Influence of Rice Husk Biochar on Soil Nematode Community under Upland and Flooded Conditions: A Microcosm Experiment

Abstract

Biochar has the potential for improving soil properties and supporting ecological functions, but it has negative impacts on soil organisms in some cases. This study aimed to assess the effect of biochar application at rates of 0 (B0), 5 Mg ha⁻¹ (B5), 20 Mg⁻¹ (B20), and 40 Mg ha⁻¹ (B40) on soil nematode community under upland and flooded conditions in a short-term microcosm experiment. After biochar application, soil was incubated for 2 to 8 weeks and nematodes were identified for community composition, trophic structures, functional guilds, maturity index and metabolic footprints. The chemical properties of the soils were also analyzed. General linear model revealed that biochar increased soil pH, EC, NO₃⁻-N, available phosphorus, total C, and C/N ratio, particularly in the highest application rate and shifted the composition of nematodes. The greatest abundances of omnivores (Mesodorylaimus, Thornenema), predator (Nygolaimus) and functional guilds of cp5 were observed in B5, resulting in greatest structure footprint and composite footprint, (omnivorous + predator) footprint and total biomass in B5. While abundances of nematodes tended to decrease with the biochar applicate rates, the abundance of Prismatolaimus was the highest in B40. During the 8-week incubation period, the abundances of Achromadora, Alaimus, Aporcelaimellus, Cryptonchus, Mononchus, and Tobrilus remained stable in upland conditions. Under flooded conditions, the abundances of almost all taxa were markedly lower than those under upland conditions irrespective of biochar application, except for Acrobeloides, Alaimus, Aphelenchoides, and Ditylenchus. We highlighted that 5 Mg ha⁻¹ of rice husk biochar can be the optimum in shaping the nematode community.

1. Introduction

Biochar is a pyrolysis product of various organic resources, such as agricultural residue biomass and livestock waste, under limited oxygen conditions [1]. In recent years, biochar has been widely used and considered as a soil amendment to improve biological and physicochemical properties and crop productivity, and to mitigate environmental impacts [2,3,4]. In general, the application of biochar increases total carbon storage and reduces greenhouse gas emission [5,6,7]. Besides, biochar contributes to several key roles in biological functions by stimulating the activities and biomass of microbial communities and organisms in higher trophic levels [5,8,9].

The addition of biochar has drawn more attention due to its potential effects on ecological functions. However, contrasting effects by biochar addition are reported on soil organisms [10], including nematodes [11,12,13,14], and higher trophic levels of consumer fauna [15]. For instance, Kamau et al. [4] reported that the application of 10 Mg ha⁻¹ wood tree (Prosopis juliflora) biochar reduced the abundance of bacterivorous nematodes in the soil by more than eight times. On the contrary, Liu et al. [16] reported that the application of 4.5 Mg ha⁻¹ peanut shells biochar increased the total abundance of nematode communities, particularly that of bacterivorous nematodes. Such contrasting effects of biochar on soil organisms may depend on the rates, material sources, processing conditions, and timing of application, and soil conditions, e.g., pH, soil moisture and temperature [17,18]. The application of biochar directly changes the soil physicochemical properties, e.g., soil pH, porosity and aggregate components [19], and water retention [20,21] that are involved in factors to promote nematode community [12,16]. For instance, Kitagami et al. [22] reported that soil pH is a key factor that shifts the distribution pattern of nematodes. In addition, biochar application changes the microbial community, which may indirectly change the soil nematode composition through their prey–predator interactions [23]. Further factors may come from the toxicity of polycyclic aromatic hydrocarbons (PAHs) [24,25], which are produced during the pyrolysis of organic materials [26]. A high amount of biochar application (20 Mg ha⁻¹) introduced a significant amount of PAHs [27,28] which reduced the abundances of fungivorous and omnivorous/predator nematodes [25] and shifted the nematode community in freshwater environment [24]. Therefore, the biochar addition needs to be carefully considered based on the overall effects on soil organisms [13].

Nematodes are a diverse group of metazoans and play key functions in an agroecosystem [29,30,31]. Soil-dwelling nematodes contribute to a wide range of the belowground food webs that support the nutrient turnover pathway [32,33]. Therefore, nematodes are a potential indicator for predicting the ecological service and for proposing good soil management techniques. For this purpose, feeding structure and functional guilds of nematodes have been investigated [34,35]. For instance, the application of compost increased the total abundance of nematodes and changed their trophic structure [36,37], and suppressed plant-parasitic nematodes in organic farming [38]. Further, Neher et al. [39] reported that crop rotation significantly changed the soil nematode community. Conventional tillage shifted the nematode community composition and diversity by changing the soil physicochemical properties in a wheat field [40,41].

