Moso bamboo (Phyllostachys edulis) invasions into broadleaf forests can cause serious ecological problems, such as reducing biodiversity and disrupting community succession. An elevated soil NH₄⁺/NO₃^- ratio has been identified as a potential mechanism that facilitates Moso bamboo invasion. However, it remains unclear whether and how this change is the result of the modification of soil nitrogen-transforming microorganisms by bamboo. Here, we collected soil from evergreen broadleaf forests that were either uninvaded or heavily invaded by Moso bamboo. We then compared the effects of soil origin on: (1) the growth of two common broadleaf tree species,Castanopsis fissa and Schima superba; (2) the soil nitrogen content and nitrogen transformation rates; and (3) the key microbial functional genes involved in nitrogen fixation, nitrification, and denitrification. By showing the significantly increased NH₄⁺/NO₃^- ratio in bamboo-invaded forest and the negative impact of this increase on the growth of the study plants, our study offers the first comprehensive synthesis of the positive feedback mechanisms of Moso bamboo invasion, with a focus on the instrumental role of nitrogen transformation processes.

Study area and site description

Our study site was located at Nankun Mountain Nature Reserve, Guangdong, China (23°30′N, 114°38′E). The area has a typical subtropical monsoon climate with an annual temperature of 12.4–28.5 ℃ and a mean annual precipitation of 2163 mm. The soil in the sampling area was acrisol. The region is covered primarily by a native evergreen broadleaf forest dominated by Schima superba, Castanopsis fissa, Castanopsis eyrei, Castanopsis carlesii, Machilus breviflora, and Castanopsis fabri. In the study area, Moso bamboo (Phyllostachys edulis) can expand as much as 16 ha/year to adjacent forests following anthropogenic disturbances (e.g., forest product harvesting). The intercept between Moso bamboo and broadleaf forests was obscure despite the astonishing speed of bamboo invasion, suggesting a rapid competitive replacement of broadleaf tree species by Moso bamboo. In the present study, we selected a broadleaf forest that was heavily invaded, a Moso bamboo forest with very few remnant broadleaf plants (referred to as Moso bamboo forest (MBF)) and an adjacent secondary evergreen broadleaf forest that had been preserved since the 1950s as our study sites (referred to as evergreen broadleaf forest (EBF)).

Soil sampling

We conducted three separate soil sample collections for three distinct purposes: determining of soil nitrogen-transforming functional genes, measuring soil nitrogen transformation rates, and preparing the growth media for controlled experiments.

To obtain soil samples for microbial DNA high-throughput sequencing and soil nitrogen transformation rate measurement, we established two parallel transects in each forest type (MBF and EBF) in June 2021. Within each transect, four plots (10 m × 10 m) were set up at 20-m intervals, resulting in a total of 16 plots for soil sampling (2 forest types × 8 replicates). In June 2021 and June 2024, we collected soil samples for DNA sequencing and soil nitrogen transformation rate measurements, respectively. For each collection, we used an auger to extract soil (20 cm depth × 10 cm diameter) from five randomly selected points within each plot, ensuring that the litter had been thoroughly removed beforehand. The soil from each plot was then mixed to create a composite sample and sieved through a 2-mm sieve to remove stones, roots and leaf litter. The soil samples were stored at -20℃ until further use.

With respect to the soil samples used in the controlled experiments, we revisited 5 of the 8 replicate plots for each forest type in July 2021 and used an auger to collect surface soil (20 cm depth × 10 cm diameter) after thorough litter removal. For each plot, at least 20 kg of soil (equivalent to 15 soil cores) was collected, mixed and sieved through a 2-mm sieve to form a composite sample. We stored a 2-kg subsample of each plot at 4℃ for the greenhouse experiment.

Soil physiochemical properties

Soil pH was measured in the field by a five-point sampling method with a soil pH detector (PHSJ-4F). Soil organic matter content was determined using an elemental analyser (model CHN-ORAPID, Heraeus Co., Hanau, Germany). Ammonium nitrogen (NH₄^+^{-N) and nitrate nitrogen (NO₃}-^{-N) were determined using a San}++^ continuous flow chemistry analyser (Skalar Analytica I.B.V, Netherlands) via the nitrogen diffusion method (Verdouw et al., 1978). Available phosphorus (P) was determined by the Mo‒Sb colorimetric method on the basis of the the Bray procedure (Bray and Kurtz, 1945). All the soil properties were expressed on an oven-dried weight basis.

Because there was a three-year time lag between the soil sampling for DNA sequencing and nitrogen transformation measurement, to ensure the compatibility of the data derived from the soil samples from the two different periods, we measured the soil NH₄^+^{-N content and NO₃}-^-N content for both periods and conducted paired t tests for the two periods.

Greenhouse experiment

From July 2021 to October 2021, we conducted a controlled experiment in a greenhouse at Sun Yat-sen University,Guangdong, China to explore the potentially different effects of soil from Moso bamboo forests and evergreen broadleaf forests on the growth performance of two dominant broadleaf species in southern China, i.e., Castanopsis fissa andSchima superba. For each species, fifty 2-month-old seedlings of similar sizes (i.e., approximately 10 cm in height) were purchased from a local plantation. We chose a random subset of 10 seedlings of each species to quantify the initial height and biomass for these two species and selected another 15 random seedlings for the greenhouse experiment.

