Effect of Banana-Waste Biochar and Compost Mixtures on Growth Responses and Physiological Traits of Seashore Paspalum Subjected to Six Different Water Conditions

Abstract

The effects of pyrolyzed agricultural waste generated from banana leaves on the development and physiological responses of Paspalum vaginatum in different water conditions were investigated. X-ray diffraction (XRD) and Fourier-transform infrared (FTIR) computations were utilized to describe the banana-waste biochar and determine the crystalline structure and functional groups. A plastic pot was used in two trials to examine the effectiveness of the studied biochar under two situations (well-watered Ww and limited-watered Lw). Seashore paspalum was cultivated in loam soil that had been modified with biochar as a single addition and a biochar compost mix. Six water scarcity scenarios were chosen (100, 80, 60, 25, 20, and 15% of water holding capacity (WHC) of the control soil). To analyze the varied responses of P. vaginatum in well-watered and limited-water environments, principal component analysis (PCA) was used. Under Ww, photosynthesis, biomass, fluorescence, and chlorophyll content increased, whereas, under Lw and control, they declined. Biochar and compost combinations enhanced the relative water content (RWC) more than biochar alone or in combination. Conversely, stomatal density in drought-stricken plants showed the reverse trend.

1. Introduction

The scarcity of water is one of the most crucial matters in arid and semi-arid regions. One of these regions is Morocco’s south, where a lack of water has impacted agriculture, golf courses, and other industries. The region is regarded as a popular tourist destination in the Kingdom, attracting visitors from all over the world and encouraging turf managers to build golf courses there. However, turfgrasses must be watered regularly to have actively growing grass, but limited water resources make this irrigation difficult [1]. Furthermore, rising water supply prices make it challenging to manage irrigated turf areas on a budget [2].

Under these conditions, management options for high-value golf courses will be limited and costly. This fact has prompted scientists to consider new technologies for irrigating more efficiently and sustainably and achieve new goals by managing the arid and semi-arid region’s water resources. Several studies had recommended organic amendments such as peat because it increases nutrients and water, but it degraded quickly. Instead of the fast degradation of peat, it will require biochar and compost combination for green spaces, public parks, and golf courses in this region. The use of charcoal as an amendment for golf courses to enhance porosity and other properties was proposed over 92 years ago [3]. A similar recent study had revealed that compost with biochar could provide nutrient uptake in the root zones of grasses [4]. Because of its porous structure, biochar could be used as a golf amendment [4,5,6].

Seashore paspalum (Paspalum vaginatum Swartz), belonging to the subfamily Panicoideae, was chosen in this survey for its virtues and resistance to drought and salinization. Water-stress turfgrass studies received less attention than saline-stress studies [7,8].

A primary objective of the present research was to find the best organic amendment combination, which would allow reduced rates of water resources in the arid and semi-arid regions, with the same optimal turfgrass growth.

This study focused on the development of seashore paspalum in loam soil modified with biochar mixes, biochar, and compost combinations under six different water conditions: 100, 80, 60% (well-watered conditions), 25, 20, and 15% (limited water conditions). A pot experiment was conducted on this concern using seven levels of organic amendments addition (1) 0% B (Biochar) (control), (2) 2% B, (3) 2% Co (Compost), (4) 3% B, (5) 3% Co, (6) 4% B + C mixture of 2% of B (biochar) + 2% Co (Compost), and (7) 6% B + C mixture of 3% of B (biochar) + 3% Co (Compost). Thirty days P. vaginatum was rinsed with three water levels well-watered (Ww) (100, 80, and 60% of soil water holding capacity, (WHC)) for the first experiment, while for the second experiment, ninety days of stress water with the three water regimes in limited-water conditions (Lw) (25, 20, and 15% of soil WHC).

