Allelopathic effects of aqueous extracts from uncomposted and composted Mexican devil (Ageratina adenophora) plants on forest fungal growth and soil nitrogen and phosphorus mobilization

Abstract

Mexican devil [Ageratina adenophora (Spreng.) R.M. King & H. Rob.], a globally invasive weed with destructive effects on forests, has spread to numerous countries. To elucidate the inhibition of tree growth by A. adenophora, a study was conducted using the fungi (Lactarius deliciosus, Ceriporia lacerata, and Fomitopsis palustris) involved in the recycling of carbon and nutrients in forests. The focus was on investigating soil nitrogen and phosphorus availability in response to aqueous extracts from uncomposted and aerobically composted A. adenophora (EUA and ECA, respectively). The samples of composted A. adenophora from different sites exhibited a significant reduction in the concentration of allelochemicals 4,7-dimethyl-1-(propan-2-ylidene)-1,4,4a,8a-tetrahydronaphthalene-2,6(1H, 7H)-dione and 6-hydroxy-5-isopropyl-3,8-dimethyl-4a,5,6,7,8,8a-hexahydronaphthalen-2(1H)-one. This reduction more than 94% when compared with the concentration of these allelochemicals in CA. The EUA solutions at 5 and 10 mg L⁻¹ (oven-dried plant biomass base) minimized L. deliciosus and C. lacerata growth, and significantly decreased F. palustris growth on the soil surface and within the soil. However, soil with ECA had no effect or promoting effect on the fungal growth. Compared with CK (only fungal inoculation in tested soil), the EUA solution reduced soil nitrogen and phosphorus, while ECA had the opposite effect; soil pH was increased by 0.01 to 0.08 under EUA treatment, while it decreased by 0.5 to 0.41under ECA treatment. Nitrogen and phosphorus availability were positively correlated with protease and phosphatase activity (r = 0.723 to 0.944), while available phosphorus was inversely correlated with pH in tested soils (r = -(0.809 to 0.978)). As such, the EUA solution decreased soil nitrogen and phosphorus supplies by inhibiting the liberation of proteases, phosphatases, and protons, which may lead to poor growth or even mortality of three fungal species. The in situ aerobically composted A. adenophora residues left behind may directly supply fungal species with nutrients and indirectly increase soil nutrient availability via the promotion of nitrogen and phosphorus mobilization.

Introduction

Mexican devil [Ageratina adenophora (Spreng.) R.M. King & H. Rob.], originally native to Mexico and Central America, has invaded numerous low-latitude countries (Tererai and Wood 2014; Zhao et al. 2019). For instance, the perennial A. adenophora invaded China through the Sino-Burmese border during the 1940s, and the species is now commonly found in southwest China. Ageratina adenophora occupies approximately 14% of the total terrestrial area in Liangshan Autonomous Prefecture, Sichuan Province, including various land types such as pastures, forests, orchards, croplands, hillsides, roadsides, and riprap crevices (Fu et al. 2018; Gui et al. 2012). The weed has had a profound impact on local ecosystems by displacing native vegetation, notably trees in forests. As a result, it has caused significant economic losses and extensive damage to forests, leading to its nickname of the “forest-killer weed” (Wang et al. 2017).