We hypothesized that nematodes may have species-specific responses to different biochar application rates under different moisture conditions. To clarify the effects of biochar on soil nematode community under upland and flooded conditions, this study aimed to find out (1) the responses of species-specific nematode community to biochar application rates, and (2) the changes in its trophic structures, functional guilds, and metabolic footprint as well as soil physicochemical properties.

2. Materials and Methods

2.1. Soil and Biochar Preparations

Soil was collected from a paddy rice field from 0 to 15 cm at harvest. After removing plant residues, the field moist soil was passed through a 5 mm sieve. Some of soil properties were pH (H₂O) 6.2, electric conductivity (EC) 0.15 mS cm⁻¹, total C 59.1 g kg⁻¹, total N of 5.3 g kg⁻¹, C/N ratio 11.1, NH₄⁺-N 0 mg kg⁻¹, NO₃⁻-N 34.5 mg kg⁻¹, available phosphorus (P-Bray II) 128 mg kg⁻¹, and 1.33 g H₂O g⁻¹ dry soil of maximum water holding capacity (MWHC).

Rice husk biochar was produced by pyrolysis in a chamber at 600–700 °C at the College of Environment and Natural Resources, Can Tho University, Vietnam. The biochar was the same as reported in our previous study [42] and had the following properties, pH (H₂O) 7.6, EC 0.50 mS cm⁻¹, total C 479 mg g⁻¹, total N 1.51 mg g⁻¹, total P 771 mg kg⁻¹, MWHC 5.8 g H₂O g soil⁻¹, water content 8.4 %, and iodine number 173 mg g⁻¹ (equivalent to 167 m² g⁻¹ as the total surface area) [43].

2.2. Biochar-Amended Soil

Five hundred grams of moist soil (corresponding to 323 g dry soil, 0.55 g H₂O g⁻¹ dry soil) and biochar were mixed thoroughly and filled into a cylindrical polyvinyl pot (14.0 cm height and 11.2 cm diameter). The following 8 treatments (4 levels of biochar × 2 levels of moisture condition) were set up in triplicate, resulting in a total of 24 pots (Figure 1). Biochar was amended at rates of 0, 5, 20, and 40 Mg ha⁻¹ equivalent to 0, 1.75 g (0.54%, w/w), 7.0 g (2.2%, w/w), and 14.0 g (4.3%, w/w) per pot, respectively, assumed the homogeneous application of biochar to 0–10 cm depth soil with a specific gravity of 0.93 g cm⁻³, a practical value of the soil. For upland conditions, the soil moisture content was adjusted to 70% MWHC, and for flooded conditions, water level was maintained at a height of 2 cm on the surface throughout incubation. All pots were placed randomly in an incubator at 30 °C under dark conditions. The moisture content in soil was monitored daily and adjusted to the initial water contents.

2.3. Sampling

Soil samples were uniformly collected from the surface to the bottom of the pot using a syringe (TERUMO 20 mL, with 2.0 cm diameter and 9.0 cm length) at 0, 2 weeks, 3 weeks, 4 weeks, and 8 weeks of the inoculation period. Fifty grams of wet soil were collected each time. Soil remained in the pot was then homogenized by a spoon to fill the holes made by sampling. Immediately after sampling, 20 g of wet soil were used to extract nematodes with the Baermann method, as described below. Fifteen grams of wet soil were air-dried and sieved through a 0.5 mm sieve for analyzing soil pH, EC, total C, N, and available P. The remaining wet soil (15 g) was used to analyze ammonium (NH₄⁺-N), nitrate (NO₃⁻-N), soil moisture and microbial activity.

2.4. Soil Physico-Chemical Properties Analysis

The air-dried and sieved soil was stored in a cold room until analysis. Soil moisture was analyzed by drying a known weight of moist soil in an oven at 105 °C for 48 h. Soil pH and EC were analyzed using the air-dry soil in a ratio of 1:5 of soil weight and deionized water after shaking for 1 h at 120 rpm and centrifuging at 8000 rpm for 5 min. A compact pH meter (LAQUAtwin-pH-22, HORIBA, Kyoto, Japan), and a compact electric conductivity meter (LAQUAtwin-EC-33, HORIBA, Japan) were used. Inorganic nitrogen (NH₄⁺-N, NO₃⁻-N) was extracted using 2 M KCl solution in a ratio of 1:10, sharking for 1 h, centrifuging at 8000 rpm, and filtering through ADVANTEC 5C filter paper. Ammonium was analyzed with the indophenol-blue method [44] and nitrate was measured with a spectrophotometer at a wavelength of 220 nm. Available phosphorus was measured with the Bray-II method [45]. Briefly, two grams of air-dry soil was used to extract with the combination of acidic 0.1N HCl and 0.03N NH₄F in a ratio of 1:7. Total C and N were analyzed using 0.5 g of air-dry soil with a CN Coder MT-700 (Yanaco Technical Science Co., Tokyo, Japan). After two months of incubation period, the remaining soil was packed into a 100-mL core and the moisture contents at pF 1.8 and pF 3.0 were measured to estimate the available water content using a multi-fold pF meter (Daiki Rika Kogyo Co., Ltd., Konosu, Japan).

