The role of chemical properties of the material deposited in nests of white stork in shaping enzymatic activity and fungal diversity

Organic debris accumulated in bird nests creates a unique environment for organisms, including microbes. Built from various plant materials that are typically enriched by animal residues, bird nest favours the development of various fungal groups. The aim of this study was to investigate the chemical properties of the material deposited in the white stork Ciconia ciconia nests and the link between extracellular enzyme activity and the diversity and composition of culturable fungi. Our findings revealed low C/P and N/P ratio values in the nest materials, which indicate a high P availability. Nest material C/N/P ratio ranged from 67/8/1 to 438/33/1. Enzymatic activity strongly correlated with the content of carbon, nitrogen, and pH of the material deposited in the nests. A total of 2726 fungal isolates were obtained from the nests, from which 82 taxa were identified based on morphology and DNA sequence data. The study indicates that white stork nests are microhabitat characterised by diverse chemical and biochemical properties. We found relationship between the fungal richness and diversity and the C/P and N/P ratios of materials from the nests. Our study showed that culturable fungi occurred frequently in materials with high levels of C, N, and P, as well as high concentrations of base alkaline elements (Ca, Mg, and K).


Introduction
Fungi exists in different types of environment and can survive across a wide range of pH and temperature (Rousk and Bååth 2011;Frąc et al. 2015) and they can help to stabilise organic matter (Treseder and Lennon 2015;Frąc et al. 2018).The accumulated organic debris present in constructed bird nests offers a unique environment for microbial assemblage (Baggott and Graeme-Cook 2002).Depending on the nest type (e.g.open-cup nests, tree cavities) or habitat location (e.g.aquatic or terrestrial), different fungal species become characteristic components of this specific microhabitat and play important roles in nutrient cycling (Apinis and Pugh 1967;Pugh and Evans 1970;Hubálek and Balat 1974;Hubálek andBalat 1974, 1976;Korniłłowicz-Kowalska and Kitowski 2009, 2013, 2017;Korniłłowicz-Kowalska et al. 2010, 2011, 2018;Jankowiak et al. 2019).The taxonomic and spatial distributions of the mycobiota in nests of wetland birds appear mainly affected by the properties of materials deposited in the nest constructions (Korniłłowicz-Kowalska et al. 2018).Most bird nests are built from various plant materials that are typically enriched by animal residues such as hairs, feathers, and insect exoskeletons, and that favour the development of various fungal groups, including cellulolytic and keratinolytic fungi (Korniłłowicz-Kowalska et al. 2010, 2018;AI Rubaiee et al. 2021).Bird nests also house potentially phytopathogenic and zoopathogenic fungi (Korniłłowicz-Kowalska andKitowski 2013, 2017;Jankowiak et al. 2019).The predominance of ascomycetes appears to be a characteristic of open-cup bird nest mycobiota (Pugh 1965(Pugh , 1966;;Apinis and Pugh 1967;Otčenašek et al. 1967;Pugh and Evans 1970;Korniłłowicz-Kowalska andKitowski 2009, 2017;Korniłłowicz-Kowalska et al. 2010, 2011, 2018).Interestingly, basidiomycetes are the dominant and co-dominant members of fungal communities in woodpeckers' cavities excavated in dead or living trees (Jusino et al. 2015;Jankowiak et al. 2019;Pozzi et al. 2020).
As fungi are key producers of the enzymes required for cellulose and lignin decomposition (Schneider et al. 2012), measurements of the activity of extracellular enzymes involved in the circulation of nutrients that originate from organic compounds provide information about their biogeochemical cycles (Adamczyk et al. 2014).Several common hydrolytic enzymes contribute to the carbon (C) (β-Dcellobiosidase, β-glucosidase, β-xylosidase), nitrogen (N) (N-acetyl-β-glucominidase), phosphorus (P) (phosphatase), and sulphur (S) (arylosulphatase) cycles, and their main function is to degrade cellulose, hemicelluloses, and chitin (Parvin et al. 2018).Substrate availability and nutrient limitation are two strong drivers of enzyme activity in soil, and consequently of the C, N, and P cycles (Stock et al. 2019).Therefore, enzyme activity has been proposed as a quality indicator because it indicates changes in organic matter status and its turnover (Błońska et al. 2017).The decomposition of organic matter depends on substance properties and accessibility of microorganisms and their enzymes (Pająk et al. 2016).
The white stork Ciconia ciconia is an iconic species from European farmlands that builds one of the largest and heaviest nests among modern birds, which are used for many years.The nests are built of organic material such as hay, straw, manure, twigs, and branches at different stages of decomposition and as well as mineral particles (sand grains) and showed considerable variation in size (range: 80-230 cm in diameter; range: 10-140 cm in height) and weight 70-1348 kg (Zbyryt et al. 2021).In addition, the substrate composition of nests may change when parents incubate eggs and feed offspring; during subsequent breeding events, nests are enriched with excrement, pellets, feathers, eggshells, and food remains of invertebrate prey mainly insects and earthworms and small vertebrates mainly mammals.Large birds created a microhabitat in their nests which was favourable to nest-mediated seed dispersal (Dylewski et al. 2021).Recently, Błońska et al. (2021a) investigated the biological and physicochemical properties of white stork nests from north-east Poland and found that deposited materials gradually form soils over the years, with distinguishable layers possessing different physicochemical characteristics and biochemical activities.The results of this study indicated that the material contained in the nests could be characterised as lignic histosols ornithic.However, to the best of our knowledge, there are no reports that link the diversity of fungi in the open-cup nests of large-bodied birds to biological properties of stocked materials.
In our study, we hypothesised that large nest constructions are a specific microhabitat, characterised by diverse chemical properties that affect the enzyme activity and diversity of the fungal community.We also predicted the C/N/P ratio of material from large nests to be similar to that known in soils.C, N, and P are commonly considered major drivers of the enzyme activities and fungal diversity in soil.However, no studies have been conducted so far to test such relationships in material deposited in nests of large birds.As we expected considerable variation in the chemical properties of deposited material, we predicted these characteristics to significantly influence the enzyme activity, diversity of the culturable mycobiota associated with large-bodied birds.

