*3.2. Percentage of Pathogenic and Saprotrophic Fungi Colonizing Grain on PDA*

Dark fungal colonies of the genus *Alternaria* were prevalent on non-disinfected kernels cultured on PDA, and they were identified in 14.81% of grain samples from treatments SP80 and Rec40 to 27.78% of grain samples from treatment Rec80 (Table 6). *Fusarium* fungi were encountered most frequently on kernels from plots fertilized with superphosphate (SP) and control plots (C0). Four *Fusarium*

species—*F. avenaceum*, *F. graminearum*, *F. poae*, and *F. sporotrichioides*—were identified on 14.82% of control kernels. Three *Fusarium* species were also abundant on grain samples from treatments supplied with the commercial phosphorus fertilizer (14.82% in treatment SP40, 12.96% in treatments SP60 and SP80). The second method of isolation from disinfected grain appeared to yield more *Fusarias*.


**Table 6.** Percentage of non-disinfected wheat grain colonized by epiphytic fungi.

No significant differences between treatments (abbreviations are explained in Table 1).

The percentage of disinfected kernels contaminated with fungi of the genus *Alternaria* ranged from 18.52% (SP60, Bio40) to 31.48% (Rec40) (Table 7). *Fusarium* fungi colonized less than 4% of disinfected kernels. The only exception was disinfected grain from treatment Bio40 which was colonized by *F. sporotrichioides* at 5.56%.

**Table 7.** Percentage of disinfected wheat kernels colonized by endophytic fungi.


Values in columns that did not differ significantly in Tukey's test (*p* < 0.01) are marked with identical letters; values not marked with letters do not differ significantly (abbreviations are explained in Table 1).

## *3.3. Structure and Composition of Fungal Communities*

The biodiversity of fungal communities was analyzed by next-generation sequencing in the Illumina MiSeq system. The sequence of the ITS region was compared with the sequences from the UNITE-BLAST database to reveal that fungi of the phylum Ascomycota predominated in all grain samples and accounted for 91.99% (Bio40) to 98.92% of OTUs (Rec40). Fungi of the phylum Basidiomycota accounted for 0.38% (Rec60) to 1.7% (SP60) of sequence reads. Species of the genus *Alternaria*, family Pleosporaceae, order Pleosporares, class Dothideomycetes accounted for 58.06% (SP80) to 95.35% (Rec60) of reading frames in the ITS2 region. A very high percentage of *Alternaria* fungi were classified as *A. infectoria* (43.41–92.79%), whereas only 2.43–16.3% were identified as *A. betae-kenyensis* (Figure 1).

Fungi of the genus *Gibberella*, family Nectriaceae, order Hypocreales were identified in all grain samples (Table 8, Figure 1). They were represented mainly by the pathogenic species *Gibberella tricincta* which was most abundant in grain samples from treatments SP80 (4.5% OTUs), Bio60 (4.5%), and Bio80 (5.48%). Grain samples from treatments C0, SP60, and Bio40 were also colonized by unidentified *Gibberella* species. Unidentified pathogenic species of the genus *Fusarium* (family Nectriaceae) were identified in grain samples from treatments SP40 and Bio40. The pathogenic species *Monographella nivalis* of the order Xylariales, class Sordariomycetes was detected in seven grain samples, excluding samples from treatments SP40, Rec40, Rec60. *Monographella nivalis* accounted for 11% reading frames in control grain (C0). The pathogenic species *P. tritici-repentis* of the family Pleosporaceae, order Pleosporales, class Dothideomycetes was detected in grain from treatments Rec40 (1.73% OTUs), Rec80 (3.33% OTUs), Bio40 (2.41% OTUs), Bio60 (5.89% OTUs), and Bio80 (2.46% OTUs). A metagenomic analysis also demonstrated the presence of biotrophic species of the genus *Ustilago*, family Ustilaginaceae, order Ustilaginales, class Ustilaginomycetes, phylum Basidiomycota (Table 8, Figure 1). These fungi were identified only on grain from treatment Rec80 (0.41% OTUs). Fungi of the genus *Ustilago* cannot be isolated on synthetic media in a laboratory. The saprotrophic species *M. tassiana* of the family Mycosphaerellaceae, order Capnodiales, class Dothideomycetes colonized seven out of the 10 analyzed grain samples, and it accounted for 0.6% (Rec60) to 8% (Bio80) of reading frames. Unidentified species of the genus *Mycosphaerella* represented 1.6% (SP80), 0.6% (Bio40), and 1% (Bio60) of reading frames.

**Figure 1.** *Cont*.

(**b**)

**Figure 1.** Heat map of operational taxonomic units (OTUs) in each experimental unit, classified at the class, order, family, genus, and species level (abbreviations are explained in Table 1). Red corresponds to high amount and green to low amount. Scale: −0.4—>5.6% OTUs, 0.1—5.7–12.6% OTUs, 0.6—12.7–27.6% OTUs, 1.1—27.7–49.8% OTUs, 1.6—49.9–58.0% OTUs, 2.1—58.1–68.2% OTUs, 2.6—68.3–93.6% OTUs, and 3.1—<93.7% OTUs. Dendrogram from hierarchical cluster analysis (Ward method using a dissimilarity matrix of Euclidean distances) on ln-transformed DNA-data of OTU 1 to OTU 10 combined for (**a**) fungi and (**b**) type of phosphorus fertilizers.

**Table 8.** Structure of fungal genera in wheat grain (percentage of OTUs).


\*—abbreviations are explained in Table 1.

The fungal community colonizing wheat grain from the treatment fertilized with superphosphate (clade 3, SP80) differed from the fungal communities identified in the remaining treatments (Figure 1). This difference was attributed to the lower amount of *A. infectoria*, sporadic appearance of *Stemphylium herbarum*, and higher amount of species of the family Nectriaceae. Fungal communities from the remaining treatments were grouped in two clades. Clade 1 was composed of fungal communities from treatments C0, Rec80, SP40, Bio60, and Bio 80, and clade 2 comprised fungal communities from treatments SP60, Rec40, Rec60, and Bio40. Clade 1 was characterized by a high frequency of *A. infectoria* and *M. nivalis* (C0, Rec80), and *G. tricincta* (Bio60, Bio80). The identified pathogens were less abundant in the communities forming clade 2. The method of next-generation sequencing in the Illumina MiSeq system allowed to identify of rare species and biotrophic fungi unable to grow on agar media.

The applied phosphorus fertilization modified the amount of fungal genera, as demonstrated by the PCA biplot (Figure 2). Treatments Rec40, Bio40, and Bio60 were grouped closest to the Tukey median (in the bagplot), and treatments SP60, SP80, Rec60, and Rec80 were located further away (in the bagplot cover region). An analysis of the PCA biplot revealed that the control treatment was separated

by a significant distance from the Tukey median, and it was located in the opposite direction from treatments SP40 and Bio80.

**Figure 2.** Principal component analysis (PCA) biplot of the microbiome in wheat grain based on fungal genera. The dark blue square denotes the Tukey median, the blue square is the bagplot, the light blue square is the bagplot cover. Alt—*Alternaria* spp., Pyr—*Pyrenophora* spp., Myc—*Mycosphaerella* spp., Ant—*Anthracocystis* spp., Lec—*Lecanicillium* spp., Bip—*Bipolaris* spp., Mon—*Monographella* spp., Fus—*Fusarium* spp.; C0, SP40, SP60, SP80, Rec40, Rec60, Rec80, Bio40, Bio60, Bio80—abbreviations are explained in Table 1.