Some plants release allelochemicals to the surrounding environment through plant decomposition, natural volatilization, rainfall leaching, and root secretion. These allelochemicals influence nearby plants and soil microorganisms, granting the releasing plant a competitive advantage in the struggle for survival. This phenomenon benefits the plant by allowing it to expand its habitat and population (Irimia et al. 2019; Scavo et al. 2019). At present, approximately 100 allelopathic chemicals have been isolated from A. adenophora, including monoterpenes, sesquiterpenes, steroids, triterpenoids, phenylpropanoids, flavonoids, and other derivatives (Yang et al. 2016). Such allelochemicals can accumulate in soils over time (Yang et al. 2013). Laboratory studies have shown these allelochemicals are toxic to other plants, such as rice (Oryza sativa L.), alfalfa (Medicago sativa L.), wheat (Triticum aestivum L.), perennial ryegrass (Lolium perenne L.), white clover (Trifolium repens L.), eucalyptus (Eucalyptus robusta Sm.), Chinese red pine (Pinus massoniana Lamb,), turnip (Brassica rapa L. ssp. rapa), sorghum [Sorghum bicolor (L.) Moench], and maize (Zea mays L.), among others (Hu et al. 2016; Jiao et al. 2021a, 2021b; Li et al. 2010; Wang et al. 2016; Yang et al. 2013; Zhang et al. 2012). Zhao et al. (2019) revealed that the invasion of new regions by A. adenophora can lead to alterations in the microbial community structure within the soil. This can result in variations in the abundance of certain microorganisms, which may be either relatively high or low (including azotobacteria and ammonia-oxidizing bacteria) in the soil. The allelochemicals 4,7-dimethyl-1-(propan-2-ylidene)-1,4,4a,8a-tetrahydronaphthalene-2,6(1H, 7H)-dione (DTD) and 6-hydroxy-5-isopropyl-3,8-dimethyl-4a,5,6,7,8,8a-hexahydronaphthalen-2(1H)-one (HHO) were found to be the main allelochemicals in the leaching pathway of A. adenophora, and they were effectively detected in the rhizosphere soil of the weeds (Yang et al. 2006, 2013). The minimum effective concentrations of DTD and HHO required to inhibit the germination of alfalfa seeds were found to be considerably low (0.714 and 0.660 mmol L⁻¹, respectively; Yang et al. 2013). These findings suggest that, following invasion into new areas, A. adenophora may release allelochemicals into the adjacent soil, potentially influencing local plants and soil microbes (Inderjit 2000).

Two macronutrients are essential for the growth and development of plants: nitrogen and phosphorus. Natural forests typically do not receive fertilization, and trees in these forests primarily rely on the soil for their nutrient supply. The limited availability of nitrogen and phosphorus in forestry soils is recognized as one of the factors that can constrain the overall health of trees in such environments (Sarmiento et al. 2006). Plant growth–promoting microbes can mobilize unavailable nutrients from either minerals or organics into available forms in soils, facilitating tree nutrient uptake and growth (Abhilash et al. 2016). Soils typically contain more than 90% of their total nitrogen within their organic matter (OM) content (Geisseler and Horwath 2008). Owing to chemical fixation, particle adsorption, and microbial utilization, the phosphorus concentration is relatively low in soil solutions (<1 μM) (Balemi and Negisho 2012). Thus, mobilization of nitrogen and phosphorus in minerals or OM by microbes in forest soils is essential for tree health and forest productivity (Nottingham et al. 2015). Considering the antagonistic effects observed for the abovementioned plants and human pathogens, it is reasonable to anticipate that the allelochemicals released by A. adenophora may also hinder the growth and activity of soil microbes responsible for nutrient mobilization. As a result, nutrient bioavailability may be decreased in forest soils, which subsequently leads to the poor growth or mortality of trees in A. adenophora–invaded forests (Fu et al. 2018; Poudel et al. 2019).

Ceriporia lacerata and Fomitopsis palustris grows on live or dead trees and in soils, participating in litter decomposition and OM mineralization in forest ecosystems. There is an increasing amount of evidence suggesting that these two fungi possess the capacity to mobilize nitrogen and phosphorus in soil through the release of protease and phosphatase (Peng and Huang 2022; Song et al. 2021; Yin et al. 2021a, 2021b). Lactarius deliciosus is a common ectomycorrhizal fungus in boreal forests, forming associations with pine trees and facilitating their water and nutrient extraction from soils, phytohormone synthesis, and disease resistance (Liu et al. 2016a). Some pine trees such as P. massoniana are ectomycorrhizal-fungus dependent (Li et al. 2014). These three fungi are commonly found in Pinus forests in southwest China and Southeast Asia. They play a crucial role in the recycling of carbon, nitrogen, and phosphorus within soil–tree ecosystems in these regions (Peng and Huang 2022; Song et al. 2021; Yin et al. 2021a, 2021b). However, there is limited understanding of how A. adenophora affects these fungi in forest ecosystems. In the present incubation experiment, the soil nitrogen and phosphorus availability were compared when exposed to aqueous extract solutions derived from both uncomposted and aerobically composted A. adenophora in relation to the three fungi.