Study sites
The investigation was carried out in north-eastern Poland, in the Podlasie province, where materials from nineteen white stork nests were selected for biochemical examination and fungi cultivation.As the collection of material led to the destruction or serious damaging of the nest structure, the number and mass of the sample material are limited due to legal restrictions.The research covered nests used by white storks for several decades, although exact age of each nest is not known.Samples for analysis were collected from occupied nests after the breeding season.Nest samples were taken from a special crane raised several meters above the ground.Samples were collected from each nest to determine their chemical and biochemical properties.Further analyses were carried out using aggregate samples, homogenised from 5 smaller sub-samples from different places of nest so that all parts of the nest were represented (Carter and Gregorich 2008).The goal of sampling was to produce a sample that was representative for materials deposited in a given nest.The samples from each nest were placed in a plastic container and mixed.The containers and tools used to collect samples from the nests were washed with water and ethanol (> 99%, w/w) for disinfection before each sampling event.The samples were immediately transported to the laboratory and stored in the dark at 4 °C for subsequent analysis.

Laboratory analysis
To determine their basic properties, 19 aggregate samples were dried to air dry condition and sieved (< 2-mm mesh) to remove artefacts (twine, plastic).Each sample was mixed in distilled water (1:5 w/w) and its pH was determined using the potentiometric method.The total N, organic C, and S contents were measured using a LECO CNS True Mac Analyzer (Leco, St. Joseph, MI, USA).Following mineralisation in a mixture of concentrated nitric acid and perchloric acid at a ratio of 2:1 v/v, the Ca, Na, Mg, K, P, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, and Zn contents were determined using ICP-OES (Thermo iCAP 6500 DUO, Thermo Fisher Scientific, Cambridge, UK).C/N, C/P, and N/P ratios were calculated on a molecular basis (Piaszczyk et al. 2019).

Fungal isolation and identification
Based on chemical properties of material from nests such as pH and carbon content, 11 nests were selected for fungal identification (Table 1).Each sample (50 g) in a state of natural moisture was stored in sterile plastic containers at 5 °C for 2 days prior to the isolation procedures.Culturable fungi were isolated using soil dilutions.A soil sample (10 g) was suspended in 90 mL of sterile distilled water and thoroughly mixed for 3 min to obtain a 10 −1 suspension.Serial dilutions 10 −2 , 10 −3 , 10 −4 , and 10 −5 were then prepared from this suspension.The methodology of the isolation procedures was previously described by Błońska et al. (2021a).Fungal taxa identified in nests of white stork and their frequencies (%) were detailed presented by Błońska et al. (2021a).

