3.1. Soil Characteristic
According to the pH range [
38], the soil reaction of all the study soil samples from the depth of 5 cm (assigned as “a”) is medium alkali, and of all the study soil samples from the depth of 50 cm (assigned as “b”) are highly alkali. The amount of organic matter is also very low. The calcium content in the soils is variable and significantly influenced by the parent rock and especially by precipitation (
Table 1). Therefore, it accumulates in areas with limited precipitation, which is evident in samples from a depth of 50 cm (A3b, A2b and A1b). The salinity content of the analyzed soil samples is shown in
Table 2. In the studied soil samples, a total of 59 elements were detected, including metals, lanthanides, alkali metals, alkali earth metals, semi-metals, non-metals and actinides. From this analysis, there is an evident increase in the values of metals in both soil samples from locality A2 (Cu, Pb, Zn, Ag, Ni, Co, Fe, V, La, Cr, Mg, Al, W Zr, Sn, Sc, Y, Nb and Ga). The same situation also occurred in the analysis of lanthanides (Ce, Pr, Nd, Dy, Er). From localities A2 and A3, increased values of Sr were detected. From the soil samples of locality A3, the highest amount of U was detected compared to the other soil samples (Table in
Supplementary Material).
3.2. Cultivable Soil Microscopic Filamentous Fungi
From the analyzed soil samples, 15 genera and 31 species of soil microscopic filamentous fungi were recovered (
Table 3). The species
Rhizopus microsporus (
Figure 4A) and
Saksena vasiformis, from the strain Zygomycota, occurred only in samples A1a, A2a and A2b. All other species belonging to the strain Ascomycota were much more abundant. The most species (seven) were recorded in the genera
Aspergillus, as
A.
flavus,
A.
fumigatus,
A.
pseudoglaucus,
A.
jensenii,
A.
niger (
Figure 4B),
A.
oryzae and
A.
proliferans (
Figure 4C). In the genus
Cladosporium, five species were identified, as
C.
cladosporioides,
C.
floccosum,
C.
herbarum,
C.
iridis (
Figure 4D) and
C.
sphaerospermum. Also, in the genus
Penicillium, five species were identified, as
P.
chrysogenum,
P.
corraligenum (
Figure 4E–H),
P.
echinulatum,
P.
expansum and
P.
rugulosum. Four species were identified in the genus
Alternaria, as
A.
alternata,
A.
atra (
Figure 5A),
A.
japonica and
A.
tenuissima.All of the identified species of the genus
Alternaria were isolated from soil sample A1a (the depth of 5 cm).
Alternaria is one of the most ubiquitous fungal genera, inhabiting nearly every environmental substrate, but most species have been recorded on an extremely wide range of plants. Among the less common species is
Alternaria japonica, occurring on seeds, which causes black spot on turnips, and head rot and leaf spots on Brassicaceae [
39]. Also,
Cladosporium sphaerospermum belongs to the group of melanized fungi isolated from the same sample.
Aspergillus fumigatus and
Rhizopus microsporus also occurred in soil sample A1a. The identification of
Rhizopus microsporus is very interesting, because of its pathogenicity on plants such as maize, sunflower or rice. It has also been isolated from pristine soils from sites in Antarctica. According to Durán et al. [
40], the extreme conditions that coexist in Antarctica produce a strong selective pressure that could lead to the evolution of novel mechanisms for stress tolerance by indigenous microorganisms. The lowest number of species (
Paraengyodontium album,
Penicillium expansum,
P.
chrysogenum and melanized
Stachybotrys chartarum) was isolated from soil sample A1b (the depth of 50 cm). All of these species are known as cosmopolitan, occurring in variable soils [
41,
42]. According to Jaouani et al. [
43], the keratinophilic species
Paraengyodontium album is halotolerant and able to grow in solid and liquid media with a salt concentration from 10% to 15% NaCl. This strain is even able to tolerate 20% NaCl and alkaline tress at pH 10. It was identified from locality A1, together with 11 species of soil microscopic filamentous fungi (
Table 3).
