**4. Discussion**

Although only a small percentage (0.1–10%) of microorganisms can be grown on synthetic media in a laboratory, they can be predominant in the analyzed microbial communities [44,45]. The results of the culture-dependent method, as well as the modern high-throughput sequencing approach, indicate that wheat grain is an ecological niche which is colonized by relatively few fungal species with low amount [1]. The genera of filamentous fungi, *Alternaria*, *Cladosporium*, *Epicoccum*, *Botrytis*, and *Fusarium*, as well as yeast genera *Cryptococcus* and *Sporobolomyces* are characteristic of this environment [1,46,47]. In the present study, the co-existence patterns could be condensed into three distinct clusters of OTUs. Clade 1 was composed of fungal communities colonizing grain from non-fertilized plants and grain from plants supplied with the recycled biofertilizer with the addition of *B. megaterium* (Bio) bacteria at 60 and 80 P2O5 ha<sup>−</sup>1, recycled biofertilizer at 80 kg ha−<sup>1</sup> (Rec), and superphosphate (SP) at 40 kg ha<sup>−</sup>1, and was characterized by higher counts of pathogenic species *Monographella nivalis* and *G. tricincta*, as well as species of the genera *Pyrenophora* and *Mycosphaerella*. Clade 3 comprised a fungal community colonizing grain from plants fertilized with the highest superphosphate rate (80 kg ha<sup>−</sup>1), characterized by above-average proportions of pathogenic species of the genus *Fusarium*, unidentified species of the class Sordariomycetes, with the possible presence of the pathogenic genus *Claviceps*, and the saprotrophic species *Stemphylium herbarum*. Clade 2 grouped fungal communities colonizing grain

from treatments with low and moderate fertilizer rates (Bio 40 kg, Rec 40 and 60 kg, and SP 60 kg P2O5 ha<sup>−</sup>1). The fungal communities in clade 2 were characterized by a very high prevalence of *A. infectoria*, while the proportions of the remaining pathogens were low. In a study by Suproniene et al. [ ˙ 48], fungi of the genus *Fusarium* were more prevalent in wheat grain grown in non-fertilized treatments and treatments fertilized with a moderate rate of NPK than in grain from treatments fertilized with a high rate of NPK. According to the literature, nitrogen fertilization exerts a negative effect on the health status of wheat plants and contributes to grain colonization by pathogens. The above can be attributed mainly to changes in stand structure: fertilized stands are dense, and they retain more moisture, which promotes the growth and sporulation of pathogenic fungi [49]. Higher rates of nitrogen fertilizers also prolong flowering and plant maturation, and wheat is most susceptible to infections during flowering [49].

The influence of phosphorus fertilizers on plant health is significantly more complex. In a study by Karimzadeh et al. [50], wheat plants fertilized with phosphorus were characterized by higher root and above-ground biomass, higher chlorophyll and proline concentrations in tissues, as well as higher yields than plants not fertilized with this nutrient. Proline is an amino acid with a secondary amine that functions as an osmolyte during stress and plays a significant role in protecting plants against stress related to the infection process [51]. Phosphorus uptake by plants from soil is also modified by bacteria and soil moisture content [51]. In the work of Arif et al. [52], phosphorus uptake was significantly higher in soybean plants inoculated with *Bacillus cereus* GS6 than in control plants. In the present experiment, recycled phosphorus fertilizers were as effective sources of plant-available phosphorus in soil as superphosphate.

Phosphorus fertilizers probably enhanced plant growth and increased stand density, but they also promoted the production of compounds which increased wheat resistance against pathogens. However, the influence of the tested types of phosphorus fertilizers, including those containing *B. megaterium* that can act as PGPM [26], on the prevalence of pathogens in the field was sometimes ambiguous and modified by other factors. Similar results have never been reported in the literature, and further research is needed to explore these ambiguities.