Statistical analysis
The Spearman's rank correlation coefficients for the physical and chemical characteristics and enzyme activity of the 19 nest materials were calculated.Principal component analysis (PCA) was used to identify the interrelationships that existed among variables and to determine how suites of variables were related.The PCA method was used to evaluate the relationships between soil chemical properties, enzymatic activity, and fungal species composition in 11 nests.Differences with p < 0.05 were considered statistically significant.All statistical analyses were performed using the Statistica 13 software (2018).
For each nest sample, the number of colony-forming units (CFU) per gram of soil was determined based on the 10 −3 dilution isolation results.The relative frequency (RF) was the ratio of the number of isolates of a certain taxon to the total number of isolates in each nest.Dominance (Y) was calculated as described by Du et al. (2020); when Y > 0.02, the genus was deemed dominant.The following ecological diversity measures used by Du et al. (2020) and characterised by Magurran (1988) were calculated for each nest: species richness index (SR), Margalef index (D′), Shannon-Wiener index (H′), Simpson diversity index (D s ), Simpson dominant index (λ), probability of interspecific encounter index (PIE), and Pielou's evenness index (J).
The studied material was also characterised by various enzyme activities (Table 2).CB activity ranged from 16.3 to 1261.1 nmol MUB .g −1 d.s. .h −1 , BG activity from 307.7 to 3,261.7 nmol MUB .g −1 d.s. .h −1 , NAG activity from 242.8 to 1772.4 nmol MUB .g −1 d.s. .h −1 , and PH activity from 285.5 to 3222.8 nmol MUB .g −1 d.s. .h −1 .We noted a lower activity   for XYL and SP (33.7-399.2nmol MUB .g −1 d.s. .h −1 and 0.0-43.3nmol MUB .g −1 d.s. .h −1 , respectively).The activity of most enzymes, except for SP, correlated with the C content (Table 3).The highest correlation with the content of C (0.83 and 0.82) was noted for the activity of CB and BG.The activity of CB, BG, and XYL correlated significantly with the content of N and S. A negative, significant correlation was noted between pH and the activity of CB and XYL (Table 3).
The nest material contained a C/N ratio of approximately 10 (in the range of 9.0 to 14.8).The mean C/P ratio was 216.9, with values ranging from 67.4 to 438.3.In the case of the N/P ratio, low values in the range of 7.1-33.4were recorded.The C/N/P ratio in the materials from the nests was lower, and ranged from 67/8/1 to 438/33/1.There was a strong positive correlation between the C and N concentrations in the materials (r = 0.89) (Table 4).The concentrations of N and P correlated with the C/N ratio (r = − 0.60).Positive correlations between C/N and C/P and between N/P and C/P were observed (r = 0.51 and r = 0.97, respectively) (Table 4).
The highest SR values were found in the M2 (SR = 28) and CZ (SR = 24) nests, while the lowest species richness values occurred in the ŁA (SR = 7) and GS1 (SR = 8) nests (Table 6).SR positively correlated with K and P contents.The Margalef index (D′) also showed a high correlation to Ca, Cd, Co, Mg, and Mn contents (Table 7).
The fungal diversity values varied widely among the sampled nests (Table 5).Fungal communities associated with SZ2, M2, GS2, CZ, D23, and D2 nests had the highest diversity (H′ and D s ≥ 0.81), while the lowest diversity was found in the ŁA nest (H′ and D s ≤ 0.31) (Table 6).PIE values were similar to D s values, and the highest values were recorded in the following nests: D23, D2, CZ, M2, GS2, and SZ2.Fungi from D2 and D23 nests possessed the highest Pielou index (J = 0.83 and J = 0.83, respectively).The fungal community in ŁA and SZ1 nests showed the highest degree of ecological dominance with a λ value of 0.64 and 0.48, respectively.Conversely, the lowest λ value was detected in D23 and D2 nests (λ = 0.11 and λ = 0.14, respectively) (Table 6).The diversity indices statistically significant correlated with a content of Ca, K, and Mn in nests (Table 7).
Two main PCA factors had a significant total impact (49.2%) on the variance of the variables.Factor 1 explained 30.9% of the variance of the examined properties, whereas factor 2 accounted for 18.3% of the variance (Fig. 1).The occurrence of Penicillium, Leuconeurospora, Hawksworthiomyces sp., Mortierella, Aspergillus, and Scopulariopsis was associated with high enzyme activity, as well as high C and N contents (Fig. 1).The higher C content and enzyme activity were noted in M1, GS1, GS2, and D37 nests (Fig. 1 and Table 1).Fungal communities associated with M1, GS1, GS2, and D37 nests were the most distant from the D2 nest, which was associated with high Pb, Mg, Cr, and Fe contents, as well as high pH (Fig. 1).The D2 nest was associated with Alternaria, Dinemasporium, Neosetophoma, and Sarocladium fungi (Fig. 1).In addition, the high content of K, Ca, Mn, and Co correlated with the presence of Acaulium, Phialophora, and Fusarium (Fig. 1).PCA analysis separated M2 nests mainly based on the prevalence of Mucor and Aspergiliaceae that were associated with high N, Cd, Zn, Na, and P contents and on the occurrence of Scopulariopsis, Pseudogymnoascus, and Chaetomium, that were associated with nests characterised by a low content of these elements (ŁA, SZ1, and SZ2 nests) (Fig. 1).

