Indophenol

The AAC was determined using a 2,6-dichloro-indophenol titration method based on CNS GB 5009.86-2016 [27 ]. The TPC was determined by Folin-Ciocalteu colorimetry as follows: 0.2 mL of the polyphenol extract was added in 2 mL of the diluted Folin-Ciocalteu solution and 1.8 mL of 7.5% sodium carbonate solution, then mixed and placed in the dark for 1 h, the absorbance was measured at 765 nm. And the results were expressed as mg gallic acid equivalents (GAE)/100 g flesh weight (FW) [28 (link),29 (link)]. The TFC was determined as described previously as follows: 0.5 mL of polyphenol extract and 0.3 mL of 0.5 mol/L sodium nitrite solution were mixed and rested for 3 min, then 0.3 mL of 0.3 mol/L aluminum chloride solution, 2 mL of 1 mol/L sodium hydroxide solution, and 70% ethanol to 5 mL were added sequentially and rested for 10 min, the absorbance was measured at 506 nm. The results were expressed as mg catechin equivalents (CTE)/100 g flesh weight (FW) [30 (link)]. The TAC was estimated using the pH differential method as follows: 0.2 mL polyphenol extract was taken twice, 3.8 mL pH = 1 hydrochloric acid-sodium chloride buffer solution was added to the first solution, and 3.8 mL pH = 4.5 acetic acid-sodium acetate buffer solution was added to the second solution, and the absorbance of the two solutions was measured at 510 nm and 700 nm. The results were expressed as mg cyanidin-3-glucoside (CGE)/100 g flesh weight (FW) [30 (link)].

During the experimental period, automatic feeding equipment (made by Institute of Animal Science Chinese Academy of Agricultural Sciences, Beijing, China and NanShang Husbandry Science and Technology Ltd. Henan, China) was used to record dry matter intake. Milking facilities (90 Side-by-Side Parallel Stall Construction, Afimilk, Israel) were applied to record milk production of each cow.
On the last day of the experiment, a gastric rumen sampler was used to collect rumen fluid samples through the esophagus at 3 h after the morning feeding. Collected samples were strained through four layers of cheesecloth to obtain rumen fluid. Rumen fluid was then divided into two parts. One part was processed to analyze the pH value, rumen volatile fatty acid (VFAs) and ammonia-N (NH₃-N). The other part was put into the liquid nitrogen immediately after adding stabilizer and then stored at −80 °C for DNA extraction. Rumen contents were strained through four layers of cheesecloth with a mesh size of 250 μm. The pH of each rumen fluid sample was measured immediately using a portable pH meter (Testo 205, Testo AG, Lenzkirch, Germany). Individual and total VFAs (TVFA) in the aliquots of ruminal fluid were determined by gas chromatograph (GC-2010, Shimadzu, Kyoto, Japan). Concentration of NH₃-N was determined by indophenol method and the absorbance value was measured through UV-2600 ultraviolet spectrophotometer (Tianmei Ltd., China).
Milk samples were collected from individual cows during the last four consecutive days in 100-mL vials in each milking period. Samples were preserved with 2-bromo-2-nitropropan-1,3-diol and stored at 4 °C, before sent to the Milk and Dairy Products Quality Supervision and Testing Center, Ministry of Agriculture (Beijing, China) for analyses of milk protein, fat, lactose, somatic cell count and milk iodine content by a mid-infrared spectroscopy (Fossomatic 4000, Foss Electric A/S, Hillerød, Denmark).