Physiological parameters and the growth of P. vaginatum (gas exchange, chlorophyll content, chlorophyll fluorescence, relative water content (RWC), leaf stomatal traits, and fresh and dry weight of aerial and underground biomass) were measured. Furthermore, the crystallinity and functional groups of banana-waste biochar were determined using X-ray diffraction and FTIR studies.

2. Materials and Methods

2.1. X-ray Diffraction and FTIR

The X-ray diffraction (XRD) analysis of banana-waste biochar was done by X-ray diffractometer (Bruker D8 Advance Twin, Karlsruhe, Germany) with Cu-Kα Radiation, (λ = 1.5405 Å) with a sweep of 5 to 100° and a step of 0.05°. In addition, Fourier-transform infrared (FTIR) (IRAffinity-1S Shimadzu, Japan) spectroscopy was used to detect the surface functional groups in the banana-waste biochar.

2.2. Experimental Design

Two experiments were carried out independently in this survey. The two works were established using the same soil. Soil samples were collected at depths ranging from 0 to 20 cm, and they were air-dried and sieved through a 2 mm mesh sieve. The soil was texturally classified as loam with sand (39.54%), silt (50.82%), and clay (9.65%). Furthermore, the soil pH was 8.62. Biochar was prepared from banana-waste feedstock at 201 °C, having pH 9.3, and detailed physicochemical properties of banana-waste biochar were reported previously [9]. The same commercial compost was used for the two experiments.

Seeds of Paspalum vaginatum Swartz were germinated in plastic trays (33.5 × 50 cm) filled with wet peat as a source of nutrients for seedling establishment. After 4 weeks, seedlings were transplanted into plastic tubes (26 cm length, 12.5 cm diameter; four plants per tube) filled with loam soil. Seven mixtures were prepared with four replications from each treatment: (1) 0% B (biochar) (control), (2) 2% B, (3) 2% C (compost), (4) 3% B, (5) 3% C, (6) 4% B + C mixture of 2 % of B (Biochar) + 2% C (compost), and (7) 6% B + C mixture of 3% of B (biochar) + 3% C (compost). Six watering treatments were applied in the two experiments: 100, 80, and 60% soil water holding capacities in the first experiment during four months and 25, 20, and 15% soil water holding capacities in the second experiment during six months.

At the beginning of each experiment, P. vaginatum was irrigated daily for eight weeks in the two experiments. For the first experiment, P. vaginatum was watered for the past 30 days, twice a week with three different water regimes: 100, 80, and 60%; the duration of the experiment was four months. The second experiment took six months, while the stress-application duration was three months with a minimum of irrigation water (25, 20, and 15%).

2.3. Gas-Exchange, Chlorophyll-Content, and Chlorophyll-Fluorescence Measurements

Photosynthetic gas-exchange variables were determined by using an Infra-Red Gas Analyzer (IRGA) (LCi-portable photosynthesis System, ADC, Hertfordshire, UK). Fresh leaves were placed inside the IRGA, and values of physiological characteristics were observed during the daytime between 9:00 am to 12:00 pm. Each treatment measurement was repeated three times. Fresh leaves of P. vaginatum seedlings were measured to extract chlorophyll a, chlorophyll b, chlorophyll a/b (chl a/chl b) ratio, and carotenoid in three biological replicates using a spectrophotometer (UV-1600PC; Shimadzu, Japan). The procedure described by Lichtenthaler was used [10].

Using the OS5p Modulator Fluorometer (Model OS5p, Opti-Sciences, Hudson, NH, USA), the chlorophyll-fluorescence parameters were measured on three leaves from the same treatment in the afternoon in the dark and in light. Before measuring photosystem-II activity, all leaf samples were kept in the dark for 30 min [11].

2.4. Relative Water Content (RWC)

The relative water content (RWC) of P. vaginatum leaves was determined using the technique [12,13,14].

