Effects of the Physicochemical Properties of Lignocellulosic Artificial Soil Containing Bacillus subtilis on the Growth of Lespedeza cyrtobotrya

Commercially available peat moss (pH 3.5–4.5, Sunshine, Canada), perlite (S company, Seoul, Korea), and carboxyl methyl cellulose (CMC, K company, Daejeon, Korea) were used in these experiments. Unseasoned Quercus mongolica chips were processed at a pressure of 25 kgf/cm² in a high-temperature/ pressure processing machine (Y company, Daegu, Korea) for 5 min. Ethanol (10%) was extracted from the wood chips at 60 °C for 3 h (1:20 w/v) and subjected to decompression filtration. The resulting solids were used in the next steps of the experiment.

Bacillus subtilis, the bacterial test strain used in this study, was acquired from H company (Jinju-si, Gyeongsangnam-do, Korea). The microbes were cultured in liquid broth at 30 °C and 100 rpm for 4 days. Subsequently, 30 mL of the culture was added to 30 mL of 2% CMC and incubated in 100 g of sterilized perlite at 30 °C for 8 days. The microbes were dried at 35 °C for 48 h and formulated with a freeze dryer to be used as a raw material for the artificial soil. Artificial soil was prepared by mixing peat moss, perlite, pre-processed Quercus mongolica chips, and the microbial formulation in a ratio of 3:1:3:3 (w/w/w/w).

Before the experimental plots were established, the control soil (wetland soil from Cheongseongsan, Yangsan-si, Gyeongsangnam-do), 25% mixed soil (75% natural soil + 25% artificial soil, w/w), 50% mixed soil (50% natural soil + 50% artificial soil, w/w), 75% mixed soil (25% natural soil + 75% artificial soil, w/w), and 100% artificial soil were prepared (based on dry weights). The plots were set up by randomly placing the soils in cube-shaped pots (7.5 × 7.5 × 7.5 cm³) on a sloped surface at a latitude of 35.14N and longitude of 128.09E. The pots were watered once every 3 days to prevent the soils from drying. Subsequently, 50 seeds of the woody plant L. cyrtobotrya were planted in the plots to analyze plant growth. The physicochemical properties and microbial densities of the control, mixed, and artificial soils were also assessed.

The control, mixed, and artificial soils were dried at 60 °C for 24 h before analysis of the physicochemical properties. To measure bulk density, a 100-cm³ core was filled with the sample, and pressure was applied for 3 min using a 500-g weight. Next, the top of the core was vertically cut from the container surface, and the sample was weighed to determine the bulk density (CEN, 1999a, 1999b; Byun et al., 2012). Porosity was calculated by applying the specific gravity (2.65) to the bulk density measurement (Inbar et al., 1993). To measure the water-holding capacity, a 100-cm³ core was filled with the sample and pressured as described above. The sample was saturated with water, which was allowed to drain under gravity over 24 h. The final weight of the dry sample was then measured to determine the water-holding capacity (Yi et al., 2012). The sample was mixed with distilled water (1:5, w/w) and then placed in a shaking incubator for 30 min, after which the pH was measured using a pH meter (HI-8417, HANNA Instrument, USA). The electrical conductivity (EC) of the suspended filtrate was measured using an EC meter (Orion 3-Star, Plus, Thermo Fisher Scientific, USA). The content of organic matter was measured by completely carbonizing the sample in a muffle furnace (NIAST, 2000) at 550 °C. The carbon-to-nitrogen (C/N) mass ratio was calculated using an elemental analyzer (Flash 2000 Series, Thermo Fisher Scientific Inc., USA).

The 50 L. cyrtobotrya seedlings were allowed to germinate for 3 months; subsequently, 5 seedlings were randomly selected for plant growth analysis. The stem heights and root lengths of the seedlings were measured with a pair of calipers (Digital calipers, Blue bird, China) (Lee et al., 2013).