2.5. Microbial Activity Measurement

Microbial activity was measured as the respiration of 5.0 g moist soil. Wet soil collected at every sampling time was mixed thoroughly and put into a vial of 10 mL volume. An amount of 0.5 mL air in the headspace of vial was taken immediately after closing the lid and injected into a TCD-GC (Shimadzu-8A). Then, the soil was incubated at 25 °C for 3 h and 0.5 mL air was taken again. A 0.5 mL gas of pure CO₂ was used as a standard. The differences in CO₂ concentrations between 0 and 3 h were considered as the microbial activity.

2.6. Nematode Community Assessment

Twenty grams of moist soil were put on a tissue-paper (Kimwipes S-200) supported by a sieve (65 mm diameter and 1 mm mesh size). Then, the sieve was placed in a plastic funnel filled with tap water and kept for 48 h at room temperature (ca. 25 °C). Then, nematodes were fixed by adding 1 mL of a 70 °C hot solution of 4% formaldehyde into a 0.25 mL nematode suspension. A drop of 1% Rose Bengal was added to stain the nematodes. The next day, the total nematodes were counted under a microscope and converted to the density per 100 g of dry soil. At least 100 individual nematodes were picked out at random into a micro Petri dish containing 1 mL solution I (99 parts of 4% formalin solution and 1 part of glycerin), then put into a desiccator saturated with ethanol at the bottom. The desiccator was kept in an oven at 40 °C for 24 h; the next day the Petri dish was taken out of the desiccator, and ¾ partially covered by a glass piece on top to allow slow evaporation of the ethanol and placed into the oven. Solution II (95 parts of ethanol 98% and 5 parts of glycerin) was added every 2 h for a total of 4–5 times, then solution III (50 parts of ethanol 96% and 50 parts of glycerin) was added. Finally, individual nematodes were mounted into a drop of glycerin on a glass slide and sealed with a paraffin ring. These slides were used during the identification process for nematodes. The nematodes were identified up to a genus level for further calculation of trophic structures such as bacterivorous, fungivorous, omnivorous, predator and herbivorous [46], functional guilds as a colonizer-persister scale (cp1–5) [47]. The specific online program, Nematode INdicator Joint Analysis (NINJA), was used to calculate the maturity index, total biomass, and metabolic footprints at https://sieriebriennikov.shinyapps.io/ninja/, accessed on 24 December 2021 [48,49].

2.7. Statistics

The general linear model was performed to analyze the effects of independent factors as biochar application rates (0, 5, 20, and 40 Mg ha⁻¹), water regimes (upland and flooded), and sampling times (2, 3, 4, and 8 weeks), and their interactions on soil chemical properties, nematode abundance, trophic structure, functional guilds, metabolic footprint and the ecological index. Abundance of nematodes, trophic structure, functional guilds, biological index and metabolic footprint were log (X + 1) transformed to obtain the normality of variables by a normal distribution by Kolmogorov–Smirnov’s test and equal variances by Levene’s test. One-way ANOVA was performed to test significant differences among biochar application rates within each water regime corresponding to each sampling time. The post hoc Tukey’s HSD test was performed to compare differences among treatments. To find out the main corresponding variables of soil chemical properties, microbial activity and nematode composition among locations, the principal component analysis (PCA) was performed. PCA was run on a full set (normalized data) (N = 48) of nematode abundance both upland and flooded conditions at sampling times from week 2 to week 8 with the corresponding soil chemical properties and microbial activity to get the same metric for all variables. Statistical analyses were performed using the statistical package STATISTICA version 7 and Minitab version 16.

3. Results

3.1. Biochar Affected Soil Physico-Chemical and Biological Properties

Biochar increased total C by 14.2% and 26.0% in B20 (p < 0.001) and B40 (p < 0.001), respectively, compared to control (Figure 2A). Biochar did not affect total N in both water regimes (Figure 2B). Biochar increased C/N ratio in B20 (p < 0.001) and B40 (p < 0.001) compared to control (Figure 2C). Biochar also significantly (p < 0.01) increased the available water content estimated by the difference between pF 1.8 and pF 3.0 from B0 (0.395 g water g⁻¹) to B40 (0.445 g water g⁻¹).

