The widespread use of crude oil and petroleum products in the world leads to an increasing contamination of the natural environment (Baran et al., 2004; Albert and Tanee, 2011; Lebrero et al., 2012; Mohsenzadeh et al., 2012; Souza et al., 2014; Nwaichi et al., 2015). One of the underlying reasons is the technical, industrial and economic progress. Each year, around 0.10 to 0.25% of petroleum products pervade the natural environment (Garcia-Lor et al., 2012). According to the International Energy Agency [IEA] (2016), the global demand for crude oil in the first quarter of 2016 was 95.0 mln barrels d^-1. The consumption of biodiesel oil in the world increases dramatically. Hence, the contamination of the natural environment with products originating from crude oil processing raises concern in both industrial and developing countries (Lebrero et al., 2012). Because petroleum products are a mixture of hydrocarbons with low bioavailability, which are often carcinogenic and mutagenic compounds (Souza et al., 2014), they are considered to be among the most toxic and dangerous pollutants in particular compartments of nature, especially in soil, which is the major pool of their accumulation (Sutton et al., 2013; Covino et al., 2016; Marchand et al., 2017). They can cause changes in of fungal biodiversity of soil. This can have an unfavorable impact on the soil health and grown plants (Alrumman et al., 2015).

The presence of petroleum products in the natural environment is a grave problem, because it causes gradual soil degradation in many parts of the world (Albert and Tanee, 2011), and sometimes leads to the permanent destruction of soil, loss of its fertility and disappearance of the plant cover (Wyszkowska et al., 2015). Petroleum substances have a high potential to accumulate in the soil environment, where they can interfere with the soil’s microbiome (Kucharski et al., 2010; Wyszkowska and Wyszkowski, 2010; Lipińska et al., 2013, 2014a,b; Kaczyńska et al., 2015; Nwaichi et al., 2015; Wyszkowska et al., 2015).

Much importance is attached to fungi in bioremediation of soils contaminated with petroleum products, one reason being their adaptability to extreme conditions in habitat (Schadt et al., 2003; Rousk et al., 2010). In general, fungi demonstrate quite a large range of pH in which they can obtain an optimal growth (Rousk et al., 2010). The biomass of fungi is positively correlated with their diversity (Setälä and McLean, 2004). They respond positively to the input of organic matter to soil (Gomez et al., 2006; Islam et al., 2011; Mohsenzadeh et al., 2012). They can be effective in the removal of petroleum products from soil, e.g., some species of the genera Aspergillus (Díaz-Ramírez et al., 2013; El-Hanafy et al., 2017) and Candida (Fan et al., 2014; Silva et al., 2015). High capacity to degrade diesel oil is also ascribed to such fungi as Alternaria alternaria, Aspergillus terreus, Cladosporium sphaerospermum, Eupenicillium hirayamae, Paecilomyces variotii, Trichoderma tomentosum, and Fusarium oxysporum (Ameen et al., 2016; Jiang et al., 2016; Marchand et al., 2017). On the one hand, the fungi present in soil polluted with petroleum products are beneficial for this environment as they contribute to the removal of PAHs from soil. On the other hand, when such fungi appear in fuel tanks, they can add to the deterioration of petroleum products (Bentoa and Gaylardeb, 2001). The ability of fungi to survive in environments contaminated with petroleum products and petroleum fuels alone suggests a reciprocal relationship between these fungi and the products mentioned (Alrumman et al., 2015). Next to bacteria, fungi affect transformations of these products, whereas petroleum products produce some influence on the growth of fungi (Kucharski and Jastrzębska, 2005; Alrumman et al., 2015).

Precise identification of the impact of petroleum products on the natural environment is still difficult. One reason is that our knowledge of the diversity of microorganisms, especially fungi, present in environments contaminated with these products is incomplete and fragmentary (Jiang et al., 2016; Marchand et al., 2017). In such soils, changes may occur in proportions between counts of fast and slow growing microorganisms (Kucharski and Jastrzębska, 2005; Wyszkowska and Kucharski, 2005), and the succession of microorganisms is a necessary condition for effective soil purification from petroleum products. In addition, PAHs contained in petroleum products can be an excellent source of energy for some microorganisms (Sun et al., 2010).

In view of the above, the following study has been undertaken in order to determine the abundance and functional diversity of fungal communities, based on an analysis of the metabolic profile in soil polluted with diesel oil. This problem was scrutinized in the context of the duration impact (from 7 to 270 days) of diesel oil on soil fungal assemblages. Also, the percentage degradation of PAHs by soil microorganisms was determined. The assessment was supported by determinations of the activity of selected enzymes participating in processes of carbon transformation.