From a mycological point of view, locality A2 is very interesting. From the depth of 5 cm (sample A2a), only six species were isolated (
Chaetomium globosum,
Cladosporium floccosum,
C.
herbarum,
Neomicrosphaeropsis italica (
Figure 5B),
Rhizopus microsporus and
Simplicillium sympodiophorum). All these species occur in soil but all of them are also plant pathogens [
44,
45,
46]. Of all the studied soil samples, the most microfungal species (11) were identified from sample A2b (from the depth of 50 cm). A total of six cosmopolitan aspergili were identified. According to Hubka et al. [
31],
Aspergillus proliferans is an economically important strain, and it seems to be relatively common. It has been isolated from variable soils in Tibet or China, from moldy wood, cave sediment, inside books from a library, from air in a living room and from unknown sources [
47], as well as from onychomycoses [
48]. The species
Auxarthron umbrinum (
Figure 5C,D), in contrast, was until now isolated only from onychomycoses [
49]. The phytopathogenic species
Cladosporium iridis has been isolated from leaf spot and blotch of
Iris sp. from many countries, such as Africa and Cyprus and also from Uzbekistan, Turkmenistan, Kazakhstan, Kyrgyzstan and others [
50]. The species
Myriodontium keratinophilum (
Figure 5E) is widespread in the environment and able to colonize keratinous surfaces of human body [
51]. It has been isolated from soil samples in India [
52] and also from the funeral clothes of Cardinal Peter Pázmany [
53].
Penicillium species are among the most widespread fungal organisms on the Earth, but marine environments and marine subaqueous soils have been poorly studied.
Penicillium coralligerum (
Figure 4E–H) is a marine species sometimes referred to as a deep-sea fungus and in some languages named the equivalent of a “deep-sea mold”, isolated from subaqueous soil in the Sakhalin shelf [
54]. According to the study of Takahashi et al. [
55], among 91 deep-sea fungal strains,
Penicillium coralligerum could produce notable anti-
Saprolegnia parasitica activity.
Saksena vasiformis is a species able to cause severe human infections. It has also been isolated from soils, driftwood or grains [
56].
Locality A3 is closest to the southern part of the Aral Sea, where there is still water. From the soil depth of 5 cm (sample A3a), three aspergili were isolated, from which
Aspergillus jensenii was described as a new species in 2012. This species belongs to the section Versicolores, and according to Jurjevic et al. [
57], variable propagules of
A.
versicolor have been recovered from the highly saline Dead Sea, showing an ability to survive conditions of salinity or drying. High tolerance to salinity may extend to other species in the section Versicolores.
Aspergillus jensenii was also isolated from an old manuscript from Indonesia [
58] and from the soil of potted plants [
59]. All other microscopic fungi isolated from this soil sample belong to ubiquitous species, including in the case of the sample from a depth of 50 cm (A3b). Among the species isolated are the phytopathogenic
Cladosporium cladosporioides,
C.
iridis and
Epicoccum nigrum and the entomopathogenic
Isaria farinosa [
60].
Based on the calculation according to Sörensen and Jaccard [
34]. (
Table 4), the similarity of mycocoenoses is the highest between samples A1a and A1b (0.64). The similarity values of 0.57 (between samples A2b and A3b) and 0.54 (between samples A1a and A3a) are on the border of similarity and difference of mycocoenoses.
The species composition of microscopic filamentous fungi of the arid and slightly halophile environment of the Aral Sea dry bottom is characterized by a big group of melanized species of the genera
Alternaria,
Cladosporium,
Epicoccum and
Stachybotrys, from which most of them are also phytopathogenic. The second group consists of keratinophilic species, such as
Auxarthron umbrinum,
Isaria farinosa,
Myriodontium keratinophilum,
Paraengyodontium album and
Saksena vasiformis, which are widespread in nature but found most abundantly in keratin-rich environments, such as insects, feathers, animal fur, nails and hair. Fungal species belonging to the genera
Alternaria,
Aspergillus,
Cladosporium,
Chaetomium and
Penicillium have been isolated from saline or hypersaline environments by many other authors [
17,
19,
22], and for this reason they have been assigned as halophiles. Halophiles are defined as organisms requiring > 3% NaCl for growth [
24]. Fungal species which can tolerate salt concentrations of 2 to 5%
w/
v are known as slight halophiles [
17]. However, the long-term adaptation of microscopic fungi to salinity requires cellular and metabolic responses that differ from short-term osmotic stress signaling [
18].
3.3. Bacterial Isolates
According to the isolated bacteria, the soil that had the richest bacterial diversity was from sample A1a. From this sample, 11 different species were isolated, mainly members of the genera
Arthrobacter,
Bacillus,
Massilia and
Rhodococcus. Soils from A3a and A3b displayed the poorest bacterial diversity, with four and three species isolated, respectively. Half of these species belong to the
Bacillus genus (
Table 5).
Bacillus species were the only isolates spread in all the samples. Members of this genus have already been isolated from Aral soils, and some of them were able to mineralize [
61] and to protect plants against pathogenic fungi [
62]. Bacteria isolated exclusively from soils at A1a were
Massilia strains; these bacteria have also been isolated in other arid soils from Uzbekistan [
63] and Morocco and show interesting hydrolytic properties [
64].