General linear model analysis revealed that biochar affected soil pH (F = 4.34, p = 0.008) (Figure 3A), EC (F = 5.49, p < 0.002) (Figure 3B), available phosphorus (F = 21.64, p < 0.001) (Figure 3E), and microbial activity (F = 2.89, p = 0.042) (Figure 3F). Among biochar application rates, pH was greater in B20 and B40 than in B0, EC was greater in B40 than in B0 and B20, NO₃-N was greater in B40 than in B0, and available P was the greatest in B40. Biochar increased microbial activity at 40 Mg ha⁻¹ both in upland and flooded conditions at week 4, and microbial activity was reduced in all biochar treatments in flooded conditions at week 8. Water regimes had strong effects (p < 0.001) to all the soil chemical properties, higher pH and NH₄⁺-N and microbial activity, and lower EC, NO₃⁻-N and available-P were observed in flooded than in upland conditions. Soil chemical properties were changed by time of incubation.

3.2. Biochar Affected Abundance of Nematodes

General linear model analysis showed that biochar did not affect the total abundance of nematodes (F = 0.18, p = 0.910) (Figure 4). Under upland conditions, an abundance of nematodes was greater (F = 3.91, p = 0.05) in B5 than in B20 at week 2, and the greatest abundance (F = 17.46, p = 0.001) was observed in B5 compared to B20 and B40 at week 8. The abundances of nematodes were markedly low in flooded conditions (F = 730.75, p < 0.001) compared to upland conditions at every sampling time. Over time, nematode abundance reached the greatest value at week 4, and the lowest value was observed at week 2 (F = 35.48, p < 0.001).

3.3. Biochar Affected Nematodes Community Composition

Total 26 nematode genera were identified (Supplementary Table S1). General linear model revealed that biochar affected Anaplectus (F = 3.66, p = 0.017) (Figure 5A), Chronogaster (F = 3.20, p = 0.029) (Figure 5B), Mesodorylaimus (F = 2.98, p = 0.038) (Figure 5C), Mesorhabditis (F = 3.90, p = 0.013) (Figure 5D), Nygolaimus (F = 5.99, p = 0.001) (Supplementary Table S1), and Prismatolaimus (F = 3.32, p = 0.025) (Figure 5E). Under flooded condition, the abundances of almost nematode genera were quite low, except for Acrobeloides, Alaimus, Aphelenchoides, and Ditylenchus. The abundances did not change in some genera among sampling times, e.g., Achromadora, Alaimus, Aporcelaimellus, Cryptonchus, Mononchus, and Tobrilus.

Within water regime conditions and each sampling time, the abundance of Anaplectus was greater (p < 0.05) in B5 than in B40 at week 8 (Figure 5A). The abundance of Chronogaster was lower (p < 0.05) in B5 than in B0 in upland conditions at week 8 (Figure 5B). The abundance of Mesodorylaimus was consistently greater (p < 0.05) in B5 than in B0 at weeks 3 to 8, while it tended to be reduced in the higher amounts of biochar (B20, B40) (Figure 5C). The abundance of Mesorhabditis was lower (p < 0.05) in B5 and B20 than in B40 at week 4 and week 3, respectively (Figure 5D). The abundance of Nygolaimus was greater (p < 0.05) in B5 than in B20 at week 8. The abundance of Prismatolaimus was lower (p < 0.05) in B20 than in B40 at week 4 and week 8 (Figure 5E). The abundance of Thornenema was greater (p < 0.05) in B5 than in B0 at week 3 and 4, and B20 at week 8 and in B40 at week 3 and week 8 (Figure 5F).

3.4. Biochar Affected Trophic Structure and Functional Guilds

The general linear model revealed that biochar did not affect abundance of bacterivorous (F = 1.57, p = 0.205), fungivorous (F = 0.62, p = 0.604), omnivorous (F = 2.42, p = 0.074), predator (F = 0.96, p = 0.415) and herbivorous (F = 0.02, p = 0.997) (

Table 1. Periodical change in trophic structures of nematodes in soil amended with different rates of biochar under upland and flooded conditions.

