Global nitrogen (N) deposition and changes in precipitation patterns profoundly influence leaf photosynthesis and carbon sequestration in terrestrial ecosystems. Foliar fungi, a critical microbial community on leaf surfaces, could simultaneously impact several leaf functions. However, the regulated effects of foliar fungi on leaf photosynthesis in response to nitrogen and water additions remain unclear.  Here, using a long-term field and a complementary pot fungicide experiment in an alpine meadow, we quantified the effects of nitrogen and water addition on leaf photosynthesis and foliar fungal communities in two dominant species.

Site description

The experiment was conducted at Haiyan County (36.92° N, 100.94° E, 3090 m a.s.l), located on the northeastern Qinghai-Tibetan Plateau. This region has a mean annual temperature of 0.94°C and mean annual precipitation of 404.66 mm, with approximately 80% of precipitation occurring during the growing season (June to August). The vegetation at this site is dominated by Carex alatauensis and Leymus secalinus. The soil at the study site is classified as Mat-Gryic Cambisol with a clay-loam texture. Additionally, the soil pH is approximately 8.09, soil total carbon concentration is 21.28 g kg^-1, and soil total nitrogen concentration is 3.33 g kg^-1.

Experimental design

Our study conducted both a long-term field experiment and a complementary pot experiment. The field experiment was separated from the adjacent pot experiment by 5 m buffer zone.

Field experiment

The nitrogen (N) and precipitation addition experiment has been established since 2017. Nitrogen was applied as urea at a rate of 10 g N m^-2 year^-1, manually distributed before the onset of the annual growing season (late April). Increased precipitation treatment was simulated by adding water equivalent to 10% of the mean annual precipitation (approximately 40 mm), distributed across eight events: twice in June, and three times each in July and August. Therefore, the experiment included four treatments: control, (CK), no nitrogen or water addition; nitrogen addition, (N, 10 g N m^-2 year^-1); water addition (W); nitrogen and water addition, (NW, 10 g N m^-2 year^-1 with water addition). Each treatment was conducted with 5 replicates. Plots were 3 × 3 m in size, which were randomly distributed with a 1 m buffer zone separating adjacent plots.

Within each plot, species composition and height were recorded in a 0.5 × 0.5 m quadrat throughout the study period. Above-ground net primary productivity was measured annually at its peak (early August) by clipping all above-ground biomass at ground level. The samples were sorted by species, oven-dried at 70 °C for 48 hours to a constant weight, and above-ground net primary productivity was calculated. Dominant species were identified based on their contribution to total above-ground net primary productivity, with Carex alatauensis and Leymus secalinus selected as the focus species for further analysis.

Pot experiment

For further verify the role of foliar fungi community, a pot experiment was conducted in 2023 nearby the experimental field, following the same treatment design as the field experiment. Polyvinyl chloride pipes with a diameter of 16 cm were cut into 20 cm height pots. Each pot was filled with 6 kg of air-dried, sieved (0.2 mm) clay-loam soil collected from the experimental field. Dominant plant species, C. alatauensis and L. secalinus, were transplanted into the pots from experimental field in mid-August 2023. To ensure sufficient plant survival after the winter and spring, five individuals of C. alatauensis and three individuals of L. secalinus were maintained in each pot. Each treatment was conducted with 5 replicates.

Before foliar fungicide treatment, we ensured that the plants were mature. Foliar fungicide treatments were applied after the first water addition in July 2024, using "Score Profi" (25.0% Difenoconazole) and "Ortiva" (10% Azoxystrobin) to investigate the role of foliar fungi on leaf photosynthetic properties.