Materials and Methods

Experimental Fungi and Soils

The three fungi included ectomycorrhizal fungus L. deliciosus JY03 (data unpublished), white-rot fungus C. lacerata HG2011 (NCBI accession no. MT675050; Song et al. 2021; Yin et al. 2021a, 2021b), and wood-decaying fungus F. palustris CQ18 (NCBI accession no. MT377823; Peng and Huang 2022). The first fungus was originally screened and isolated from a light purple soil and the latter two from a dark purple soil (the two types of soil can be attributed to Eutric Regosol according to the FAO Soil Taxonomic System) in local P. massoniana forests (FAO-UNESCO 1990). The three tested fungi were cultured on potato dextrose agar medium for 14 d at 25 ± 2 C in a constant-temperature incubator (preserved in the China General Microbiological Culture Collection Center).

The plant residues were removed from the two forest soil samples collected from 0 to 20 cm to isolate the fungi. Thereafter, the soils were air-dried and ground through a 0.5-mm sieve. The selected soil properties are listed in Table 1.

Table 1. Selected properties of experimental soils.

Experimental Procedure

Ageratina adenophora (leaves and shoots) were individually collected from a field at five sites (Xichang City, Huili County, Huidong County, Dechang County, and Mianning County) in Liangshan Autonomous Prefecture, Sichuan Province, China (26º34'N to 29º16'N, 102º10'E to 102º30'E, 1,186 to 1,305 m above sea level) during July 2021 (Figure 1). Ageratina adenophora (registration no. 20060712) was deposited at the College of Chemistry, Beijing Normal University. The collected weeds were subjected to in situ aerobic composting, following the method proposed by Jiao et al. (2021a, 2021b). The aboveground parts of A. adenophora were harvested using both artificial and mechanical methods. One part of the uncomposted A. adenophora (UA) was dried and prepared for analysis, and the other part was piled up nearby with the decomposed microbial agent prepared using high-temperature Clostridium fibrium. To compost 1,000 kg of UA, 2.0 kg of compost inoculants and composting aids were needed. A total of about 1,500 kg of the mixture was piled into a cone (about 5 m in diameter and 1.5 m in height) and covered with plastic film to reduce water and heat loss during composting. Ten kilograms of uncomposted A. adenophora (UA) was collected from the five sites of the study. The samples were then dried at 35 C, ground using a 1-mm sieve, thoroughly mixed to ensure homogeneity, and stored at 4 C for subsequent use in the fungal experiment. The in situ composted A. adenophora (CA) samples for the fungal experiment were prepared and stored in the same manner as the UA samples. Samples of UA and CA (10 g) were mixed with 100 ml sterilized deionized water and then constantly shaken for 30 min at 150 rpm at 40 C. The suspension was filtrated through 20-μm filter paper. In the aqueous extract solution of UA (EUA), a total of 0.31 g L⁻¹ of nitrogen and 0.23 g L⁻¹ of phosphorus were detected. In contrast, the extract solution from CA (ECA) contained higher concentrations of nutrients, with 1.61 g L⁻¹ of nitrogen and 1.42 g L⁻¹ of phosphorus. The EUA and ECA solutions were diluted, respectively, with sterile deionized water to reach 0 (CK), 2.5, 5, and 10 mg L⁻¹ (oven-dried CA or UA per liter; the same below (NH₄)₂SO₄ and NaH₂PO₄ were added into the no-inoculation treatment, CK (0 mg L⁻¹), EUA (2.5, 5, and 10 mg L⁻¹), and ECA (2.5 and 5 mg L⁻¹) such that the nutrients were equal to those of the ECA (10 mg L⁻¹). The pH values of CK, EUA, and ECA solutions ranged between 6.92 and 7.01. As previously reported that the optimum soil pH values for C. lacerata, F. palustris, and L. deliciosus are 6.20 to 7.58 (Song et al. 2021; Yin et al. 2021a, 2021b), 5.53 to 8.67 (Peng and Huang 2022) and 6.89 to 7.04 (data unpublished), respectively. Therefore, the effect of solution pH could be ignored. The EUA and ECA solutions were stored at 4 C and used for soil experiments within 24 h to prevent the degradation of allelochemicals.