Time (T)	Water (W)	Biochar (B)	Ba	Fu	Om	Pre	Her
Week 2	Upland	B0	2.07 ± 0.04	1.12 ± 0.3 ab	1.77 ± 0.27 ab	1.06 ± 0.2	0.86 ± 0.79
		B5	2.09 ± 0.19	1.31 ± 0.25 a	1.88 ± 0.13 a	1.18 ± 1.05	1.16 ± 0.15
		B20	1.72 ± 0.29	1.14 ± 0.28 ab	1.21 ± 0.28 b	0.82 ± 0.76	0 ± 0
		B40	1.84 ± 0.30	0.32 ± 0.56 b	1.75 ± 0.25 ab	0.96 ± 0.83	0.76 ± 0.68
	Flooded	B0	1.92 ± 0.15	0 ± 0	0.37 ± 0.63	0.89 ± 0.83	0.37 ± 0.63
		B5	1.87 ± 0.04	0.85 ± 0.74	0.95 ± 0.86	1.33 ± 0.16	0 ± 0
		B20	1.80 ± 0.19	0.36 ± 0.62	0.45 ± 0.78	0.9 ± 0.8	0 ± 0
		B40	1.83 ± 0.22	0 ± 0	0.82 ± 0.71	0.34 ± 0.59	0.63 ± 0.55
Week 3	Upland	B0	2.67 ± 0.16	2.00 ± 0.24	2.26 ± 0.07 b	1.90 ± 0.12	1.58 ± 0.28
		B5	2.59 ± 0.11	2.14 ± 0.18	2.69 ± 0.12 a	1.77 ± 0.37	1.59 ± 0.28
		B20	2.62 ± 0.10	2.02 ± 0.52	2.44 ± 0.18 ab	1.87 ± 0.09	1.75 ± 0.07
		B40	2.86 ± 0.13	2.21 ± 0.41	2.27 ± 0.03 b	1.93 ± 0.23	1.59 ± 0.30
	Flooded	B0	1.66 ± 0.19	0 ± 0	0.8 ± 0.72	1.14 ± 0.21	0.26 ± 0.45
		B5	1.18 ± 1.03	0.71 ± 0.62	0.81 ± 0.7	0.8 ± 0.69	0 ± 0
		B20	1.92 ± 0.13	1.03 ± 0.90	1.39 ± 0.28	0.29 ± 0.51	0.75 ± 0.65
		B40	1.84 ± 0.07	1.27 ± 0.24	0.81 ± 0.70	0.47 ± 0.82	0.33 ± 0.56
Week 4	Upland	B0	2.84 ± 0.12	2.28 ± 0.13	2.42 ± 0.03 b	1.64 ± 0.16	1.18 ± 1.03
		B5	2.79 ± 0.03	2.22 ± 0.12	2.75 ± 0.07 a	1.81 ± 0.22	1.60 ± 0.25
		B20	2.79 ± 0.08	2.15 ± 0.12	2.62 ± 0.1 a	1.69 ± 0.17	1.69 ± 0.17
		B40	2.96 ± 0.05	2.19 ± 0.17	2.44 ± 0.0 b	1.72 ± 0.23	1.77 ± 0.07
	Flooded	B0	1.94 ± 0.12	1.10 ± 0.01	0.93 ± 0.85	0 ± 0	0.37 ± 0.64
		B5	1.84 ± 0.24	0.37 ± 0.64	1.35 ± 0.23	0 ± 0	0.37 ± 0.64
		B20	2.11 ± 0.18	0.52 ± 0.89	0.76 ± 0.65	0.84 ± 0.75	0.74 ± 0.64
		B40	1.64 ± 0.44	0.93 ± 0.81	0.99 ± 0.86	0.37 ± 0.64	0 ± 0
Week 8	Upland	B0	2.59 ± 0.12	2.13 ± 0.15	2.85 ± 0.05 ab	1.62 ± 0.21	1.38 ± 0.25
		B5	2.37 ± 0.16	2.17 ± 0.24	3.08 ± 0.11 a	1.98 ± 0.16	1.31 ± 0.21
		B20	2.35 ± 0.07	1.72 ± 0.17	2.69 ± 0.15 b	1.97 ± 0.45	0.88 ± 0.81
		B40	2.47 ± 0.10	1.75 ± 0.15	2.25 ± 0.05 c	1.70 ± 0.33	0.92 ± 0.86
	Flooded	B0	1.43 ± 0.43	0 ± 0	0.32 ± 0.55	0.32 ± 0.55	0 ± 0
		B5	1.22 ± 0.21	0.28 ± 0.48	0.34 ± 0.59	0.62 ± 0.54	0 ± 0
		B20	1.48 ± 0.41	0.38 ± 0.66	0 ± 0	0.45 ± 0.78	0 ± 0
		B40	1.70 ± 0.30	0.47 ± 0.81	0 ± 0	0 ± 0	0 ± 0
p		B	ns	ns	ns	ns	ns
		W	***	***	***	***	***
		T	***	***	***	ns	***
		BxW	ns	ns	ns	ns	ns
		BxT	ns	*	ns	ns	ns
		WxT	***	**	***	***	**
		BxWxT	ns	ns	ns	ns	ns

Values are mean of log(X + 1) ± standard deviation (n = 3). The different letters after a mean value indicate significant differences between biochar application rates in the corresponding water regime and sampling time by Tukey’s HSD test at p < 0.05, while global differences of the biochar (B), water (W) and time (T), and their interaction are also shown by three-factor generalized linear mixed model test, * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant. Ba, bacterivorous; Fu, fungivorous; Om, omnivorous; Pre, predator; Her, herbivorous.

