultimately result in limited improvements of plant growth. Indeed, significant decreases in total, marketable yields and leaf transpiration in different lettuce cultivars were observed by Orsini et al., when salinity stress associated with excessive minerals was experienced [42]. Accordingly, lettuce has been considered a moderately salt-sensitive crop with a threshold electrical conductivity (EC) of 1.3 dS m −1 and a negative slope of 13.0 for each unit added salinity above this threshold value [43,44]. Finally, the three experiments highlighted the importance of using the natural resource available accurately, considering the impact of some technical choices also on climate change. The HM-mix substrate used in the three experiments has proven to be a suitable local substrate for seedling production. Considering the growth potential of the horticultural sector in Myanmar to contribute to food security [8], the adoption of such a substrate will have a lower impact on climate change by limiting the use of substrates commonly used for seedling production, such as peat. Peat comes from peatland ecosystems, which are very important for carbon sequestration. Hence, when peat is used as a substrate, the stored carbon is released, negatively affecting the environment and CO 2 balance [45]. The same is also true for the use of mineral fertilizers. Indeed, N 2 O is considered to be highly damaging to the ozone layer, and about 80% of anthropogenic N 2 O emissions are attributed to agriculture [46][47][48][49]. The second and third experiments provide indications on how to obtain good quality seedlings without exceeding in the use of mineral fertilizer, which, besides determining an economic loss for the growers, negatively impacts the environment.

Conclusions
The three experiments highlight the importance of adopting accurate substrates and nutrient solution concentration to improve vegetable seedlings production in semi-arid areas such as the Central Dry Zone of Myanmar. The study confirmed that proper substrate selection associated with a proper nutrient solution management might increase seedling quality and improve physiological parameters of lettuce and Chinese cabbage seedling production. Under limited resources, the substrate named HM-mixed and nutrient solution NS 1.2 allowed for obtaining high quality seedlings. An interdisciplinary approach and appropriate dissemination and knowledge transfer will be essential to guarantee that the seedling management methodology and technology for nursery purposes will be put in place adequately by local institutions and farmers.