Figure 1. Distribution map of sampling sites.

Twenty grams of soil were steam-sterilized and placed on a plate. Subsequently, four different treatments were applied individually to the soil samples: no inoculation, CK (control), EUA, and ECA, each at various concentrations as previously noted. The aim was to achieve a soil moisture level equivalent to 65% of the maximum field water capacity. Three small fragments of fungal mycelium, aged 2 wk and measuring 6 mm in diameter, were excised from a newly cultured source. The fragments were cultivated on distinct soil surfaces treated with C. lacerata and F. palustris on dark purple soil and L. deliciosus on light purple soil. Subsequently, the fragments were incubated in darkness at a temperature of 25 C for a period of 14 d (refer to Figure 2). To facilitate inoculation, the soil water content was adjusted to 65 ± 2% of the maximum field water capacity by the addition of sterilized water over the course of the 14 d. There were four replicate plates for each concentration. Blank control plates were set up in the same way but with no inoculation.

Figure 2. Influence of aqueous extracts from uncomposted (EUA) and composted (ECA) Ageratina adenophora plants on fungal growth on the soil surfaces: (A) Lactarius deliciosus; (B) Ceriporia lacerata; and (C) Fomitopsis palustris.

Chemical, Enzymatic, and Microbial Analysis

Aliquots of EUA and ECA (plant sample/water = 1:10, weight/volume) were analyzed by high-performance liquid chromatography (HPLC; Jiao et al. 2021a, 2021b). DTD and HHO in the sample solutions were calculated using the external standard method. A 20-μl aliquot of the sample solution was injected into a GS-310 analytical column (C18 column; inner diameter: 4.6 mm; length: 150 mm). The chromatographic separation was conducted using a mobile phase consisting of a mixture of methanol and water in a 70:30 volumetric ratio. The elution was performed at a flow rate of 1 ml min⁻¹ with a pressure of 1.5 MPa. Under the specified conditions, which included a column temperature of 30 C and a UV detector set to a wavelength of 254 nm, a 10-ml volume of the extract was introduced into the HPLC system, and the concentrations of DTD and HHO in UA and CA were determined according to the standard peak area. The retention times of DTD and HHO were 3.1 and 8.2 min, respectively. The standard methods for DTD and HHO preparation were from the Laboratory of Entomological Ecology, Yangzhou University, Jiangsu Province, China (Yang et al. 2006).

Following the completion of the incubation period, soil samples from each plate were collected for various analyses. The pH of the soil was determined using a 1:2.5 soil-to-water suspension and a pH meter (Thermo Scientific Orion Star A211, Wilmington, Massachusetts, American, 01887). The concentration of NH₄ ⁺-N was extracted using a 2 M KCl solution and measured via spectrophotometry (Mulvaney et al. 1997). Water-soluble phosphorus (P) and Olsen P were extracted using 0.5 M NaHCO₃ and quantified using molybdenum blue colorimetry (Pansu and Gautheyrou 2006). Additionally, 1 M NaOH-hydrolyzed N was assessed using the alkali solution diffusion method (Pansu and Gautheyrou 2006). To measure microbial biomass nitrogen (MBN) and phosphorus (MBP), the total nitrogen was determined using the K₂SO₄ extraction method and the inorganic phosphorus was determined using the NaHCO₃ extraction method in chloroform-fumigated soil (Brookes et al. 1982, 1985). The soil was incubated in a buffer solution (pH 6.5) containing 0.115 M of p-nitrophenyl phosphate (pNPP) for 60 min at 37 C to measure phosphatase activity (Marin et al. 2005; Tabatabai and Bremner 1969). The activity of soil protease was determined by means of spectrophotometry with casein as substrate according to the method of Ladd and Butler (1972).