Other soil-specific isolates were members of the genera
Rhodococcus and
Nocardiopsis, which occurred in the soils at A1 and A2, respectively. The
Rhodococcus species was previously isolated in Aral Sea areas in association with halophytes [
62] and detected by metagenomics analysis in water samples [
65]. However,
Rhodococcus strains are able to adapt to different soil conditions, including high concentrations of salts and pollutants. These characteristics permit them to be versatile bacteria for bioremediation applications [
66]. Several
Nocardiopsis species have also been isolated from halophytes of the west Aral Sea basin, and they display strong proteolytic and cellulolytic activities [
62]. They can be considered as free-living entities occurring in a variety of soils and other saline or hypersaline habitats, and they are producers of diverse bioactive compounds and extracellular enzymes [
67].
Typical strains of the soils from A3 were as follows:
Sphingomonas sp.,
Rathayibacter sp. and
Paenisporosarcina sp. Wicaksono et al. [
68] found that
Sphingomonas were part of the bacterial community in the rhizosphere of the plant
Suaeda acuminata, rather than being present in Aral soil samples, and Osman et al. [
63] frequently found
Sphingomonas in soil samples from the Kyzyl-Kum desert in Uzbekistan.
Rathayibacter sp. and
Paenisporosarcina sp. were not previously identified in Aral-related samples. However,
Rathayibacter species are known mainly as opportunistic phytopathogenic bacteria that frequently occur in arid areas [
69].
The highest similarity of bacteria is between samples A1a and A3b (0.76), A1a and A3b (0.75), A2b and A3b (0.69), A1a and A3b (0.67), A1a and A2a (0.65) and A2a and A3b (0.64). Much less samples show dissimilarity of bacteriocenosis (
Table 6).
3.4. High-Throughput Sequencing Analysis
To see the differences in alpha-diversity between bacterial fungal and archaeal communities, we performed the Mann–Whitney U test, which did not show significant differences among communities. However, the highest Simpson’s index was found for bacterial communities and the lowest for archaea (
Figure 6).
3.4.1. Fungi
The detected fungi belonged mainly to the phylum Ascomycota, namely the classes Saccharomycetes, Eurotiomycetes, Schizosaccharomycetes, Leotiomycetes and Sordariomycetes, while only two classes, Tremellomycetes and Malasseziomycetes, represented Basidiomycota (A3b 0.5%–A3a 5.1%). The PCR of sample A2b did not produce any amplicon; therefore, no data are available.
Nonetheless, 19 fungal genera were identified (
Figure 7), of which
Aspergillus, represented by
Aspergillus oryzae and
Aspergillus fumigatus,
Colletotrichum higginsianum and
Botrytis cinerea, comprised over 50% of all detected taxa (A1a—57%; A1b—73%; A2a—71%; A3b—82%). While sample A3a was uniquely dominated by
Saccharomyces cerevisiae (59%), followed by
Schizosaccharomyces pombe (16%), the latter was also detected in samples A1a (20%) and A1b (9%). Other species were also detected, such as
Neurospora crassa (not found in A1a) and
Malassezia restricta (not found in A3b), which belonged to the widely spread fungi.
Practically speaking, only a few links occurred between the cultivation and sequencing analysis. The correlation regarded only the high presence of Aspergillus species, and some members belonged to the order Sordariales (Neurospora/Chaetomium).
Considering the most abundant genera detected by high-throughput sequencing, new taxa were added to the isolated ones. In fact, the well-known phytopathogens
Colletotrichum and
Botrytis [
70] were revealed. Members of the genera
Saccharomyces and
Schizosaccharomyces are not usually considered as halophilic microorganisms, and perhaps their detection is more associated with their ability to grow in the presence of toxic contaminants [
71].
3.4.2. Bacteria
A total of 30 different bacterial phyla were identified (
Figure 8), of which Pseudomonadota (A1b—49%, A3a—89%, A3b—28%), Bacteroidota (A2a—88%) and Actinomycetota (A2b—53%) were the most abundant, followed by Cyanobacteria (A1a—34%) and Bacillota (2–8%). The highest bacterial diversity at the phylum level was detected in samples A2b (26 phyla) and A3b (23 phyla).
The most abundant bacterial family of the A1a sample, Hymenobacteraceae (8%), was also identified in A2a (11%), while Chitinophagaceae, Rubrobacteraceae, Cytophagaceae and Oscillatoriaceae (from 7% to 5%) were unique. For each sample, a different profile of the most abundant bacterial families was typical: A1b (Marinobacteraceae 45%, Iamiaceae 9%), A2a (Flavobacteriaceae 57%, Hymenobacteraceae 11%, Fulvivirgaceae 9%), A2b (Iamiaceae 23%, Egicoccaceae 17.5%, Borreliaceae 5%), A3a (Comamonadaceae 33%, Burkholderiaceae 27%, Pseudomonadaceae 14%) and A3b (Flavobacteriaceae 17.5%, Egicoccaceae 11.3%, Iamiaceae 7.5%).