For measurements of extractable C and N pools fresh soils (aliquots of 4 g) were extracted for 30 min with 30 ml 0.5 M K₂SO₄, subsequently filtered through ash-free cellulose filters and extracts kept frozen at −20°C for later analyses. Total dissolved N and dissolved organic carbon (measured as non-purgeable organic carbon) were measured by high temperature catalytic oxidation on a Shimadzu TOC-VCPH with TNM-1 unit and ASI-V autosampler (Shimadzu Austria, Korneuburg, Austria). Ammonium and nitrate were determined with colorimetric methods, NH₄⁺ with a modified indophenol reaction and NO₃⁻ with the VCl₃/Griess assay, as described in detail by Hood et al. [88] . Amino acid concentrations were quantified by a modified fluorometric OPAME procedure based on Jones et al. [89] , optimized for free amino acid measurement in protein hydrolysates. The modified OPAME reagent was made up of 10 mg o-phthaldialdehyde dissolved in 1 ml of HPLC grade methanol to which 20 µl of 3-mercaptopropionic acid was added. This reagent was mixed with 40 ml potassium borate buffer (0.2 M; pH 9.5) and left to stand overnight. Sample aliquots (50 µl) were mixed with 200 µl reagent in the microtiter plate and measured after 10 min at an excitation wavelength of 340 nm and emission wavelength of 450 nm with a fluorescence microplate reader (Tecan Infinite M200, Tecan Austria GmbH, Grödig, Austria). As NH₄⁺ in the soil extracts also gives a fluorescence signal, NH₄⁺ concentration had to be determined [88] and its fluorescence contribution was determined via a NH₄⁺ concentration series and subtracted from the total fluorescence of the samples. Total free amino acid-N concentration was then calculated using glycine as a standard and assuming that each mol of amino acid contains 1.37 mol N. Proteins were quantified in K₂SO₄ extracts by acid hydrolysis and subsequent amino acid analysis (modification of [89] –[90] ; see amino acid determination above). Protein hydrolysis to free amino acids was performed in 6 M HCl and heating was performed for 15 hours at 100°C in a drying oven with Bovine serum albumin (BSA containing 15.8% N) as internal standard. Samples were then dried in an N₂ stream and redissolved in deionized water. Determination of the NH₄⁺ concentration to correct the amino acid fluorescence was done as in the case of amino acid determination, with the exception that the sodium nitroprusside solution (the color reagent) contained 1.5 M NaOH instead of the original 0.3 M NaOH to neutralize residual acid in the hydrolysates.

Almost 10 hetares of harvested sites of eucalypt plantation were divided into four equal blocks, and three planting patterns were randomly arranged within each block in July 2016. The first planting pattern (E) was the continuous planting of pure Eucalyptus. urograndis (hybrid strain of Eucalyptus urophylla and Eucalyptus grandis) plantations of the third generation at a density of 1,667 plants/ha. The second planting pattern (EC) was the creation of mixed plantations of E. urograndis and Cinnamomum camphora (mixed pattern: inter-row, mixed density: 1667 plants/ha). The third planting pattern (EH) was the creation of mixed plantations of E. urograndis and Castanopsis hystrix (mixed pattern: inter-row, mixed density: 1667 plants/ha). Simultaneously, four unmanaged first-generation E. urophylla plantations in Luogangling Forest Park were selected as controls (CK). Information on forestland preparation, seedling specifications of eucalypts and native trees, and later plantation tending can be found in Xu et al. (2022) (link).
Sixteen mixed topsoil samples in the 10-cm layer were collected in December 2019 by removing the humus and litterfall from four different planting patterns. Soil samples for fungal community structure analysis were preserved with dry ice in centrifuge tubes and transferred to a-80°C freezer as soon as possible. Other soil samples for analyses of soil chemical properties and enzyme activities were stored in a portable refrigerator at 4°C.
The pH of each sample was determined with an electronic pH meter (soil: water, 1:2.5). Soil OM was determined by the potassium dichromate-sulfate colorimetric method (Sims and Haby, 1971 (link)). Total nitrogen (TN) and total phosphorus (TP) were measured with the Kjeldahl method (Tsiknia et al., 2014 (link)) and sodium hydroxide fusion-molybdenum antimony colorimetric method (Liu H. et al., 2017 (link)), respectively. Nitrate nitrogen (NO¯ 3_N) was determined by 2 mol·L⁻¹ KCl leaching-indophenol blue colorimetric method and ammonium nitrogen (NH+ 4_N) was determined by UV spectrophotometry (Lu, 1999 ). Available phosphorus (AP) was measured by the hydrochloric acid-ammonium fluoride extraction-molybdenum antimony colorimetric method (Lu, 1999 ). Soil available zinc (AZn) and available calcium (ACa) were measured by hydrochloric acid extract, atomic absorption spectrophotometry and ammonium acetate exchange, atomic absorption spectrophotometry, respectively (Liu J. et al., 2017 (link)). For soil enzyme activities, acid phosphatase (ACP) was determined by Phenylphosphonium-4-amino-antipyrine colorimetric method (Guan, 1986 ), urease (URE) by alkaline dish diffusion-HCL titration method (Guan, 1986 ), and invertase (INV) by 3,5-Dinitrosalicylic acid colorimetric method (Lu, 1999 ).