A total of 10-g (dry weight) samples of control, mixed, and artificial soil was collected before establishment of the plots, and equivalent 10 g of samples were collected taken after 3 months. The soils were mixed with 90 mL of sterilized 0.85% NaCl solution and then placed in a shaking incubator (Vision Co., Bucheon, Korea) at 200 rpm for 30 min. The suspension was serially diluted, smeared, and cultured on R2A agar (NA, Difco, Detroit, MI) badge containing cycloheximide (0.05 g/L). The plates were incubated at 28 °C for 2 days, and the number of colonies was counted. For each sample, the microbial number (CFU/ml) was calculated by averaging the counts from three Petri dishes (Oh et al., 2014).

All statistical analyses were performed using IBM SPSS statistics v25 (IBM Corp., Armonk, NY, USA). The Duncan’s multiple range test (p < 0.05) was conducted to determine significance levels between the means of the experimental plots. Pearson’s correlation coefficient analysis was used to investigate the correlation between the physicochemical properties of artificial soil and root/stem growth.

Comparisons of the physical properties (e.g., bulk density, porosity, and water-holding capacity) of different soils are important because these parameters affect plant growth significantly (Boodley, 1998). Table 1 summarizes the bulk densities, porosities, and waterholding capacities of the control, mixed, and artificial soils.

The bulk densities of the 25% and 50% mixed soils were 0.24 and 0.16g/cm³, respectively. These measurements were significantly lower than that of the control soil (0.51 g/cm³). The observed gradual decrease in bulk density was associated with an increase in the percentage of artificial soil. The level of organic matter in soil is thought to be negatively correlated to bulk density (Ju et al., 2015). The decrease in bulk density due to the presence of artificial soil has the advantage of improving water-holding capacity and air permeability (Nelson, 2003). Porosity has been shown to affect the drainage and water-holding capacity of soils and the growth of plant roots (Schoenholtz et al., 2000; Liebig and Doran, 1999). Plants are reported to grow well in soils with a porosity of ≥85% (De Boodt and Verdonck, 1972; Gruda and Schnitzler, 2004). Although the control soil had an inadequate porosity of 80.7%, this porosity increased to 90.8%–95.5% by increasing the mixing ratio of the artificial soil. Although an increase in porosity can be beneficial for plant growth, it may also lead to an increase in permeability because moisture migrates through the pores, thereby subjecting plants to significant dehydration-induced damage (Shin, 2002). Therefore, natural and artificial soils must be mixed according to the soil conditions at the planting site, rather than solely using artificial soil with a high porosity. The water-holding capacity of a soil is the amount of water that it can store and is known to affect the distribution of water into the plant, a process that is essential for growth (Yang et al., 2013). The water-holding capacities of the control, 25% mixed, 50% mixed, 75% mixed, and artificial soils were found to be 59.9%, 66.1%, 76.5%, 79.8%, and 83.3%, respectively. These findings demonstrate the positive correlation between water-holding capacity and the mixing ratio of the artificial soil. The water-holding capacity of the soils in all the experimental plots (except the control) was > 60%, which is considered adequate (Yeager et al., 1997). Basso et al. (2013) and Lawes et al. (2009) have reported that high water-holding capacity is beneficial for long-term plant growth and soil loss prevention. These benefits can be achieved by the on-site application of artificial soil as prepared in this study. In addition, increases in the bulk density, porosity and water-holding capacity of the soils were achieved by increasing the ratio of the artificial soil, relative to the control soil.

The chemical properties of soils are important determinants of plant growth and appropriate environment (Gabriels et al., 1986). Accordingly, a comparative analysis of the soil components should be conducted with reference to ratios of control to artificial soil. Table 2 summarized the pH, EC, C/N ratio, and organic matter content of the control, mixed, and artificial soils.