). Under upland conditions, abundance of omnivorous was greater (p < 0.05) in B5 than in B0 at week 3 and week 4, in B20 at week 2 and week 8, and in B40 at week 3 to week 8. The abundance of fungivorous was greater (p < 0.05) in B5 than in B40 at week 2.

Biochar did not affect the abundance of functional guilds of cp1 (F = 0.63, p = 0.596), cp2 (F = 1.55, p = 0.210), cp3 (F = 0.24, p = 0.866), cp4 (F = 0.88, p = 0.458), and cp5 (F = 2.16, p = 0.101) (Supplementary Table S2). Under upland conditions, nematodes of cp5 were greater (p < 0.05) in B5 than in B0 at week 3 and week 4, in B20 at week 2 and week 8, and in B40 at week 3 to week 8. The abundance of functional guilds cp2 was greater (p < 0.05) in B0 and B5 than in B40 at week 8. By contrast, the abundance of functional guilds cp3 was lower (p < 0.05) in B5 than in B40 at week 3. At week 8, the abundance of cp3 was lower (p < 0.05) in B20 than in B0 and B40.

3.5. Biochar Affected Maturity Index, Total Biomass and Metabolic Footprint

Global analysis showed that biochar increased maturity index (MI) (F = 3.22, p = 0.028), total biomass (F = 2.99, p = 0.038), and total predator and omnivore footprint (F = 2.83, p = 0.046) in B5 (Figure 6). Among biochar treatments, a greater value of MI (p < 0.01) was observed in B5 than in B0 and B40 at week 4, and a lowest value (p < 0.01) was observed in B40 compared to other treatments at week 8 (Figure 6A). Total biomass of nematodes (p < 0.001) (Figure 6B), structure footprint (p < 0.001) (Figure 6C), total omnivore and predator footprints (p < 0.001) (Figure 6D), and composite footprint (p < 0.01) (Figure 6E) were greatest in B5 than in the other treatments at week 8.

3.6. Relationship between Nematodes Community Composition and Soil Properties

Principal component analysis (PCA) and correlation analysis were performed based on the entire nematode community composition in upland (Figure 7A) and flooded conditions (Figure 7B) and soil properties. Results showed that Thornenema was negatively correlated with soil moisture (p < 0.001), pH (p < 0.01), and NH₄⁺-N (p < 0.01). Mesodorylaimus was positively correlated with EC (p < 0.05), NH₄⁺-N (p < 0.05), inorganic N (p < 0.01), and negatively correlated with available phosphorus (p < 0.05) (Supplementary Tables S3 and S4). Nygolaimus was positively correlated with EC (p < 0.01) and NO₃⁻-N (p < 0.05). Prismatolaimus was positively correlated with microbial activity (p < 0.05), and available phosphorus (p < 0.05). Mesorhabditis was positively correlated with soil EC (p < 0.05), microbial activity (p < 0.01), NH₄⁺-N (p < 0.05) and inorganic N (p < 0.05). Anaplectus was positively correlated with EC (p < 0.01), NH₄⁺-N (p < 0.01), NO₃⁻-N (p < 0.05) and inorganic N (p < 0.001). Acrobeloides, Chronogaster, Dorylaimus, Filenchus, Monhystera, Panagrolaimus were negatively correlated with pH (p < 0.05).

4. Discussion

4.1. Effects of Biochar on Soil Physico-Chemical Properties

This study demonstrated that rice husk biochar amendment increased total soil C by 14% and 26% by the rates of 20 Mg ha⁻¹ and 40 Mg ha⁻¹, respectively. It is well known that the application of biochar increases soil C, pH and EC in cultivated soils, depending on the amount [50,51]. In addition, biochar provides essential available nutrients such as phosphorus and potassium [19]. In our study, the application of 40 Mg ha⁻¹ biochar increased soil pH, EC, NO₃-N, and available P, like our previous study [42] and also the available water content.

4.2. Effects of Biochar on Nematode Community Composition upon Water Regimes

In this study, nematode abundance rapidly decreased at 2 weeks in both water regimes, and they gradually increased at 3 to 8 weeks in upland conditions, not in flooded conditions. This result suggested that moisture content is a primary factor affecting the nematode community. Previous studies reported that soil moisture and temperature are the most important parameters as driving forces to belowground organisms, including nematodes [52,53]. In addition, our result is supported by Gebremikael et al. [54], who reported that the increase in moisture content from 50% water-filled pore space (WFPS) to 80% WFPS killed significant numbers of nematodes in the soil.