Chemical properties of nests and their enzyme activities
We observed substantial differences in the chemical properties of materials deposited in the nests of white stork, which produced a high variability in enzymatic activity and composition of the fungal community.The nests were characterised by a high organic C and N contents, which led to the classification of white stork nests as lignic histosol ornithic (Błońska et al. 2021a).Birds transfer nutrients because the excreta of birds are rich in N and other nutrients such as P and Ca (Osono 2012).Organic matter is the basic parameter that influences soil biochemical activity by providing substrates for enzymatic reactions (Uzarowicz et al. 2020).Our findings confirmed the correlation between the extracellular enzyme activity involved in the C, N, and P cycles and the organic C content in white stork nests.The nests were also characterised by a high N content, which enabled the organic matter in the nests to decompose at a favourable rate.We recorded a suitable C/N ratio, which indicated the availability of N to soil microorganisms.The C/N ratio of material from white stork nests fluctuated to approximately 10.According to Robertson and Groffman (2015), microorganisms can easily obtain N during the decomposition of a relatively N-rich detritus (with a C/N ratio of approximately 20) because mineralisation outweighs the processes of N immobilisation and absorption.Organic C content in the nest material was the driver of all the tested hydrolytic enzyme activities that contributed to the C cycle (CB, BG, XYL), the N cycle (NAG), and the P cycle (PH).Additionally, β-Dcellobiosidase, β-glucosidase, and phosphatase activity significantly correlated with the pH of the tested material.pH influences the activity of soil enzymes by controlling ionisation caused by conformational changes in enzymes, substrate availability, and enzymatic cofactors (Błońska et al. 2021b).Kreyling et al. (2012) claimed that the organic matter in soil results from microbial degradation and the presence of nutrients such as N and P. According to WRB (2014) ornithogenic material contains at least 0.25% phosphorus pentoxide (P 2 O 5 ) (in a 1% citric acid extract) and the tested nesting substrate met the criteria for onithogenic material (P 2 O 5 content > 0.25%) (Błońska et al. 2021a).The obtained P value results meet the criteria defined in the WRB (2022) classification.
The samples collected from white stork nests were characterised by narrow C/N/P ratio ranging from 67/8/1 to 438/33/1, similar to that known for soils.Piaszczyk et al. (2019) noted that the C/N/P ratio for soils strongly influenced by decomposing deadwood ranged from 395/27/1 to 1592/81/1.The C/N/P ratio of the studied materials from nests was similar to that of mineral soils.The C/N/P ratio values obtained from the present study further confirm the possibility of soil formation in white stork nests.The C/N, C/P, and N/P ratios are good indicators of nutrient content and its potential availability (Piaszczyk et al. 2019).High C/N ratios indicate a slow decomposition of organic matter (Tian et al. 2010;Fazhu et al. 2015).The C/N ratio values obtained in the present study confirm the efficient decomposition of organic residues delivered to the nest.Negative correlations were noted between the C/N ratio and N and P, confirming that P was one of the key regulators of C and N circulation in the nest profile.Zhao et al. (2017) report that P can increase the labile fraction of C by increasing soil microbial activity.P prevails in nutrient limiting and controls the microbial community structure and succession (Knelman et al. 2014).According to Zhang et al. (2017), C, N, and P are the three main macroelements for biomass building, and the C/N/P ratio is therefore important to recognise the relationships between organisms and their environment.

