A poisoned apple: First insights into community assembly and networks of the fungal pathobiome of healthy-looking senescing leaves of temperate trees in mixed forest ecosystem

Despite the abundance of observations of foliar pathogens, our knowledge is severely lacking regarding how the potential fungal pathobiome is structured and which processes determine community assembly. In this study, we addressed these questions by analysing the potential fungal pathobiome associated with the senescing leaves and needles of 12 temperate tree species. We compared fungal plant pathogen load in the senescing leaves/needles and demonstrated that healthy-looking leaves/needles are inhabited by diverse and distinct fungal plant pathogens. We detected 400 fungal plant pathogenic ASVs belonging to 130 genera. The fungal plant pathogenic generalist, Mycosphaerella, was found to be the potential most significant contributor to foliar disease in seedlings. The analyses of assembly process and co-occurrence network showed that the fungal plant pathogenic communities in different tree types are mainly determined by stochastic processes. However, the homogenising dispersal highly contributes in broadleaf trees, whereas ecological drift plays an important role in coniferious trees. The deterministic assembly processes (dominated by variable selection) contributed more in broadleaf trees as compared to coniferous trees. We found that pH and P level significantly corresponded with fungal plant pathogenic community compositions in both tree types. Our study provides the first insight and mechanistic understanding into the community assembly, networks, and complete taxonomy of the foliar fungal pathobiome in senescing leaves and needles.


Physiochemical analyses
To obtain water-leachable components, senescing leaf and needle samples were shaken in 30 mL milliQ water for 1 h in falcon tubes, centrifuged for 5 min at 3500 rpm, decanted, and filtered. The remaining leaf/needle material was dried for two weeks at 40 °C to determine dry weight, which was used as reference for all subsequent qualifications. Leachate pH was determined using pH paper with a scale precision of 0.2 units. TN was analyzed using a sum parameter analyzer with high temperature combustion and chemiluminescence detection (Mitsubishi TN-100; a1 envirosciences, Düsseldorf, Germany). All samples were measured as triplicates. Norg was calculated as the difference: Norg = TN-NMin. For NMin quantification, a flow injection analyzer (Quikchem QC85S5; Lachat Instruments, Hach Company, Loveland CO, USA) was used with corresponding manifolds to measure ammonium nitrogen N NH 4 + , nitrite nitrogen N NO 2 − , and nitrate-plus nitrite nitrogen N NO 3 − +NO 2 − content. N NH 4 + was determined by the gas diffusion method. N NO 3 − was reduced to nitrite using a cadmium column in the manifold prior to the chemical reaction to form an azo dye. The nitrate reduced by cadmium and the nitrite originally present in the sample were analyzed using the Griess reaction by diazotization with sulfanilamide and coupling with N-(1-naphthyl) ethylenediamine dihydrochloride. The deep pink color of the resulting dye was measured at λ = 520 nm. N NO 2 − alone was determined after the same reaction, without using a cadmium column. DOC was quantified as non-purgeable organic carbon (NPOC) with a sum parameter analyzer using high-temperature combustion and infrared detection (vario TOC cube, Elementar Analysensysteme GmbH, Langenselbold, Germany). Each sample was measured as triplicate. A sample volume of 200 µL each was automatically injected into the ash finger of the combustion tube which contains platinum as catalyst. The samples were combusted at 850°C in synthetic air, a hydrocarbon-free mixture of nitrogen and oxygen. After removing moisture from the combustion gas, NPOC was quantified by IR detection of CO2 formed from the organic carbon compounds in the sample.
The determination of nutrient content, Ca, Fe, K, Mg, and P of leaves and needles followed two processes. First, the sample digestion, in which 100 mg of sample material were submitted to a microwave-assisted high-pressure digestion (Multiwave 3000, Anton Paar, Graz, Austria) at a maximum microwave power of 1200 W and a maximum pressure of 60 bar after addition of 3 -5 mL 65% HNO3, supra-pur, (Merck, Darmstadt, Germany). A rotor 8SXF100 with reaction vessels made of TFM (tetrafluor-modified polytetrafluoroethylene) was used. Overall digestion time was 50 min, including 20 min of cooling at zero microwave power. A blank, consisting of nitric acid only was run to check for possible contamination of reagents and vessels. After accomplishment of digestion, the solutions were filtered and transferred to 50 mL PE vessels which were filled to the mark with ultrapure water (Millipore, Eschborn, Germany). Secondly, the sample solution analyses were carried out using inductively coupled plasma-optical emission spectrometry (ICP-OES) "Arcos" (Spectro, Kleve, Germany) equipped with a 27.12 MHz free-running LDMOS generator and ORCA optical system. A three-point-calibration based on single-element standards issued by Merck, Darmstadt, Germany, was carried out at the following concentration levels: 10, 50, 100 mg/L for Ca, K, Mg, P and 0.5, 2.5 and 5 mg/L for Fe, respectively.

Goodness-of-fit statistic and variance partitioning analyses
(a) ANOSIM and NPMANOVA based on relative abundance data and the Bray-Curtis distance measure.   Table S4 Latitude and longitude of each tree replicate (please see in a separate excel file).

Figure S1
NMDS ordinations of plant pathogenic community compositions based on presence/absence data and Jaccard distance similarity.

Figure S2
Network between tree species and plant pathogenic fungal ASVs. Large circles indicate each tree species. Tree species name can be found at the outer layer of the network cycle. are Smaller circles in light purple color refer to plant pathogenic fungal ASVs.

Figure S4
Goodness-of-fit statistics (R 2 ) of environmental variables fitted to NMDS ordination of fungal plant pathogenic community based on presence/absence data and Jaccard distance measure (a), Venn diagrams showing the contributions of the factors shaping fungal plant pathogenic community (b-c). The locations (latitude and longitude) of each tree replicate are provided in Supplementary Table  S4. Nutrient evaluated in the analysis of broadleaf trees was NH4 + -N. Nutrients evaluated in the analysis of coniferous trees were DOC, Norg, K, Mg, and P. The number and percentage in the parentheses in the Venn diagram indicate the explained variance and its percentage in the total explainable variance.