In this study, rice husk biochar did not affect the total abundance of nematodes, although it tended to increase in B5 treatment at week 8. This result was supported by Domene et al. [13], who reported that biochar did not affect the total abundance of nematodes at the application rates of 5 Mg and 30 Mg ha⁻¹ applied, and that the abundance of some genera, such was Cervidellus, was higher in 30 Mg ha⁻¹ than in 5 Mg ha⁻¹. In this study, the application of 5 Mg ha⁻¹ biochar had the potential to increase the total abundance of nematodes, as represented by the data in B5 at 2 and 8 weeks in upland conditions. Liu et al. [16] reported that the application of 4.5 Mg ha⁻¹ peanut shell biochar increased the total abundance of nematodes in field conditions. However, these results are inconsistent with the study by Kamau et al. [4], i.e., the application of wood tree biochar at 5 Mg and 10 Mg ha⁻¹ reduced the abundance of some species. In soil habitats, the total nematode abundance under the effects of biochar may vary because biochar exerts some negative effects on soil organisms, including the nematode community, possibly due to PAHs compounds [11,55]. Therefore, we hypothesized that the effects of biochar on soil nematode community may depend on the application amount and soil properties in particularly because biochar could have species-specific effects on the occurrence of nematode community assemblage in the bulk soils.

4.3. Effects of Biochar on Nematode Trophic Structure and Functional Guilds

In this study, the species-specific effects of biochar on nematode community composition have been observed in upland conditions depending on biochar application rate and timing, which affect the trophic structure and functional guilds. Overall, the low rate of biochar increased the abundance of a taxon that belongs to omnivorous and predator nematodes, while the high amounts promoted the abundance of bacterivorous nematodes. In our study, biochar at 5 Mg ha⁻¹ promoted the abundance of omnivorous taxa Mesodorylaimus, Thornenema, and predator taxon Nygolaimus. It is well known that Mesodorylaimus has a wide distribution in soil habitats, including high salinity conditions [56,57]. In our study, a high rate of biochar application tended to increase soil EC. However, since soil EC values were not different among treatments within each sampling time in upland conditions, thus it may not be the main factor affecting their abundances. PCA showed that Mesodorylaimus has significantly negative correlations to nitrate and phosphorus, particularly in flooded conditions. We assumed that these factors might be involved in the decrease in the abundance of omnivores, including Mesodorylaimus and Thornenema. This observation is agreed to Zhao et al. [58], who reported that the application of phosphorus fertilizer suppresses the abundance of omnivores/predator nematodes. Another possibility may come from soil moisture, one of the most sensitive factors to Mesodorylaimus [59]. The higher moisture content tended to increase the abundance of Mesodorylaimus in the highest biochar application rate, while it had a significantly negative correlation to Thornenema. Biochar increases water holding capacity which may well remain water in soil particles and biochar itself. Therefore, the 40 Mg ha⁻¹ biochar application may induce some unsuitable habitats for omnivores in general and some specific species in particular. Our previous study found that Thornenema was only present in a non-salt-affected paddy field and absent in all the soil samples from salt-affected paddy fields with high EC values. In this study, a 40 Mg ha⁻¹ biochar application increased EC, and thus it might reduce Thornenema. Moreover, Thornenema had significantly negative correlations to pH and NH₄⁺-N. The results are supported by Zhao et al. [58], who reported that phosphorus addition was detrimental to total abundance, particularly abundance of omnivorous and predator nematodes, while nitrogen addition stimulated soil nematodes, mainly bacterivorous and fungivorous nematodes. Contrary to omnivorous and predator nematodes, a high rate of 40 Mg ha⁻¹ tended to increase the abundance of bacterivorous taxa Mesorhabditis and Prismatolaimus. This result can be explained by the fact that a high rate of biochar addition promotes bacteria and fungi [60,61], resulting in the increase in smaller size nematodes such as Mesorhabditis and Prismatolaimus, as opportunistic nematodes. In addition, biochar addition at a high rate tended to increase microbial activity in upland conditions that significantly positively correlated to the abundances of Mesorhabditis, Prismatolaimus, and Chronogaster (Figure 7). We assumed that a high application rate of biochar likely brings preferable conditions for bacterivorous nematodes.

In our study, water regimes had stronger effects on the nematode community, in which the abundances of almost all taxa were significantly reduced in flooded conditions. There were only a few taxa that did show no change between water regimes, likely Acrobeloides, Alaimus, Aphelenchoides, and Ditylenchus. Most of the taxa in our study were identified as free-living nematodes that frequently occur in the terrestrial ecosystems, particularly in agroecosystems like paddy fields and uplands [30,57,62]. It is well known that free-living nematodes are dominant in aerobic soils, which have the suitable conditions for their growth and production compared with anaerobic soils [54,62,63]. On the contrary, the extreme flooding may induce anoxic conditions [64,65] or toxic conditions [66] that may change the soil organisms, including microbial community [64,67] and nematode community; Okada et al. [62] and Liu et al. [63] reported that flooded condition reduces the abundance of nematodes in soils. We suggested that keeping flooded conditions over time induces adverse environmental factors that can decline the abundance of almost nematode communities, particularly in unplanted soil.