The pH of the control, 25% mixed, 50% mixed, and 75% mixed soils were 4.8, 4.7, and 4.6, respectively. These values are lower than the optimal pH range for plant growth, i.e., 5.3–6.5 (Abad et al., 1992). Significant differences in pH were not observed between the soils containing different percentages of artificial soil. The artificial soil was found to have the lowest pH at 4.3. Yamazaki (1982) have reported that plant roots are easily damaged at a pH of <four. These observations highlight the need to adjust the pH of soils according to local conditions to optimize plant growth. The EC of the control, mixed, and artificial soils were measured between 0.061 and 0.101 ds/m. ECs of ≤0.5 ds/m are generally considered adequate for plant growth (Abad et al., 2002). High ECs can result in salt accumulation and poor conditions for root growth (Aliasgharzadeh et al., 2001). As such, we suggest that the low EC of the artificial soil prevents salt-induced growth inhibition. A high C/N ratio promotes microbial absorption of nitrogen in the root-zone around the plant, thereby hindering early growth (Choi et al., 2011). For this reason, nitrogen fertilizers should be added if the C/N ratio of the artificial soil is ≥ 50 (Lee and Sang, 1991). Sou et al. (2011) have reported a C/N ratio of 17.4 in a habitat containing L. cyrtobotrya. In the present study, the 25% mixed, 50% mixed, and 75% mixed soils showed C/N ratios ranging from 15.0 to 19.8, thus suggesting that varying amounts of artificial soil are unlikely to inhibit early growth. The control soil showed the lowest organic matter content (23.6%); however, as the mixing ratio of the artificial soil increased, the organic matter significantly increased from 27.6% to 43.2% possibly because of the addition of pre-processed oak and peat moss, which are rich in organic materials, to the artificial soil. High levels of organic matter provide large amounts of nutrients to the plant and improve the cation exchange capacity necessary for small nutrient exchanges (Wood et al., 2018).

Table 3 presents the microbial densities before and after the on-site application of the control, mixed, and artificial soils in small outdoor pots.

Prior to the on-site application, the control soil had the lowest microbial density of 3.5 × 10⁷ CFU/g. This microbial density increased from 1.57 × 10⁸ to 6.24 × 10⁸ CFU/g as the ratio of the artificial soil increased.

After 3 months, the microbial density of the control soil had decreased by 13.4%. Conversely, the microbial numbers of the 25%, 50%, and 75% mixed soils and the artificial soil had increased by 61.1%, 30.8, 21, and 36.9%, respectively. These increases can be explained by the ability of microbes to use organic carbon and nitrogen sources as macronutrients for plant growth. Moreover, the artificial soil prepared in this study contained peat moss and wood materials comprising organic matter that is beneficial for microbial growth (Barber and Lynch, 1997; Lynch, 1982; Krieg and Holt, 1984). An increase in microbial density is known to positively affect plant growth (Nautiyal et al., 2013).

Fig. 1 illustrates stem heights and root lengths of L. cyrtobotrya planted in small outdoor pots containing the control, mixed, and artificial soils.

Fig. 1. Effect of mixing ratio of artificial soil with soil on growth of Lespedeza cyrtobotrya.

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The longest stem was 5.1 cm in the 25% mixed soil. Stem lengths in the 50% and 75% mixed soils and the artificial soil were 4.8, 4.4, and 3.5 cm, respectively. Stem growth decreased in the 50% (or above) mixed soil. The longest root length was 11.9 cm in the 50% mixed soil, followed by 10.5, 8, 7, and 5 cm in the 25% mixed, control, 75% mixed, and artificial soils, respectively, although artificial soil is thought to improve plant growth by improving water and air permeability (Warkentin, 1984; Kang et al., 2004); however, this association was not observed between stem and root lengths and the ratio of artificial soil in the potting mix. This suggests that the appropriate mixing ratio of artificial soil must be determined for the target plant.

Table 4 shows a correlation analysis of the effects of the physicochemical properties of soils on the stem and root growth of L. cyrtobotrya.

A positive correlation between the physicochemical properties of the soils and stem/root growth was observed in terms of microbial density, C/N ratio, organic matter content, and water-holding capacity (p < 0.01). Microbial density and stem growth showed the most significant positive correlation with a correlation coefficient of 0.599. Our finding that porosity correlates with stem growth is in agreement with the report of Jo et al. (2017). The root growth of L. cyrtobotrya was found to positively correlate with bulk density (p < 0.01) but negatively correlate with porosity, water-holding capacity, C/N ratio, and organic matter content.