Focusing on the genus level, Rubrobacter tropicus, Oscillatoria nigro-viridis, Rhodocytophaga rosea and mainly Hymenobacter oligotrophus (4–6%) dominated from the 139 bacterial genera in sample A1a, while Marinobacter (46%; M. sp. Arc7-DN-1, M. salarius, M. sp. LPB0319), the oversized Actinomarinicola tropica (6.5%) and another 75 bacterial genera dominated in the A1b sample. Antarcticibacterium (32%; A. flavum, A. sp. 1MA-6-2, A. arcticum) dominated over Fulvivirga (11%; F. sp. W9P-11, F. sp. SS9-22) and another 80 genera with an abundance of less than 10% in the A3a sample, while of the 214 bacterial genera observed in the A2b sample, Egicoccus halophilus, Actinomarinicola tropica and Aquihabitans sp. Kera 3 altogether represented over 40% of all bacteria. Sample A3a was very scarce in terms of bacterial diversity, since “only” 47 bacterial genera were detected, mainly representatives of the phylum Pseudomonadota (Acidovorax sp. YS12, Lautropia mirabilis, Rubrivivax gelatinosus, Schlegelella thermodepolymerans and Pseudomonas spp.), which formed almost 70% of all classified sequences. In contrast, the environment represented by the A3b sample was dominated by Egicoccus halophilus and Aquihabitans sp. Kera 3 (14% and 6%; Actinomycetota), Antarcticibacterium sp. 1MA-6-2 (7%; Bacteroidota) and the Candidatus Promineofilum breve (7%, Chloroflexota), followed by another 174 bacterial genera.
Comparing the culture-dependent analysis with the high-throughput sequencing makes it clear that the two approaches are complementary. There is no link between the genera isolated by cultivation and those detected by Illumina sequencing. An example is given by Bacillus species which were isolated from each sample but which were not among the most abundant bacteria according to sequencing. Some small similarity between the two strategies was seen only at the phylum level regarding Pseudomonadota and Actinomycetota.
It is difficult to compare our results with the previous studies related to Aral Sea samples. Other authors oriented their taxonomical analysis at the phylum or maximally at the class level [
65,
72]. Correlations with earlier investigations only regarded the genera
Rubrobacter,
Hymenobacter,
Marinobacter and
Pseudomonas [
63,
68].
However, considering the most abundant detected genera,
Antarcticibacterium,
Egicoccus,
Actinomarinicola and
Aquihabitans, they are typical of marine environments, and they can tolerate a high concentration of salt [
73].
3.4.3. Archaea
Almost all of the analyzed samples were found to be positive for Archaea, mostly belonging to the phylum Euryarchaeota (
Figure 9). While the A1a sample was exclusively inhabited by
Thaumarchaeota, especially with
Candidatus Nitrosocosmicus franklandus (class Nitrososphaeria), the A1b sample was also represented by
Halocatena (11%) and
Haladaptatus (29%), in addition to
Candidatus Nitrosocosmicus (32%). The latter, together with
Halalkalicoccus and
Halorussus, was also found to be the main representative of the A2a and A2b environments. At the species level, A2a and A2b were found to be dominated by
Halalkalicoccus jeotgali (A2a 35.2%; A2b 22%), followed by
Halorussus halophilus (A2a 10%; A2b 6%), while
Natronomonas salina was present both in A2b (8%) and A3b (12%). The highest diversity could be observed in the A3b sample, which was represented by several abundant genera belonging to the order Halobacteriales (
Haladaptatus,
Halobacterium,
Haloarcula,
Halapricum,
Halorhabdus,
Natronomonas and
Halorussus), as well as a representative of Nitrosopumilaceae—
Nitrosopumilus. We were not able to detect any archaea in the A3a sample, because the PCR did not work with this sample.
It is evident that the archaeal community in all the analyzed samples is composed mainly of halophilic genera. Members of the order Halobacteriales are the most widespread; they were previously detected in samples of water [
74] and soil [
68] from the Aral Sea. These archaea in this habitat are involved in denitrification processes [
68].
Archaea of the class Nitrososphaeria have also previously been detected in this kind of environment [
72]. Members of this class harbored genes encoding a methane/ammonia monooxygenase and are involved in denitrification, dissimilatory nitrate reduction to ammonium and ammonia oxidation [
68].