Culturable fungal diversity
In the present study, the 2,726 culturable fungi isolated from White Stork nests were divided into 82 taxa, 38 genera, 17 orders and 3 phyla, showing abundance and high species richness.This finding was consistent with those from studies conducted on other birds that build open-cup nests (Hubálek 2000;Korniłłowicz-Kowalska and Kitowski 2009, 2013, 2017;Korniłłowicz-Kowalska et al. 2010, 2011, 2018) and suggested that large birds' nests that are occupied for long periods are a natural habitat for several fungal populations.For example, the nests of marsh harriers Circus aeruginosus yielded 63 fungal species belonging to 37 genera, among which Asperigillus falvus, Asperigillus fumigatus, Penicillium spp., and Scopulariopsis brevicaulis were the dominant ones (Korniłłowicz-Kowalska et al. 2018).Consistent with our results, most of the identified fungal species that dominate soil systems globally belong to phylum Ascomycota (Egidi et al. 2019).The abundance of fungi detected in nests could be due to a favourable C/N ratio in the nest material, which promoted the growth and activity of these microorganisms.
Our study revealed marked differences in the composition of fungal communities among white stork nests.The differences in the total number, species richness, and diversity indices of the communities that colonised the nests could be attributed to a variety of substrate characteristics, including the various chemical properties of the nest material and the nutrient availability, especially C and N sources.Despite the high content of organic C, N, and P in the nests, we observed considerable variation in the content of C, N, P, macroand micro-nutrients.PCA revealed that the occurrence of Penicillium and Aspergillus fungi strongly correlated with