2.5. Leaf Stomatal Traits

On the reverse side of the leaflet, stomata imprints were formed. After cleaning the leaf of P. vaginatum with a delicate brush, a thin layer of nail polish was applied to the surface and allowed to dry for 10–15 min. The region was then properly bandaged with transparent tape and removed. The samples were immediately inspected under an optical microscope (BX51, Olympus, Tokyo, Japan).

The number of stomata, density, and dimension parameters (LS: stomata length, WS: stomata width, Lo: ostiole length, and Wo: ostiole width) were all measured [14].

2.6. Statistics

A two-way analysis of variance (ANOVA) was used to evaluate the banana-waste biochar effect and the three water regimes (25%, 20%, and 15%) on the relative water content (RWC) at 6 am, and 12 am, to compare the relative water content in the two periods. Mean separation was accomplished using a Fisher test. Based on physiological, fresh, and dry-weight data collected during the two experiments, principal component analysis (PCA) was used to identify relationships between the different treatments (well-watered and limited water). The PCA was performed on 126 individuals and 17 response variables to evaluate the effect of banana-waste biochar and the three water regimes (25, 20, and 15%). On stomatal parameters, a PCA was carried out. All statistical analyses were performed with RStudio Version 1.4.1717.

3. Results

3.1. Characterization of Banana-Waste Biochar

X-ray Diffraction and FTIR

Biochars are frequently identified by measuring element concentrations in order to determine their potential as a nutrient. Identifying specific minerals and their abundance improves the understanding of element behavior. Powder X-ray diffraction (XRD) was used to identify minerals associated with pyrolysis residues to this end. Figure 1 depicts an XRD pattern of activated biochar made from banana waste and leaves. The mineralogical composition and mineral phases of banana-waste biochar were characterized by X-ray diffraction analysis. The mineral composition consisted of a long hump with such a centroid between 28 and 30 degrees, confirming the formation of the material of the main crystals. The major crystalline phase in the biochar was whewellite (Ca₈.₀₀C₁₆.₀₀O₄₀.₀₀H₁₀.₂₈), and the second phase was Calcite, syn CaCO₃. Whewellite (a calcium oxalate mineral; prominent peaks at 2θ: 26.15°, 28.60°, 29.71°, 36.72°, 38.31°, 39.81°, 40.82°, 43.48°, 47.69°, 48.71°, and 50.51°) was found in biochar made from banana feedstock (Figure 1). Indeed, calcite was found in banana-waste biochar (peaks at 2θ: 65.59° and 73.72°). At a value of 2θ = 50.60°, whewellite structures vanished and were totally replaced by calcite.

According to the FTIR spectra of banana-waste biochars (Figure 2), the following functional groups were present: OH alcohols appeared between 3500 and 3000 cm⁻¹ (3404 cm⁻¹) [15,16], C=O or C=C (1630 cm⁻¹), C-H alkanes (1426 and 1322 cm⁻¹), and C-O alcohols (1100 cm⁻¹) derived from cellulose component of banana waste. Similar trends were found by Monir et al. [15,16]. Aromatic C-H out of bending vibrations (878 and 785 cm⁻¹) associated with lignin was also detected. The peak at 1833 cm⁻¹ is attributed to C-H stretching, whereas the peak at 2363 cm⁻¹ corresponds to the alkyl C-H stretch. Phenol corresponds to the peak 3662 cm⁻¹. On the same spectra, the peaks (662 and 515 cm⁻¹) correspond to aromatic C-H bending functional groups [17].

3.2. Physiological Traits of P. vaginatum

A comparative study was done to assess the impact of different mixtures of banana-waste biochar and water conditions (well-watered and limited-water) and the effect of the duration of biochar (4 months for WW and 6 months of LW) on physiological traits of P. vaginatum grass (photosynthesis, fluorescence, and chlorophyll pigment) and plant growth (aerial and underground biomass).

The amount of variation explained by uncorrelated principal components (PCs) decreased in PCA as PC1 > PC2 > PC3.