Statistical Analysis

An ANOVA was conducted using SPSS v. 20.0 software (SPSS, Chicago, IL, USA) for all data. Linear correlations between available nitrogen (NH₄ ⁺-N and NaOH-hydrolyzed N) and protease activity, between available phosphorus (including Olsen P and water-soluble P; the same below) and phosphatase activity, and between pH and available phosphorus in the soils were calculated. Tukey’s multiple-range test was performed to detect the difference between treatments at a significance level of P < 0.05.

Results and Discussion

Main Allelochemicals and Nutrients of UA and CA

The DTD and HHO concentrations were 2,653.3 to 1,924.6 mg kg⁻¹ and 684.6 to 792.1 mg kg⁻¹ in the uncomposted plant samples from the five sites, respectively (Table 2). Compared with UA, CA had lower DTD and HHO contents (100.1 to 163.1 mg kg⁻¹ and 59.25-72.13 mg kg⁻¹, respectively), as well as considerably higher nitrogen and phosphorus contents (32.53 to 72.13 g N kg⁻¹ and 6.15 to 7.96 g P kg⁻¹, respectively).

Table 2. DTD, HHO, and nutrients in uncomposted (UA) and composted (CA) Ageratina adenophora plants. ^a

^a DTD, 4,7-dimethyl-1-(propan-2-ylidene)-1,4,4a,8a-tetrahydronaphthalene-2,6(1H,7H)-dione; HHO, 6-hydroxy-5-isopropyl-3,8-dimethyl-4a,5,6,7,8,8a-hexahydronaphthalen-2(1H)-one. In each column, means ± SD followed by different lowercase letters are significantly different at P < 0.05.

In recent years, various organic compounds such as tartaric acid dimethyl ester, cyclohexanamine N-cyclohexyl, diethylene glycol dibenzoate, and others have been extracted and isolated from aqueous leachates of A. adenophora (Yang et al. 2016; Zhang 2005). The primary allelochemicals in the aqueous extract of the aboveground parts of A. adenophora were identified as DTD and HHO (Yang et al. 2016; Zhu et al. 2011). It was found that DTD and HHO content decreased sharply after composting, which may be related to the addition of thermophilic Clostridium cellulosum during composting (Jiao et al. 2021a, 2021b). Therefore, C. cellulosum may effectively reduce the contents of these two allelochemicals (Jiao et al. 2021a, 2021b).

Fungal growth on Soil Surfaces and in the Soils

After incubation for 2 wk, the hyphae of C. lacerata covered nearly all soil surfaces in the CK _{C. lacerata} (only C. lacerata inoculation) and inoculation plus ECA treatments (Figure 2). Compared with CK _{L. deliciosus} (only L. deliciosus inoculation), providing the soil with ECA stimulated the growth of L. deliciosus on the surfaces. Fomitopsis palustris colonies also exhibited increased growth on the soil surfaces as the concentrations of ECA increased. However, the difference in growth was not statistically significant compared with the CK _{F. palustris} group (only F. palustris inoculation) at a concentration of 2.5 mg L⁻¹. Apart from a marginal growth at 2.5 mg L⁻¹, no visible growth of both C. lacerata and L. deliciosus was observed on the soil surfaces with the EUA amendment at 5 and 10 mg L⁻¹. As the concentrations of EUA increased, the size of the F. palustris colonies on the soil surfaces decreased. When the fungi grew on the soil surfaces, hyphae were observed in the soils near and under the fungal colonies.