One of the most severe threats to the natural environment in central and south-eastern Europe is soil degradation (Günal et al., 2015), in which a large role is played by persistent organic pollutants, including PAHs (Wang et al., 2010; Agnello et al., 2016). Petroleum products which permeate into the soil profile cause changes in the physicochemical properties of soil (Kucharski and Jastrzębska, 2005; Wyszkowska and Kucharski, 2005; Griffiths and Philippot, 2013). In consequence, soil clumping may occur. Thus, petroleum products contribute to the deterioration of physicochemical characteristics of soil (Semrany et al., 2012), which has a negative influence on the growth and development of plants (Sivitskaya and Wyszkowski, 2013; Wyszkowska et al., 2015), but – on the other hand – they are an excellent energy source for certain microorganisms (Kucharski and Jastrzębska, 2005; Wyszkowska and Kucharski, 2005). For these reasons, when describing the quality of soil it does not suffice to determine physical and chemical parameters, even though they are determinants of the growth and development of plants as well as the dynamics of soil metabolism (Aparicio and Costa, 2007; Plaza et al., 2013), but it should be borne in mind that the above paradigm does not account for real-time changes in the soil quality (Lagomarsino et al., 2012; Nannipieri et al., 2012). In our study, the fact that diesel oil could be used by fungi as a source of carbon and energy (Wu et al., 2014; Alrumman et al., 2015; Marchand et al., 2017) probably explained why the number of fungi in DO polluted soil increased and so did the activity of soil oxireductases (Figure 1), which are a sensitive indicator of changes in soil quality in real time (Hawrot-Paw et al., 2010; Mohsenzadeh et al., 2012; Kaczyńska et al., 2015). A change in the activity of dehydrogenases and catalase in soil was reflected by the indicators showing the effect of diesel oil on these enzymes. The determination of these indices is fundamental to an objective assessment of the stability of a soil ecosystem exposed to the pressure of a petroleum product. Our experiment lasted for 270 days, which was long enough to observe a tendency for a lowering pressure of the petroleum product tested on dehydrogenases and catalase. However, it should be added that deposition of diesel oil into the soil was performed only once, at the onset of the experiment.

Some petroleum products stimulate the proliferation of fungi (Fan et al., 2014; Silva et al., 2015; El-Hanafy et al., 2017), and bacteria in soil (Sutton et al., 2013; Jiang et al., 2016). Hence, large changes in the activity of the enzymes were determined in soil polluted with diesel oil. For instance, the activity of dehydrogenases increased by as much as 6.2-fold at day 30 of the experiment, while that of catalase was 1.4-fold higher ar day 7 (Figure 1), The main reason being the fact that microorganisms are the major source of soil enzymes (Ameen et al., 2016; Stręk and Telesiński, 2016; Zaborowska et al., 2016).

