3.1 Seedling morphology and physiological activity
As shown in Fig. 1, seedlings growing in substrate with SMC showed their height, stem diameter, leaves number, leaves area, strong seedling index、chlorophyll content、root length、root activity、aboveground dry weight and root dry weight, respectively in the range of 17.45–20.6 cm, 2.32–3.02 mm, 3.52–3.99、 2.86-4.92cm2, 0.48–0.68, 10.38–34.21 mg g− 1, 90.85-158.49 cm, 0.33–0.95 mg g− 1 h− 1, 2.37–3.26 g and 1.18–1.41 g. They showed significant higher values in height、 stem diameter、 strong seedling index、chlorophyll content、root activity、aboveground dry weight and root dry weight than those in CK. Among treatments R1、R2、R3 and R4, R2 showed the highest values in stem diameter, leaves number, root length, root activity and root dry weight, thus exhibiting the largest CEC value (Table 2).
Table 2
Comprehensive evaluation on rice seedlings growth traits by subordinate function values analysis.
Treatment | Height | SD | LN | LA | RL | RA | Chl | ADW | RDW | CEC |
CK | 0.00 | 0.00 | 0.49 | 0.31 | 0.66 | 0.00 | 0.00 | 0.00 | 0.00 | 0.16 |
R1 | 0.47 | 0.54 | 0.00 | 0.33 | 0.02 | 0.42 | 1.00 | 0.22 | 0.17 | 0.35 |
R2 | 0.89 | 1.00 | 1.00 | 0.79 | 1.00 | 1.00 | 0.50 | 0.89 | 1.00 | 0.90 |
R3 | 0.42 | 0.41 | 0.17 | 0.00 | 0.00 | 0.84 | 0.02 | 0.26 | 0.17 | 0.26 |
R4 | 1.00 | 0.21 | 0.24 | 1.00 | 0.02 | 0.65 | 0.69 | 1.00 | 0.43 | 0.58 |
Note: SD: stem diameter; LN: leaves number; LA: Leaves area; Chl: Chlorophyll content; RL: Root length; RA: Root activity; ADW: Aboveground dry weight; RDW: Root dry weight; CEC: comprehensive evaluation coefficient. |
Statistical analysis showed significantly higher values (P < 0.05) for properties of SMC substrates than those in pure soil substrates, except for acid phosphatase (Table 3). The activities of enzymes for carbon metabolism (αG, βG, CHB, βX and SL), nitrogen metabolism (NAG, UE) and the antioxidant enzyme CAT activity grew with the increasing SMC ratio.
Table 3
Effects of different compost substrates on enzyme activity.
Treatments | αG (nmol g− 1 h− 1) | βG (nmol g− 1 h− 1) | CHB (nmol g− 1 h− 1) | βX (nmol g− 1 h− 1) | NAG (nmol g− 1 h− 1) | UE (mg NH3 g− 1d− 1) | PHO (nmol g− 1·h− 1) | SL (U g− 1) | CAT [ml(0.1mg l− 1 KMnSO4) g− 1 h− 1] |
CK | 9.11c (2.08) | 25.44c (2.71) | 24.3d (0.63) | 35.55c (0.85) | 7.17c (1.16) | 3.55d (0.11) | 257.92a (20.87) | 0.86e (0.13) | 0.55c (0.09) |
R1 | 12.84c (1.02) | 35.61bc (2.14) | 36.27c (0.97) | 37.47c (0.65) | 7.79c (0.18) | 3.99d (0.08) | 219.38a (27.89) | 3.25d (0.19) | 0.75c (0.07) |
R2 | 20.45b (1.86) | 51.93b (11.4) | 49.57b (0.98) | 47.85ab (3.38) | 11.27bc (0.64) | 5.90c (0.85) | 210.45a (11.50) | 7.41c (0.20) | 1.78ab (0.34) |
R3 | 20.75b (2.81) | 54.94b (13.6) | 49.69b (0.62) | 44.83b (0.58) | 21.7b (13.7) | 7.62b (0.29) | 257.88a (109.98) | 9.44b (0.12) | 1.48b (0.06) |
R4 | 31.46a (2.23) | 87.61a (8.94) | 73.2a (6.24) | 50.2a (0.15) | 44.49a (1.86) | 17.42a (1.28) | 232.2a (36.81) | 14.14a (1.25) | 2.02a (0.01) |
Note: The values shown in the brackets were standard errors. Different letters mean a significant difference (P < 0.05). The values shown were the mean (SD) (n = 3). |