Biochar affected functional guilds, particularly the nematodes with high c-p values depending on application rates. In our study, a high rate of biochar amendment tended to increase the abundances of Prismatolaimus (cp3) and Mesorhabditis (cp1), which were positively correlated with available phosphorus and inorganic nitrogen, respectively. Previous studies reported that nutrient input increased the abundance of bacterivorous and fungivorous by increasing microbial activity [68,69]. Wu et al. [70] reported that adding biochar significantly increased the proportion of nematodes in the functional guilds cp1 and cp2. Specifically, the bacterivorous nematodes genera Rhabditis and Acrobeloides belonging to cp1 and cp2 were dominant in soil amended with biochar [71]. A high amount of available phosphorus and nitrogen may also contribute to the shifts of the nematode community.

4.4. Effects of Biochar on Maturity Index and Metabolic Footprint of Nematode Community

In this study, a low rate (5 Mg ha⁻¹) of biochar application did not affect the soil physicochemical properties but significantly affected ecological indices of nematodes. Previous studies also remarked that a low amount (3 Mg ha⁻¹ to 10 Mg ha⁻¹) of rice husk biochar amendment did not affect soil pH, EC, exchangeable Ca, total N, available K and soluble organic carbon in field conditions [6,72] and these amounts belong to a recommended range (5 to 50 Mg ha⁻¹) by International Biochar Initiative. However, biochar can quickly shift soil microbial community [5,73] and nematodes [4,13], even at amounts that do not affect the soil chemical properties. Indeed, this study revealed that 5 Mg ha⁻¹ of biochar amendment increased maturity index, composite footprints, structure footprint, and particularly the metabolic footprint of omnivorous/predatory nematodes. In addition, biochar application tended to increase soil pH, EC, NO₃⁻-N, and available P and these changes may be factors that affected the omnivorous and predatory nematodes because their groups are most sensitive to environmental disturbance [29,31,39]. The results in PCA that Thornenema had negative correlations with soil moisture and pH, and Mesodorylaimus had a negative correlation with available phosphorus might explain the low densities of the two nematode genera in biochar-amended soil at the high rate. Liu et al. [16] reported that the application of 4.5 Mg ha⁻¹ peanut shell biochar increased the abundance of omnivorous-predatory nematodes in a five years experiment, but a high application rate of biochar showed a possibility in reduction in omnivorous/predatory nematodes. Specifically, Liu et al. [11] reported that wheat straw biochar at a high amount (60 Mg ha⁻¹) decreased the abundance of omnivorous/predatory nematodes, resulting in a decreased metabolic footprint. Another reason that might reduce omnivorous/predatory abundance in our study is that the application of a high amount of biochar may increase suitable habitats for microbial community and provide a shelter to escape predator activities by bacterivorous and fungivorous nematodes [74], which contributed to fewer food resources for omnivorous/predatory nematodes. Recent studies revealed that the application of 40 Mg ha⁻¹ eucalyptus waste biochar reduced the enzyme activity and total microbial activity (fluorescein diacetate hydrolysis activity) of soil [75], which correlated with the reduced abundance of bacterivorous and fungivorous nematodes [23,76]. Biochar has several million pores varying in size and large surface areas [74,77], leading to improved aggregation and porosity of soil and stimulated microbial activity and biomass [78,79]. Therefore, the low rate of biochar application increased the omnivorous and predator footprints, and thereby structure and composite footprints, which particularly caused the greatest maturity index. Such an altered nematode community may bring the stable conditions of soil for their functions.

5. Conclusions

Biochar increased available water, total C, C/N ratio, and pH, EC, NO₃⁻-N, and available P in high application rates. Biochar had contrast species-specific effects on nematodes; Mesorhabditis, Prismatolaimus tended to increase in the higher biochar application rate of 40 Mg ha⁻¹, while the abundance of the nematodes in higher trophic levels and functional guilds such as omnivores and predators, Mesodorylaimus, Thornenema, and Nygolaimus tended to decrease in 40 Mg ha⁻¹ biochar addition. This study clarified that a low application rate of 5 Mg ha⁻¹ favored the omnivorous, predatory, and functional guilds of cp5, resulting in the greatest maturity index, total nematode biomass, and their composite footprints, reflecting structured soil conditions. We suggested that a low rate of biochar can be applied to stimulate omnivores and nematodes with high functional guilds that are most sensitive and have difficulty in recovery in problem soils.