Soil with EUA inhibited fungal growth on the soil surfaces at all nominal concentrations, except at 2.5 mg L⁻¹ for F. palustris, which remained unchanged. EUA inhibition of fungal growth suggested that several compounds (including DTD and HHO) present in the EUA were toxic to the tested fungi. In research conducted by Liu et al. (2016b, 2017), methanol was employed to extract A. adenophora. The research revealed that the resulting extracts displayed a potent antifungal effect against certain soilborne plant pathogens. The 50% effective concentration (EC₅₀) was more than 749 mg L⁻¹ (a liter of fresh plants was calculated based on oven-drying). Ageratina adenophora is a potential fungicide for controling plant diseases during crop production (Das and Devkota 2018). Notably, an increase was observed in the populations of azotobacteria and ammonia-oxidizing bacteria in soil containing A. adenophora (Zhao et al. 2019). Therefore, A. adenophora contains allelochemical compounds that may selectively influence the local soil microbes and phytopathogens, depending on the species. However, the results in the present study revealed similar or better growth of the test fungi on the soil surfaces and in the soils with ECA amendment, which suggests that the toxins in ECA were low enough to not influence fungal growth. It could be that aerobic composting decomposed allelochemicals. The CA sample was rich in soluble, low-molecular organics (31.61 g kg⁻¹ of humic acid; Jiao et al. 2021a), which can be used by the test fungi as a carbon source and nutrient, facilitating their growth in the soils and on the soil surfaces.

Nitrogen Mobilization

After the 2-wk incubation, soil NH₄ ⁺-N and NaOH-hydrolyzed N of CK contained 17.72 to 20.70 mg kg⁻¹ and 59.57 to 67.68 mg kg⁻¹. Notably, no protease activity or microbial biomass nitrogen (MBN) was detected in this soil (Table 3). The fungal inoculations (CK _{L. deliciosus} , CK _{C. lacerata} , and CK _{F. palustris} ) increased NH₄ ⁺-N, NaOH-hydrolyzed N, and protease activity, all of which were higher than those in the L. deliciosus– and C. lacerata–inoculated soils with the EUA amendments at all concentrations. The addition of EUA at concentrations of 5 and 10 mg L⁻¹ resulted in a decrease in soil indexes in the F. palustris–inoculated soil compared with CK _{F. palustris} . However, the addition of 2.5 mg L⁻¹ EUA increased the soil indexes. Soils inoculated with ECA showed comparable or higher NH₄ ⁺-N, NaOH-hydrolyzed N, protease activity, and MBN levels compared with soils with only inoculation.

Table 3. Influence of extracts from uncomposted (EUA) and composted (ECA) Ageratina adenophora plants on NH₄ ⁺-N, NaOH hydrolyzed-N, protease activity, and microbial biomass nitrogen (MBN) in the soils with fungal inoculation. ^a

^a In each column, means ± SD followed by different letters are significantly different at P < 0.05 (LSD test) in a fungal species.

^b CK _{L. deliciosus} , only L. deliciosus inoculation; CK _{C. lacerata} , only C. lacerata inoculation; and CK _{F. palustris} , only F. palustris inoculation.

^c Protease activity (U): μg Tyr g⁻¹ h⁻¹ (Tyr, tyrosine).

^d ND, not detected.

Almost 40% of total nitrogen in soils was present in the form of proteinaceous materials (Kieloaho et al. 2016). Hydrolytic reactions of these materials are vital for soil nitrogen mobilization. The proteinaceous mobilization into inorganic nitrogen can be divided into protein hydrolysis and ammonification. Soil protease activity plays a crucial role in breaking down proteins and peptides into amino acids. Along with urease, it contributes to soil nitrogen transformation, which is a key indicator of soil’s nitrogen supply capacity (Jan et al. 2009). In the present experiment, soil protease activity was decreased by EUA but increased by ECA. There was a positive correlation between protease activity and available nitrogen (including NH₄ ⁺-N and NaOH-hydrolyzed N). Such results are consistent with those of previous reports on the significance of proteases in organic nitrogen mobilization in soils (Liu et al. 2008; Sharma et al. 2013). The mechanisms behind the slow nitrogen mobilization observed in EUA may be attributed to either reduced production by the test fungi or the denaturation of proteases in the soil.