Generally, using SMC in the substrate has been reported to improve the height, stem diameter and biomass of seedlings (Paula et al. 2017; Meng et al. 2018). In addition, recent research showed that the compost substrate enhance root development of seedlings (Xu et al. 2022). Similar in our results, rice seedling qualities, root development and their nutrient absorption amount were greatly enhanced by using SMC (Table S1-S3). These can be ascribed to the improved properties of SMC substrate for better nutrients (organic matter and N, P, and K) and environmental conditions (such as the water retention and EC, pH, air pores and total porosity) which meet the seedling growth requirements (Table S4). However overloaded pH and EC increments can inhibit germination and growth of rice seeds and can harbor the growth of disease-causing microorganisms (Feng et al. 2020). These may happen when high content of SMC was used, such as in treatment R3 and R4.
Plant health is often associated with soil microbes (Timmis and Ramos 2021). SMS can enhance soil enzymes activities in rice fields and improve soil productivity (Li et al. 2020b). Moreover, it is commonly recognized that SMC is rich in extracellular enzyme and microbial populations (Zhu et al. 2021). Commonly, Amylase and cellulase are important indicators of organic matter degradation and carbon metabolism (Liu et al. 2022). NAG, UE is a significant indication of urea to ammonia conversion, and its activity is strongly connected to nitrogen metabolism in compost (Li et al. 2020a). CAT reflects the intensity of microbial activity and respiration, reducing the toxic effects of the composting process on microorganisms (Ukalska-Jaruga et al. 2019). It may be because CM contains enough cellulase, which assists in the decomposition of cellulose, hemicellulose, and lignin in SMS (Liu et al. 2021b). In addition, it is possible that the addition of chicken manure provided more abundant microorganisms, accelerating the mineralization of nitrogenous organic matter in the compost and producing large amounts of urea (Song et al. 2020). Whereas, SMC did not significantly affect the activity of soil acid phosphatase. This may be because SMC provides abundant available phosphorus and inhibits acid phosphatase activity (Kwiatkowska and Joniec 2022).
3.2 Microbial communities in rhizosphere soil
The composition of microbial communities in the SMC varied at the phylum and genus level (Fig. 2). Proteobacteria (37.0-43.72%), Bacteroidota (7.17–14.89%), Firmicutes (3.63–22.23%), Actinobacteriota (2.99–18.52%), and Gemmatimonadota (2.22–8.84%) were the top five bacterial taxa that appeared in all substrates (Fig. 2a). The relative abundance of Firmicutes, Verrucomicrobiota, and Bacteroidota, the dominant bacterial phylum in the SMC treatment, was much higher than in the CK treatment (p < 0.05). Multiple comparisons of genera showed that the relative abundances of Sphingomonas, Gemmatimonas, and Flavisolibacter differed significantly among treatments. Relative abundance of Pseudomonas (2.17–2.77%) and Bacillus (0.64–0.65%) in R2 and R4 were appreciably higher than those in CK (0.77% and 0.17%, respectively).
Ascomycota (67.71%), Chytridiomycota (19.79%), Mortierellomycota (3.05%) and Basidiomycota (2.84%) were the highest phylum in CK, while Ascomycota (67.07–93.09%), Chytridiomycota (0.85–12.13%), and Basidiomycota (2.62–11.75%) were the dominant fungal phylum in the SMC treatment (Fig. 2b). The fungal composition at the genera differed significantly between CK and SMS treatments, where Mycothermus (29.86–68.25%), Aspergillus (0.92–7.03%), Lobaria (1.4-10.92%), Myceliophthora (0.37–2.03%) were the most abundant genera in the SMC (p < 0.05).