The PCA illustrated relationships among variables and treatments. Biplots were used in this research to describe the similarities and differences in P. vaginatum responses to six different water conditions.

In addition, the biplot of PC1 and PC2 was able to explain 43.4% of the total variance. PC1 had high positive scores for the fresh weight of the aerial biomass (FWBA), the fresh weight of the underground biomass (FWBU), the dry weight of the aerial biomass (DWBA), and the dry weight of the underground biomass (DWBU), whereas the eigenvector for PC2 had high positive scores for E, A, chl a, chl b, and chl a/b (transpiration, photosynthesis rate, chlorophyll a, chlorophyll b, and chlorophyll a/b ratio) (Figure 3). For instance, PC1 and PC2 showed that 4BC_15, meaning (96% of dry soil mixed with 2% of banana-waste biochar and 2% of commercial compost at 15% of WHC) treatment had the high values of E, A, chl a, chl b, chl a/b, as well as developed aerial and underground biomass, even under 15% WHC.

For the PC1 and PC3 biplot, which explained 35.4% of the total variance, it exhibited that the electron transport rate (ETR) and the effective photochemical quantum yield of PSII Y(II) were higher for deficit water treatments compared to well-watered treatments (Figure 4).

Figure 4 shows that the maximum photochemical quantum yield of PSII (Fv/Fm) was greatly higher in amended water-limited treatments compared to amended well-watered treatments. The same result was found for intercellular CO₂ concentration, on which amended treatments under drought conditions showed higher value compared to amended well-watered treatments.

Hence, the PC2 and PC3 biplot, which explained 32.6% of the total variance (Figure 5) showed that all well-watered treatments had a higher fluorescence, chlorophyll pigment, and photosynthetic values, except for the control and the 2% biochar under 60% WHC (2B_60), compared to limited-water treatments, which had a low value.

3.3. Effect of Agricultural Biochar on Relative Water Content (RWC)

Two parameters were measured, RWC and stomata, to test the effect of the burial of biochar made by agricultural waste during six months. Figure 6 and Figure 7 show significant water stress and biochar on leaf relative water content at predawn and midday periods. Indeed, a tremendous increase was recorded in the amended treatments with the banana waste and compost combination (4% B + C and 6% B + C) at the three water regimes. The lowest RWC was observed in the control treatment in all water supplies (15, 20, and 25%).

The highest mean of RWC at 6 am (95.22%) was recorded in 6% B + C at 25% WHC treatment, while the lowest (40.39%) was found at the control under 15% WHC treatment (Figure 6).

Concerning the RWC at midnight, the highest mean value (73.96%) was recorded in the 4% B + C treatment at 25% WHC in Figure 7.

3.4. Effect of Agricultural Waste on Leaf Stomatal Parameters

Five stomatal parameters’ data were subjected to PCA to assess the effect of banana-waste biochar and compost combination application, biochar as a singular addition, and unamended treatments under drought. This study used the biplot and the hierarchical cluster to visualize how different levels of organic amendments’ application and three water conditions’ (15, 20, and 25%) information can impact stomatal parameters (Figure 8 and Figure 9).

The PC1 and PC2 biplot, which explained 63.7% of the total variance, provided a better subjective separation visualization among the three water regimes (15, 20, and 25%). Indeed, the ostiole length (Lo) was higher in treatments amended by biochar and compost combinations than unamended treatments. (Figure 9). The same was found for the width of stomata (WS) and the length of stomata (LS), which means that stomata grow in amending plants.