Phosphorus Mobilization

At the conclusion of the incubation period, the soil without inoculation exhibited pH levels ranging from 6.72 to 7.41, Olsen P concentrations between 3.24 and 3.65 mg kg⁻¹, and water-soluble P contents ranging from 1.08 to 1.44 mg kg⁻¹. Notably, no phosphatase activity or MBP content was detected in this soil (Table 4). The soils with fungal inoculations only (CK _{L. deliciosus} , CK _{C. lacerata} , and CK _{F. palustris} ) contained lower pH but higher available P, phosphatase activity, and MBP levels than those with no inoculation. The differences in the pH between no inoculation and CK _{C. lacerata} were not significant. Compared with fungal inoculation only, soil pH was unchanged by EUA at all concentrations except in the F. palustris–inoculated soil at 2.5 mg L⁻¹, in which pH decreased. Compared with CK (only fungal inoculation in tested soil), soil pH was increased by 0.01 to 0.08 under EUA treatment, while it decreased by 0.5 to 0.41 under ECA treatment.

Table 4. Influence of extracts from uncomposted (EUA) and composted (ECA) Ageratina adenophora on water-soluble P, Olsen P, pH, phosphatase activity, and microbial biomass phosphorus (MBP) in the soil with fungal inoculation. ^a

^a In each column, means ± SD followed by different letters are significantly different at P < 0.05 (LSD test) in a fungal species.

^b CK _{L. deliciosus} , only L. deliciosus inoculation; CK _{C. lacerata} , only C. lacerata inoculation; and CK _{F. palustris} , only F. palustris inoculation.

^c Phosphatase activity (U): μg pNP g⁻¹ h⁻¹ (pNP: p-nitrophenyl phosphate).

^d ND, not detected.

Available phosphorus, phosphatase activity, and MBP were decreased by EUA at all concentrations in the L. deliciosus– and C. lacerata–inoculated soils and in the F. palustris–inoculated soils at 5 and 10 mg L⁻¹ but remained unchanged at 2.5 mg L⁻¹. However, no significant difference was identified in the water-soluble P between CK _{L. deliciosus} and the EUA treatment. Similarly, the differences in water-soluble P among 0, 5, and 10 mg L⁻¹ were not significant. The addition of ECA, especially at a concentration of 10 mg L⁻¹, led to a substantial increase in available phosphorus, phosphatase activity, and MBP in the inoculated soils. However, it also resulted in a minor decrease in pH, although this decrease was not statistically significant in certain instances.

Most soils are deficient in available phosphorus (Ingle and Padole 2017). The mobilization of soil insoluble phosphorus into soluble forms refers to the solubilization of phosphorus-bearing minerals and the dephosphorization of macro-organics. Protons released from phosphorus-solubilizing microbes acidify the surrounding environment and eventually replace the cations combined with phosphate by H⁺, resulting in the release of H₂PO₄ ⁻ from phosphate minerals (Xing et al. 2021). The reduction in available phosphorus observed in the inoculated soils due to EUA can likely be attributed to the inhibition of fungal proton release, as indicated in Table 4. In the present study, similar to other researchers (Dick et al. 2000; Sardans et al. 2008), we employed pNPP as a substrate to measure phosphatase activity. The enzymes detected were phosphomonoesterases (often simply termed “phosphatases”) that are nonspecific for organic phosphorus mobilization in soils. Soil with EUA led to a decrease in phosphatase activity, while ECA resulted in an increase. Further, in the studied soils, available phosphorus levels exhibited an increase in conjunction with phosphatase activity. Thus, phosphatase activity may be involved in organic phosphorus mobilization, supporting previous reports (Liu et al. 2008; Sharma et al. 2013). The mechanism utilized by EUA for immobilizing organic phosphorus likely involved the inhibition of phosphatase secretion by the tested fungi. Soil with ECA promoted fungal growth, probably resulting in more phosphatases being produced by the fungi.

Pearson Correlation of Nitrogen and Phosphorus Mobilization Indexes

A positive correlation was observed between protease activity and NH₄ ⁺-N (r = 0.753 to 0.931, n = 7 to 14, P < 0.05) and between protease activity and NaOH-hydrolyzed N for each of the soils (r = 0.723 to 0.944, n = 7 to 14, P < 0.05; Figures 3 and 4).