We used the LEfSe tool to compare and identify the core microbiomes (biomarkers) which were significantly larger in all treatments (Fig. 3). The bacterial community LEfSe analysis (LDA score ≥ 4) detected 57 (CK = 15, R1 = 21, R2 = 1, R3 = 11, R4 = 10) taxa, while the fungal community LEfSe analysis (LDA score ≥ 3) detected 60 (CK = 27, R1 = 12, R2 = 4, R3 = 14, R4 = 3) taxa. The most abundant bacterial biomarkers for CK, R1, R2, R3 and R4 were Gammaproteobacteria, Bacilli, Stenotrophomonas, Alphaproteobacteria and Xanthomonadales, respectively. It is noteworthy that the relative abundance of Stenotrophomonas maltophilia in R2 (0.58%) manifested much higher than CK (0.004%) (p < 0.01). The most abundant fungal biomarkers in CK, R1, R2, R3 and R4 were Ascobolus., Mortierellomyco, Glomeromyco, Lecanoromycetes and Aspergillus_chlamydo_sp.
Microorganisms are widely involved in soil biochemical processes (Martínez-García et al. 2018). Some microorganisms in soil can be utilized as "probiotic supplements" to boost plant nutrient absorption and general vigor (Kandasamy et al. 2019). According to previous research Actinobacteriota, Azotobacter, Bacillus, Burkholderiales, Firmicutes, Flavobacteriales, Pseudomonas, Rhizobiales, Sphingomonadales, Stenotrophomonas and Verrucomicrobiota are powerful PGPR that can stimulate the growth of crops, which some of them can effectively inhibit pathogenic bacteria (Verma et al. 2019; Liu et al. 2021a). They were observed in our treatments with SMC. In addition, SMC limited the growth of Flavisolibacter, Gammaproteobacteria, a common pathogen in soil (Li et al. 2020c). However, integrating too much SMC, such as in R4 treatment, increased microbes in Xanthomonadaceae family, involving a wide range of pathogens to many crops (Costa et al. 2021). It indicates that high salinity or nutrient surpluses in the rhizosphere provide good conditions for pathogenic bacteria to multiply (Li et al. 2021).
Fungi such as Ascomycota, Basidiomycota, Mortierellomycota and Chytridiomycota interact with plants through symbiotic associations (Guo 2019; Chen et al. 2022). Arbuscular mycorrhizal fungi in Glomeromyco are the most extensively distributed symbiotic fungi and are often employed as a soil-borne pathogenic fungal inhibitor as well as plant root nourishment (Vukicevich et al. 2016). Aspergillus, Mycothermus and Myceliophthora use carbohydrate-active enzymes in the co-composting of CM and plant residues, which play a vital role in the decomposition of cellulose and hemicellulose, and thus promote the growth of probiotic bacteria (Xie et al. 2021).
The aforementioned PGPR and PGPF are important in suppressing soil-borne pathogens and plant growth, suggesting that SMC can effectively improve soil quality. In addition, even if SMC reduced fungal abundance, bacterial diversity did not change significantly (Table S6). It indicates that the application of SMC can enhance PGPR and PGPF while reduce the abundance of pathogenic microorganisms.
Our study found that SMC may influence soil fungal diversity (Fig. S2). The main microbial communities and bacterial abundance were mainly influenced by soil properties and rice seedling (Fig. 5 and S1). The fungal community structure strongly correlated with TK, seedling root vigor and soluble protein, suggesting that the plant growth may affect the inter-root microbial community based on increased plant physiological activity and root growth. Nutrient availability in the substrate can alter the microbes on host plants by changing the species composition of the microbial community (Leff et al. 2015). In general, SMC promotes plant growth by releasing plant available bioactive materials to the soil and/or by promoting the growth of microorganisms that accelerate mineralization processes (Margalef et al. 2017). Our results showed that 20%-80% of SMC treatment was able to stimulate effective recruitment of PGPR and PGPF at the rhizosphere of rice seedlings, suggesting that SMS can promote plant growth indirectly or directly through increasing beneficial microorganisms.