Accordingly, the hierarchical clustering distinguished three groups of treatments. It highlighted the overall performance of the treatments obtained by analyzing the stomatal traits’ data (Figure 9). The first group contained four treatments (3C_15, 6BC_15, 6BC_25, and 4BC_20): 97% of dry soil mixed with 3% compost at 15% of WHC, 3% compost plus 3% of banana-waste biochar added to 94% of dry soil at 15% of WHC, 94% of dry soil mixed with 3% of compost, 3% of banana-waste biochar at 25%, 2% of compost, 2% of banana waste added to 96% of dry soil at 20% of WHC had the highest values of Lo, WS, and LS, respectively. The second group (2B_20, CTR_15, CTR_20, 3B_20, 2B_15, 3B_25, 3C_20, 2C_25, 4BC_15, and 3B_15) contained treatments amended with biochar or compost as a singular application or unamended treatments (CTR: control that is only 100% of dry soil). All those ten treatments revealed a high value of stomatal density even under drought. The third group (2B_25, 3C_25, 2C_15, 2C_20, CTR_25, 6BC_20, and 4BC_25) had the greater values of ostiole width.

4. Discussion

In the present study, both banana-waste biochar and drought significantly altered seashore paspalum grass growth and plant biomass. In fact, the banana biochar has a crystalline character, as seen by the XRD graphs (Figure 1). Whewellite structures did indeed disappear at 2θ = 50.60° and were entirely replaced by calcite. Moreover, the temperature had increased at the end of the pyrolysis, which is why the calcite phase was visible. The calcite form had assisted the P. vaginatum establish its growth responses even in hard conditions (15% of WHC) due to the crystalline character of the banana-waste biochar, and this was because the calcite form is more soluble by plants than whewellite (calcium oxalate, Ca(C₂O₄). H₂O). Singh et al. [18] discovered that when the pyrolysis temperature of eucalyptus char increased, the change in calcium oxalate to calcite became more noticeable. Indeed, the ability of biochars to neutralize acidic soils may be influenced by the presence of various Ca-containing minerals. When compared to carbonate, oxalate may be less successful at neutralizing acidic soils [19]. The calcite formed, as a result, contributed to the alkalinity of the banana-waste biochar investigated, as seen by their high pH values of 9.30 [9]. In addition, the FTIR technique’s results can be utilized as an effective tool for controlling biochar quality in production circumstances, and, as a result, its impacts on plant growth. Indeed, the FTIR spectra of banana-waste biochar were used to deduce functional groups (Figure 2). In fact, it showed the existence of the following functional groups, with aromatic groups being the most prevalent in the FTIR spectra when compared to other functions. Banana-waste biochar may be resistant and have carbon-sequestration potential due to the preponderance of aromatic carbon groups. This FTIR result could explain why banana-waste biochar has a favorable effect on P. vaginatum even when it is underwater stress [20]. Photosynthesis metrics including chlorophyll concentration, chlorophyll fluorescence, and gs are frequently employed to assess plant adaptability to various environmental conditions. Figure 3 showed that treatments amended with the combination of banana-waste biochar and compost revealed the greatest result in physiological traits of the P. vaginatum. Despite the grass being exposed to severe water conditions, this result can be explained by the fact that the P. vaginatum grass has been adapted to the drought conditions, and, as a result, it developed its roots and had a high photosynthetic response. This means that amending soils with biochar and compost combination can provide grass nutrients and organic matter even in limited-water conditions. These findings are consistent with previous studies on which they found that biochar increased plant growth under drought conditions [21,22]. Katuwal et al. [23] reported that under drought, seashore paspalum limited the gas-exchange process by maintaining its stomatal conductance and closing its stomata. Furthermore, Pompeiano et al. [8] found that seashore paspalum and bermudagrass could increase roots even in salinity intermediate stress. Figure 3 also shows that the two treatments of soil amended with compost at 80% and 100% of WHC (2C_80 and 2C_100, respectively) had a higher physiological response. The current outcomes are in line with the study of Vaughn et al. [4] on which they reported that adding compost or peat as organic amendments do not persist on soils and degraded quickly, while the combination of biochar and compost had more longevity persisting and aids soils to retain nutrients and water due to the porosity of charcoal. Figure 4 revealed an increase in ETR and Y(II) under the three water regimes considered regarding the stress conditions for this study (15, 20, and 25%). This result was surprising for us; in fact, the increase in the two-fluorescence activity can be explained by amending with banana-waste biochar and compost mixtures. When banana-waste biochar was studied with a scanning electron microscope in a prior study, it revealed a number of pores [9]. Indeed, the porosity of the studied biochar helped soils to maintain more water and nutrients even in harsh conditions. Moreover, its richness with calcite mineral phases due to the increase in the temperature at the end of the pyrolysis enabled soils to retain more water, even when water was scarce, which explains P. vaginatum‘s ability to withstand harsh conditions. As the inverse of these findings, Abideen et al. [24] found that ETR and Y(II) decrease under drought.