Figure 3. The Pearson correlation coefficients between indices for Ceriporia lacerata and Fomitopsis palustris.

Figure 4. The Pearson correlation coefficients between indices for Lactarius deliciosus.

A positive correlation was observed between phosphatase activity and water-soluble P (r = 0.753 to 0.908, n = 7 to 14, P < 0.01), and between phosphatase activity and Olsen P (r = 0.826 to 0.842, n = 7 to 14, P < 0.05) for each of the soils. However, pH was inversely correlated with water-soluble P (r = −(0.879 to 0.928), n = 8 to 16, P < 0.05) and Olsen P in each soil tested (r = −(0.809 to 0.978), n = 8 to 16, P < 0.01; Figures 3 and 4).

The fungal inoculation on the soil surfaces resulted in an increase in soil available nitrogen and phosphorus. Such findings suggest that the tested fungi played a role in making previously unavailable nitrogen and phosphorus nutrients accessible. The findings support the notion that C. lacerata, L. deliciosus, and F. palustris play significant roles in nutrient recycling within forest ecosystems (Adhya et al. 2015; España et al. 2011; Yin et al. 2021a, 2021b). Providing soil with ECA stimulated the mobilization of unavailable nitrogen and phosphorus by the test fungi grown on the soil. Microbial nitrogen and phosphorus mobilization is a metabolism-dependent process, in which protons and a series of hydrolases and low-molecular-weight organic acids are produced (Cui et al. 2018). Thus, the improved fungal growth and metabolism could be the reason for the increased nitrogen and phosphorus availability in the test soils. Moreover, the contents of allelochemicals (DDT and HHO) experienced a notable reduction during decomposition. This reduction indicates that these allelochemicals would no longer hinder fungal growth and metabolic processes. Previous research by the present authors demonstrated that the aerobic composting can decompose the allelochemicals harmful to plants and thus eliminate the phytotoxicity of A. adenophora (Jiao et al. 2021a, 2021b). Therefore, if A. adenophora residues in the wilderness can be fully composted, these CA will directly supply nutrients to trees and microorganisms. Furthermore, composting eliminated A. adenophora’s toxicity to the three fungi, so CA may indirectly enhance the availability of soil nutrients by facilitating the mobilization of nitrogen and phosphorus. However, if fungal growth and metabolism in forests are inhibited by allelochemicals like DTD and HHO in EUA, the available nitrogen and phosphorus in soils will be decreased, resulting in the decrease in soil nitrogen and phosphorus supplied to trees. The mean concentrations of DTD and HHO in forest soil were 0.353 and 0.049 mg g⁻¹, respectively. The degradation rate of these two allelochemicals in forest soil was more than 70% within 1 wk under lab conditions (Yang et al. 2016). However, the time full decomposition and accumulation of allelochemicals released by plants into the soil throughout the growing season are different and may also be affected by external conditions (Yang et al. 2016). The invasion of A. adenophora into forests results in the continuous release of allelochemicals into the soil (Fu et al. 2018). These allelochemicals may inhibit the mobilization of nitrogen and phosphorus, rendering these nutrients unavailable to some microbial. Further, A. adenophora can survive in barren soil, extracting nutrients from soils that are less available for trees through their large root systems (Niu et al. 2007). Therefore, the mechanism A. adenophora employs in the invasion of forests may be its strong ability to extract nutrients from soils. At the same time, it inhibits the mobilization of nitrogen and phosphorus by releasing allelochemicals, consequently diminishing the supply of soil nitrogen and phosphorus. Further, the concentration of DTD and HHO is more impactful in inhibiting the three fungi.

The in situ CA residues left in soil may directly supply microorganisms with nutrients and may indirectly increase soil nutrient availability via the promotion of nitrogen and phosphorus mobilization. These significant findings provide initial insights into the mechanisms utilized by A. adenophora during its invasion of forests, particularly concerning plant nutrition. Additionally, the study highlights the role of aerobic composting in eliminating the toxicity of A. adenophora against microorganisms.