The absolute cover of vegetation community and C. filispica in each transect was measured before the trampling. The pictures of each transect were taken and imported into Photoshop 2020, where both the absolute cover of vegetation community and C. filispica were measured using a 5 × 20 grid, which proportionately covered each transect (0.5 m wide, 2 m long) with 100 intersections, each intersection of the grid with vegetation was recorded as a “hit,” and then multiplied by 100 to generate absolute cover values. The responses to damage had been stabilized after 2 weeks (Cole & Bayfield, 1993 ), when the absolute cover was measured again with the same method.
Two weeks after the trampling, morphological traits of C. filispica were measured: The thickness of leaves was measured with a vernier caliper, main veins included; the length, average width, and maximum width of leaves were measured by LI‐COR portable leaf area meter. The plants and surrounding soil plot of 10 × 10 × 10 cm each were excavated, which, owing to the shallow‐rooted situation, nearly contains the whole root system. All 378 individuals of C. filispica were then gently separated from the soil, washed, and sorted by whether they had DRs, after which 177 individuals with DRs were observed under a stereoscopic microscope, and the amount, density, size, color, and hair presence of DRs were recorded. The color of DRs was rated on a 5‐point scale, brightest to darkest (1 = White, 2 = Light yellow, 3 = Tawny, 4 = Dark brown, 5 = Black). The formula of DR density is as follows: total amount of DRs/ the dry weight of roots. Using a LA‐S root analyzer, we measured the total length, surface area, volume, and average diameter of the whole root system of 3 individuals with and without DRs per transect, respectively.
The plants were dried and the average biomass of the aboveground parts (leaves and fruits) and belowground parts (roots and rhizomes) were measured, respectively, after which they were ground up separately to measure the organic carbon (OC), total nitrogen (TN), and total phosphorus (TP) content. OC was determined by the potassium dichromate wet‐oxidation method, TN was determined by the Indophenol blue colorimetric method after digested with H₂SO₄‐H₂O₂, and TP was determined by the vanadium molybdate yellow colorimetric method after digested with H₂SO₄‐H₂O₂.

The urease inhibitory experiment was performed on the extract fractions of P. cubeba and their respective NPs of P. cubeba. With minimal changes, the previously stated technique was used for this investigation (Weatherburn, 1967 (link)). The experiment was performed in triplicates (n = 3) for each sample. The fractions of the crude extract and their associated NPs were separately added to 96-well plates and incubated for 30 min at 30°C using 5 µL of standard solutions (0.5–0.00625 mM concentrations). The experiment materials (NPs and fractions) were put into reaction mixes that included 55 µL buffer (pH 6.8), jack bean urease (25 µL), and 100 mM urea. Various concentrations of the samples, 0.5 mM (control), 0.625, 1.25, 2.5, and 5 mg of P. cubeba crude extract, and 0.6 mM (control) and 0.05 mg of P. cubeba AgNPs were employed to investigate the kinetics. For that purpose, each well received 70 µL of alkali (0.1% w/v NaOCl and 0.5% w/v NaOH) and 45 µL of phenol reagents (0.005% w/v sodium nitroprusside and 1% w/v phenol). After 1 h, the absorbance was measured at 630 nm. Using the indophenol method and thiourea as a standard, the production of ammonia (NH₃) was investigated. Finally, MS-Excel, SoftMax Pro (Molecular Devices, CA, United States), and EZ-FIT software applications were used to analyze the data. The following formula was used to calculate the percentage urease inhibition of each sample (Weatherburn, 1967 (link)): $\%\mathit{I}\mathit{n}\mathit{h}\mathit{i}\mathit{b}\mathit{i}\mathit{t}\mathit{i}\mathit{o}\mathit{n}=\mathbf{100}-\mathit{O}.\mathit{D}.\mathit{t}\mathit{e}\mathit{s}\mathit{t}/\mathit{O}.\mathit{D}.\mathit{c}\mathit{o}\mathit{n}\mathit{t}\mathit{r}\mathit{o}\mathit{l}\_\times \mathbf{100}.$