Table 7
Spearman's rank correlation coefficients between material properties and diversity indices of fungi obtained from in the nests of white stork Ciconia ciconia.the organic C content and high enzymes activities.These fungi are known for their high ability to secrete plant cell wall-degrading enzymes (Passos et al. 2018).The elevated organic C content in some nests could probably be linked to the higher proportion of plant debris used for its construction.The high activity levels of cellulolytic fungi may also result from raised N concentrations, as cellulase production is greatly influenced by the level and source of N in vitro (Lynd et al. 2002).Although we found no important effects of N content on fungal diversity, a previous study showed that the biomass and species richness of ectomycorrhizal fungi in pure culture increased under high N concentrations (Wilkinson et al. 2012).On the other hand, recent studies have indicated that the concentration of nitrogen fertiliser strongly modifies the composition but not the taxon richness of fungal communities in the soil (Paungfoo-Lonhienne et al. 2015).The abundance of ascomycetes is generally higher under high N concentrations and their growth is correlated with N availability (Fontaine et al. 2011).Soil microbes play a crucial role in C, and P cycling in terrestrial ecosystems by mineralising organic matter (Zhou et al. 2018).Our findings show a strong relationship between fungal richness and diversity and the C/P and N/P ratios of the nest material.The stoichiometry of fungal biomass in response to the species taxonomy and ecological status, as well as to geographic and abiotic environmental factors, has been reported.According to Zhang and Elser (2017), Ascomycota fungi have lower C/P and N/P ratios than Basidiomycota fungi, which may explain their higher demand in relation to P. According to Camenzind et al. (2020), the stoichiometric data derived from nutrient gradient tests suggested a differential allocation of N and P in fungal mycelia during growth, indicating that fungi possess certain adaptive abilities when cultured on a substrate with different N and P contents.One of the major nutrients that influence microbial growth is P, and our study revealed that most of the material from white stork nests contained high amounts of P. Certain fungal species, especially saprotrophic ascomycetes (Zhang and Elser 2017) can accumulate P (Dietrich 1976;Ceci et al. 2018).However, zygomycetes (e.g.Mucor spp.) are also noted for their strong ability to mobilise P from different forms (Ye et al. 2015;Ceci et al. 2018;Domka et al. 2018).This strong affinity of Mucor spp. to survive in P-enriched environmental conditions was also confirmed by our results.In addition, unknown ascomycetes from the Aspergillaceae family showed an association with high P content.These results may reflect those from other studies where Aspergillus, Penicillium, and Trichoderma fungi are referred to as phosphate-solubilising fungi (Morales et al. 2011;Gaind 2016;Wang et al. 2018).It appears that fungi especially those with cellulolytic activity effectively uptake a large amount of organic P from the nest material.P is an important component that induces catabolic transformations and cellular synthesis (Griffin 1993).The species richness and diversity of culturable fungi were positively correlated with the level of Ca, K, and Mg in the nest material, with high levels significantly stimulating fungal growth.According to Jones (1965), Ca, K, and Mg cations promoted the growth of different fungi in vitro.High concentrations of these cations additionally reduced the negative impact of  1 and 2, respectively), and fungal species composition (for list of culturable fungi, see Table 5) were used for diagram preparation Na on fungal development Jones (1965).This is especially important given that the material from white stork nests sampled in our study contained relatively high amounts of Na.
The organic matter content in the soil is the primary factor that influences the abundance and diversity of fungi in Technosols (Stępniewska et al. 2020).Conversely, the concentration of trace elements plays a less important role in shaping the fungal variation.The results from the present study are consistent with those reported by Stępniewska et al. (2020).Despite the generally high concentrations of the elements, these were only recorded for Zn and Cu in several tested nests and did not reduce enzymatic activity and fungal diversity.Organic matter in the nest material probably reduced the toxicity of heavy metals in relation to enzymatic activity and fungi.Lasota et al. (2020) suggested that organic matter masks the negative impact of metals on the enzyme activity in the humus soil type.Soil organic matter influences metal binding and their impact on soil microorganisms (Wolińska et al. 2018).Nevertheless, the present study demonstrated that certain fungal species correlated with the concentration of heavy metals in the material of white stork nests.PCA revealed that Mucor spp.strongly correlated with Cd and Zn content, while Co, Cr, Pb, and Fe correlated with Acaulium sp., Alternaria sp., Fusarium sp., or Sarocladium sp.The high tolerance of Mucor sp. to elevated levels of Zn, Cd, and Pb was also recently described (Domka et al. 2018).Our study confirmed that fungi may occur very frequently in different environments with high concentrations of heavy metals (Lenart-Boroń and Wolny-Kładka 2015).The composition of culturable fungal communities detected in white stork nests resembled those found in different soil ecosystems (Frąc et al. 2018).The following fungal species with strong affinities to the soil environment were detected, among others, in the white stork nests: Alternaria, Chaetomium, Fusarium, Penicillium, Sarocladium, and Trichoderma. According to Egidi et al. (2019), these species represent well-known wind-dispersed fungal genera that are globally distributed and soil related.Our study also showed that the fungal community composition of white stork nests is more similar to that of agricultural ecosystems than that of forest ecosystems (Frąc et al. 2018).This composition corresponds to the natural habitats of the white stork, which are closely linked to the agricultural landscape (Butler et al. 2010).

Conclusions
We observed notable differences in the chemical properties of materials deposited in the nests of the white stork, which produced a high variability in enzymatic activity.The results of the present study showed that the chemical properties of the material collected from white stork nests were correlated with the diversity of the culturable fungal community.Our findings revealed that fungi occurred frequently in materials with high levels of C, N, P, and trace elements.Our research confirmed low C/P and N/P ratio values in the studied nest materials, which indicate a high availability of P and, at the same time, favourable conditions for the development of fungi.Finally, we determined that C/N/P ratio was the main driver of fungal diversity in the studied nests.

Fig. 1
Fig. 1 PCA diagram with projection of variables describing properties of material and fungal taxa present in the nests of white stork Ciconia ciconia on a plane of the first and second factors and the position of the samples in the plane formed by first two axes.The

Table 1
Basic chemical properties of the material obtained from the nests of white stork Ciconia ciconia

Table 5
The dominance (Y) values of fungal genera or families obtained from the nests of white stork Ciconia ciconia*

Table 6
Diversity indices for the assemblage of culturable fungi obtained from the nests of white stork Ciconia ciconia