In this study, wheat grain was mainly colonized by fungi of the genus *Alternaria*. High-throughput sequencing in the Illumina MiSeq system revealed that *Alternaria* fungi accounted for 45–95% of OTUs (subject to treatment). The colony counts of *Alternaria* grown on PDA ranged from 0.35 to 1.48 Log(CFU + 1) per 1 g of grain. *Alternaria* fungi were also isolated from 14.81–31.48% of wheat kernels plated on PDA. Dark colonies growing on PDA and the Martin medium were identified as *A. alternata*, and similar observations were made by other authors [46,47]. *Alternaria alternata* is a ubiquitous saprotroph which infects cereal spikes and causes black scab and black point disease in cereals [53]. The species produces more than 10 allergizing proteins (www.allergen.org). The most frequently described protein Alt a 1 (AAM90320.1. NCBI. Protein Database [54] has been linked with asthma. Alt a 1 is a glycoprotein with a molecular mass of 29 kDa. *Alternaria alternata* also produces around 70 secondary metabolites, including mycotoxins that are potentially dangerous for humans and animals [55].

In traditional analyses of the plant microbiome, microorganisms are isolated and cultured on various media with the use of different methods. However, microbial communities isolated from wheat by culture-dependent methods are characterized by lower diversity than those detected with the use of culture-independent molecular techniques [53]. In the present study, a higher number of pathogenic fungi, in particular pathogens of the genus *Ustilago*, were obtained by next-generation sequencing in the Illumina MiSeq system. *Ustilago tritici* causes loose smut which is widely distributed with grain and can decrease wheat yields by up to 40%. The disease is particularly dangerous for seed farms and undressed grain [56].

In the current study, several pathogenic species that are sporadically carried by wheat grain or are less frequently isolated from grain were obtained with the use of culture-dependent methods. *Rhizoctonia cerealis*, a fungus which causes sharp eyespot, was first identified in Poland in the late

1990s [57]. *Pyrenophora tritici-repentis*, the causal agent of tan spot, was isolated from 21.31% of kernels by Bankina et al. [46]. Next-generation sequencing also supported the identification of the slow-growing pathogen *M. nivalis* which is not detected with the use of culture-dependent methods. Kernels infected with *M. nivalis* and *Fusarium* species are characterized by lower plumpness and pink discoloration. *Fusarium* fungi can cause head blight and stalk rot when distributed with infected grain. *Fusarium* fungi obtained by the culture-dependent method in this study are characteristic of the cooler regions of north-eastern Europe and Canada, and *F. culmorum* was the predominant species [3]. *Fusarium graminearum* is most prevalent in warmer, humid areas of the world such as North America, Europe, and South America [58], and it was also relatively frequently isolated in this study. The growing season of 2016 was characterized by favorable weather conditions for the growth of spring wheat, but high precipitation during grain setting and filling (total precipitation in July was 71% higher than the long-term average) delayed ripening. The above contributed to the spread of infections caused by *Fusarium* fungi.

Fungi colonizing crops can exert both positive and negative effects on the growth of host plants. The former include secreting plant growth hormones and producing compounds that inhibit the development of pathogens and increase plant resistance to infections [59,60]. In the current study, the cultured yeast communities were not significantly influenced by the tested fertilizers. The authors' previous research demonstrated that yeasts inhibit the development of *Fusarium* pathogens [3].

High-throughput sequencing in the Illumina MiSeq system supports more detailed analyses of the structure and diversity of microbial communities than conventional isolation techniques. Fungi respond more rapidly to environmental changes than other living organisms [61,62], and changes in the structure and diversity of microbial communities influence plant health. In this study, the structure and diversity of fungal communities colonizing spring wheat grain were influenced by changes in soil P content caused by the tested fertilizers. However, the observed changes were determined mainly by the P-rate rather than fertilizer type. The highest rate of commercial fertilizer induced the most adverse changes in the balance between pathogenic and non-pathogenic fungi. In a study by Eschen et al. [61], the composition of endophytic fungal communities colonizing the leaves and stems of *Cirsium arvense* varied subject to soil P content. The above authors attributed these changes to differences in fungal species' demand for leaf nutrients which can be affected by the availability of soil nutrients. Pellissier et al. [62] analyzed the composition of fungal communities in grain dust and aerosols released during wheat harvest and did not report significant correlations between total soil P and the taxonomic and phylogenetic beta diversity of fungi.