3.3 Based on FUNGuild predict Fungal function
Overall, based on FUNGuild, a total of 29.14% of OUTs were classified, while a relatively larger number of fungal Trophic modes were not yet identified in SMC (Fig. 4). SMC altered the proportion of Saprotroph, Symbiotroph, and Pathotroph in favor of symbionts. The fungal Trophic mode was dominated by Saprotroph (3.73–35.49%) for all treatments, followed by Pathotroph (6.57–12.44%) for CK inter-rhizosphere fungi, and then Symbiotroph (1.03–14.71%) for SMC treatments. As SMC increases, one guild that decreases is Dung Saprotroph-Wood Saprotroph (belongs to Saprotroph), but that increases are Lichenized (belongs to Symbiotroph). The relative abundance of fungal pathogen decreased dramatically in treatments with SMC, where Plant Pathogens dominate. The relative abundance of Pathotroph and Plant Pathogens was separately 0.31% and 0.28% in R2, 1.07% and 1.04% in R3, while reaching 8.86% and 9.35% in CK.
Saprotroph is the main trophic mode of fungal communities (Zhang et al. 2020). The addition of SMC to the substrate can create a plant symbiont-rich fungal soil community. Thus, the addition of SMC contributes to the functional changes in the fungal community. The addition of SMC significantly reduced the abundance and OTU levels of soil fungi in this experiment and reduced fungal diversity (Table S5 and S6). Previous studies have shown that diseased soils have more unique bacterial OUTs, and higher fungal and bacterial communities diversity than healthy soils (De Corato et al. 2020).
Soil-borne plant pathogens account for approximately 90% of the 2000 major diseases of major 9 crops and contribute to 50–75% of total agricultural losses of cash crops (Ziedan 2022). Changes in microbial community composition can predict whether plants remain healthy earlier than significant changes happen in pathogen density (Gu et al. 2022). SMC significantly reduced the abundance of pathogenic bacteria, especially pathogenic fungi. There were 62 probable pathogen fungi OUTs found from 31 genera, those dominated in CK, including Powellomyces (10), Magnaporthaceae (5) and etc by Guild (Table S7). The most notable plant pathogens was Magnaporthe grisea, inducing rice blast, the most dangerous fungal diseases to rice (Ahmad et al. 2017). In this study fungal pathogens were mainly affected by environmental factors (TK, TOC, TP, TN) and seedling physiological activity (LSP, TKC). A strong correlation between seedling quality and most soil and plant parameters, with the strongest correlation between plant phosphorus content (r = 0.724, p = 0.001) and seedling root activity (r = 0.603, p = 0.001) (Fig. 5). This suggests that SMC may promote plant growth by stimulating beneficial microorganisms and potentially suppressing pathogenic fungi through substrate physico-chemicals. Secondary metabolites in SMS, including phenolic compounds (coumaric acid, cinnamic acid, salicylic acid), saponins, etc., produced by mycelium can have an inhibitory effect on pathogens (Porselvi and Vijayakumar 2020). There is a strong negative correlation between bacterial community diversity and fungal community richness (Fig. S2b). It is possible that beneficial microorganisms compete with harmful microorganisms for nutrients and produce biostatic compounds (Panth et al. 2020). Also plant exudations are able to influence the compositions and activities of their inter-rhizosphere microbiome (Timmis and Ramos 2021). In general during the thermophilic phase of composting, pathogenic fungi in raw materials are exterminated, whereas some pathogenic bacteria in chicken manure are likely to survive in the manure composted and soil amended with compost (Lemunier et al. 2005). This means that SMS can supp ess or eliminate pathogens that survive in the compost.