On the other hand, not all hydrocarbons contained in diesel oil were an optimal source of carbon for the community of microorganisms. This most probably stems from the fact that two- and three-ringed PAHs have the highest share in the total polycyclic hydrocarbons (Nganje et al., 2007; Wyszkowska et al., 2015). Also, in this study, throughout the whole experiment, that is 270 days, the prevalent share of all PAHs consisted of 2- and 3-ringed compounds, while six-ringed hydrocarbons made up the smallest proportion (Supplementary Table S4). According to Baran et al. (2004), 2- to 4-ringed PAHs are responsible for about 70% of the global PAH contamination. The principal determinants of the diverse composition of PAHs in soil are: the type of pollution caused by petroleum products (Wyszkowski and Ziółkowska, 2013), physicochemical properties of soil (Gałązka et al., 2012) and species of grown plants (Wyszkowski and Ziółkowska, 2013; Nwaichi et al., 2015). In this research, tetravalent hydrocarbons were oxygenated to a higher degree than hexa-, penta-, or di-trivalent ones (Figure 2). After 270 days of degradation, the percentage of degraded compounds was 64% of four-ringed, 28% of five-ringned, 21% of 2–3-ringed and 16% of six-ringed PAHs. Alrumman et al. (2015) point to a significant correlation between the degree of degradation of PAHs in soil and the number of fungi This was one of the reasons why the structure and diversity of fungi changed. In polluted soils, the K strategy fungi decreased in number while the r strategy ones became more numerous. Changes in the structure of fungi observed during this study were a response to the biotic stress induced by the pollution with diesel oil. This conclusion is supported by values of the indices CD and EP. The CD and EP indices provide information on changes in ratios between slowly (K-strategists) and rapidly growing microorganisms (r-strategists). The CD index value ranges from 0 to 100. If it approximates 100, a rapid growth of a microbial population in a short time span is implicated. The EP index ranges from 0 to 1, and reflects the evenness of the growth of microorganisms in a given time period. If the value of this index is 1, the growth of microorganisms in a given environment is very uniform (De Leij et al., 1994). The EP index equal 0 suggests a very low functional diversity as all microorganisms grow at the same time. In our study, communities of copiotrophic fungi, quick at responding to the supply of fresh organic matter, including diesel oil, reproduced more intensively. Copiotrophic fungi are essential for efficient utilization of pollution, including contamination with diesel oil. However, they are less representative to individual types and kinds of soils than K-strategy fungi, which can be called oligotrophic fungi. This explains the difference in the dominant species of fungi between the control and DO-polluted soil. The theory of r-strategists and K-strategists presumes that there are genetic differences between microorganisms, which are responsible for the adaptation of fungi to different environments (De Leij et al., 1994). In DO-contaminated soil, some species of fungi can undergo lysis, and are then replaced by new populations of fungi, which are able to degrade PAHs owing to their intensive metabolism (Semrany et al., 2012; Wyszkowska et al., 2015). In unpolluted soil, the dominant species were Microsporum equinum and Candida lipolytica (Figure 5), whereas the dominant species in DO-contaminated soil was Fusarium solani, followed by much less dominating species, such as Candida lipolytica and Microsporum equinum. The differentiated response of autochtonous soil microorganisms to the applied diesel oil could have been caused by the succession of microorganisms (Vazquez et al., 2013; Wu et al., 2014). In the authors’ own studies, no presence of Penicillium chrysogenum, Penicillium digitatum, or Penicillium discolor was detected in soil exposed to the pressure of diesel oil. In turn, diesel oil was conducive to the development of Fusarium solani (Figure 5).

Tests on plates from the FF MicroPlate^® system by Biolog guarantee obtaining a faithful description of both pure cultures of microorganisms (Mishra and Nautiyal, 2009; Bourdel et al., 2016; Frąc et al., 2016) and their communities in the soil environment (Frąc et al., 2014; Pająk et al., 2016). The functional diversity of fungi isolated from the soil not polluted with DO, determined on FF MicroPlates^®, was higher than that of fungi isolated from the soil polluted with DO, especially at day 270. This finding is supported by the values (Figure 6) of the indices AWCD, substrate Richness (R) oraz Shannon–Weaver (H). There are two reasons behind, one being the depressed ability to use carbon substrates by fungi isolated from polluted soil, and the other consisting of a change in the preference for a carbon source. Soil microorganisms mostly used carboxyl acids and amino acids for the first 90 days and carbohydrates as well as polymers in 270 days (Figure 8). The latter can be connected with the need to adapt to the environment. The observation is in accordance with the report of Frąc et al. (2014), who concluded that carbon sources located in the FF MicroPlate^® provide a wide range of compounds, which can be used to estimate the functional diversity of soil.

Noteworthy is the coincidence between our evaluation of the functional diversity of fungi identified with the Biolog^® system and the assessment of the physiological diversity (EP), based on the culture method carried out on Petri dishes for 10 days. The EP index (Figure 3) thus obtained, similarly to the Shannon–Weaver (H) index (Figure 6C), was the lowest for fungi isolated from the soil polluted with diesel oil for 270 days.

In conclusion, it is worth underlining that any disturbances in the metabolism of soil and fungal biodiversity should be viewed in the context of soil and plants health and possible recovery of the balance in an ecosystem. This conclusion is in agreement with the European Union Biodiversity Strategy to 2020, which imposes an obligation on all EU member states to make assessments of the condition of soil ecosystems (European Commission, 2014). The initiative is further supported by the Sustainable Development Goals.

AB: conceived and designed the experiments; performed the experiments; isolation and identification of hyphal fungi, determination of: functional diversity of fungi (FF MicroPlates^®), dehydrogenases and catalase activity, analyzed the data; wrote the manuscript. JW: methodical consultation, participation in the creation of concept the manuscript, verification of the manuscript editorial. KO: participation in the determination of functional diversity of fungi (FF MicroPlates^®).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.