In this investigation, the comparative analysis of the pigment content showed an increase with well-watered treatments in a short time-course experiment (4 months) compared to stressed treatments in the experiment of six months (Figure 5). These findings on pigment responses under the two water conditions are comparable to previous research [25] in response to drought on the photosynthetic pigments of various leaves species.

Under drought conditions, Abideen et al. [24] reported that biochar application increased leaf chlorophyll content and photosynthetic activity in Phragmites karka. The same observation to these results was revealed in cucumber and other plant experiments [26]. Moreover, Abideen et al. [24] revealed that a greater concentration of chlorophyll in leaves is an indicator of stress resistance.

Water stress caused a considerable reduction in RWC. In fact, with the increasing severity of water stress, RWC decreased more. Figure 6 and Figure 7 revealed that with the two regimes (20 and 25%), RWC increased when biochar and compost were added to the soil compared to non-biochar treatment, which means that RWC increased considerably in amended treatments. The current findings of this study are in line with the study of Hafez et al. [27], on which they found that RWC improved prominently with vermicompost-biochar mixture application than biochar as a particular application. Farhangi-Abriz and Torabian [28] noticed that using 5 and 10% biochar increased the relative water content of the bean significantly. Kammann et al. [29] realized that biochar reaches a level of plant water status by implying a better susceptibility to prospective water and saline environmental stress by continuing to increase these same osmotic values of the leaf and reducing transpiration, which is consistent with the present findings.

In contrast, Abideen et al. [24] found that RWC had decreased with adding 2.5% biochar under drought, which diminishes relative water content for plants under drought.

The PCA results showed that the amount of biochar is the key to determining the stomatal morphology of P. vaginatum leaf. Figure 8 shows that even with the addition of compost and biochar, stress levels diminished stomatal density (SD). Previous studies have found that drought increases leaf stomatal density, such as in Solanum melongena [30]. SD decreased in ginger, which is consistent with these findings [31]. Furthermore, Xu and Zhou [32] observed that under modest drought, stomatal density continued to improve with reducing overall water, whereas under severe drought, SD decreased, implying that grass has the ability of leaf plasticity in response to environmental changes.

5. Conclusions

Overall, this study highlighted the comparison between the different P. vaginatum responses with six water conditions, depending on the effect of banana biochar and compost combination and the duration of amendment application. The results obtained in this survey indicated that the 4% biochar and compost under 15% WHC treatment had a more incredible response for the photosynthetic activity and developed the roots under drought, which is a quality to resistance. Furthermore, the seashore tended to positively affect specific parameters, mainly through increased relative water content for amended treatments compared to unamended treatments and increased stomatal density under the three water stresses (15, 20, and 25%). P. vaginatum had to resist the drought conditions by limiting gas-exchange processes and closing stomata. Therefore, the banana biochar and compost addition had succeeded in improving plant growth in poor soils with 4% B + C. This addition had led to increased biomass production of seashore paspalum by improving plant water status, photosynthesis, and chlorophyll pigment. These physiological parameters responded positively to biochar and compost mixtures’ application, leading to enhanced biomass production and conserved water. This work could be a prominent reason for managing luxury golf courses under arid and semi-arid zones.