For the growth medium, we filled each 3-L pot with 3500 g of γ-ray-sterilized base soil (radiation dose: 25 kilogray), which was collected from the non-rhizospheric area in the evergreen broadleaf forest in July 2021. We subsequentlyinoculated 300 g of live soil inoculum from either the Moso bamboo forest or the evergreen broadleaf forest described in Section 2.

One seedling of each species was transplanted to a pot, with 5 replicates for each combination of species and the origin of the soil inoculum, resulting in a total of 20 pots (Fig. 1b). All the pots were watered every other day. After 3 months of cultivation, all the plants were harvested, and their heights and biomasses were measured after 72 h of oven-drying.

Soil nitrogen transformation rate measurement

Nitrogen fixation rates were measured by the changes in total nitrogen before and after cultivation. To do this, the field water holding capacity and soil water content of each sample were assessed. A 20 g equivalent of fresh soil (oven-dried basis) was placed into a 150 ml flask. To provide a sufficient carbon source, 1 mg C/g dry soil of glucose and disodium malate were added separately. Additional distilled water was added to achieve 60% of the water holding capacity. The flasks were sealed with filter paper and incubated at 25°C for 10 days in the dark, with moisture promptly replenished as needed. The Kjeldahl digestion method was employed to measure the change in total nitrogen content, thereby indicating nitrogen-fixing activity (Li et al., 2010; Sainju et al., 2017).

Nitrification rates were inferred from the changes in NO₃^-^{-N content before and after cultivation. A 20-g equivalent of fresh soil (oven-dried basis) was placed into a 150-ml flask. We added glucose (0.5 mg C/g dry soil), glutamic acid (0.5 mg C/g dry soil), and 4 ml of (NH₄)₂SO₄ solution (200 µg N/g dry soil) separately. The water content was adjusted to 60% of the water holding capacity. The flasks were incubated at 25°C for 24 h in the dark. NO₃}-^-N was extracted and its concentration was measured to determine the nitrifying activity (Patra et al., 2006; Li et al., 2024).

Denitrification rates were inferred from the changes in NH₄^+^{-N before and after cultivation. A 15 g equivalent of fresh soil (oven-dried basis) was placed into a 150 ml flask. We added glucose (0.5 mg C/g dry soil), glutamic acid (0.5 mg C/g dry soil), and 5 ml of KNO₃ (200 µg N/g dry soil) separately. The water content was adjusted to 100% of the water holding capacity. The flasks were sealed with rubber stoppers and incubated at 25°C for 24 h in the dark. NH₄}+^-N was extracted, and its concentration was measured to determine its denitrifying activity (Luo et al., 1996).

DNA extraction, high-throughput sequencing and bioinformatic analysis

Total DNA was extracted from fresh samples (0.5-g dry soil basis) using a TGuide S96 Soil DNA Extra Kit (TiangenBiotech, Beijing) following the manufacturer’s instructions. DNA quality and concentration were measured with a synergy HTX microplate reader (GeneCompang Limited). A total of seven nitrogen-transforming functional genes (nifH, archaeal amoA, bacterial amoA, nirS/K, narG and nosZ) were amplified using 7 pairs of primers (POI F and POI R, amoA26F and amoA417R, amoA-1F and amoA-2R, Cd3aF and R3cdR, nirKF and nirKR, narG-F and narG-R, nosZ-F and nosZ-R) using a Veriti 96-well Thermal Cycler (Applied Biosystems,Thermo Fisher) (Braker et al., 1998; Bru et al., 2007; Park et al., 2008; Poly et al., 2001; Rösch et al., 2002; Rotthauwe et al., 1997) (Table S1). The PCR conditions were as follows: initial denaturation at 95 °C for 5 min; 25 cycles of denaturation at 95°C for 30 s; annealing at 50°C for 30 s; elongation at 72°C for 40 s and a final 7 min extension at 72°C. Individual reactions consisted of 50 ng of extracted DNA, 0.3 μL of upstream and downstream primers, 5 μL of KOD FX Neo Buffer (TOYOBO) (Biolink Biotechnology Co. Ltd, Beijing, China), 2 μL of dNTPs (2 mM each), and 2 μL of KOD FX Neo and ddH₂O diluted in a final volume of 10 μL.The PCR primers used are listed in Table S1. The PCR products were examined with a 1.8% agarose gel. The bands with the correct sizes were excised and then purified with an OMEGA DNA purification kit (Omega Bio-Tek Inc., GA, USA)and recycled with a Monarch DNA Gel Extraction kit (T1020L, New England BioLabs Inc., MA, USA). The functional gene community composition was assessed using the Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA), following standard protocols.

The raw read data obtained from sequencing were trimmed primarily by Trimmomatic (version 0.33) to omit low-quality reads (Bolger et al., 2014), and the paired-end reads were assembled by FLASH (version 1.2.11) with a minimum 10 bp overlap and a <0.2 allowable error ratio (Magoč and Salzberg, 2011). Using Cutadapt (version 1.9.1) (Martin, 2011)to identify and extract primer sequences, a clean sequence without primer sequences was obtained. Then, Usearch (version 10) (Edgar, 2010) was used to splice the clean sequences of each sample, and UCHIME (version 4.2) (Edgar, 2016) was used to identify and remove the chimeric sequences, resulting in the final valid high-quality data sequence. The high-quality sequences were subsequently clustered at the 97% similarity level using the Usearch UPARSE algorithm to generate operational taxonomic units (OTUs) (Edgar, 2013). The taxonomic database of each functional gene was established using the corresponding gene sequences derived from the Functional Gene Pipeline and Repository (Fish et al., 2013).