Pea (Pisum sativum L.) is one of the major legume crops, which are bred mainly for their high content of proteins present in pea seeds [1] and their valuable amino acid composition. Fungal diseases are frequently occurring factors limiting yield and quality of seed used for food and feed. Still, Fusarium pathogens are considered to have minor significance [2,3], while Ascochyta sp. and Alternaria sp. play major roles in this field [4,5,6]. However, Fusarium proliferatum and F. verticillioides should not be overlooked as both species are able to produce efficiently a group of the most dangerous Fusarium mycotoxins—fumonisins [7,8,9,10]. They are a family of polyketide derivatives, structurally related to sphinganine, compounds disrupting sphingolipid metabolism, causing different toxicological effects in humans, animals, as well as plants [7]. The most abundant fumonisin produced in nature is fumonisin B₁ (FB₁), a suspected risk factor for esophageal [11] and liver [12] cancers, neural tube defects [13] and cardiovascular problems [14]. Taking into consideration the available toxicological evidence, the International Agency for Research on Cancer classified FB₁ as probably carcinogenic to humans (class 2B carcinogen) [15].

Numerous studies have confirmed the presence of fumonisins in plant material contaminated with their producers, vast majority was focused on maize [16,17,18,19]. However, rice and sorghum are also often infected with Fusaria belonging to the Gibberella fujikuroi species complex: F. fujikuroi, F. proliferatum, F. verticillioides and F. andiyazi [20,21]. Several reports describing the contamination of crop plants with fumonisin-producing Fusarium species included wheat [22,23], garlic [24,25,26,27], asparagus [26,28,29,30,31], pineapple [26,32] and soybean [33].

Until now, little information was provided on pea plants serving as hosts for fumonisin producers and a potential serious threat to human health posed by the contamination of seeds. On the contrary, F. oxysporum, F. solani, F. avenaceum and F. poae have been considered as major pathogens of this crop [2]. None of these species is capable of synthesizing fumonisins [7,9]. Many recent studies were concentrated on the variability of F. proliferatum and F. verticillioides populations occurring in the environment, especially in the geographical and ecological context [8,34,35,36,37]. The latest findings suggest that mycotoxin biosynthetic genes represent good targets to design molecular tools for evolutionary and phylogenetic research with the essential genes from the trichothecene, fumonisin and enniatin/beauvericin metabolic pathways being exploited particularly frequently [26,28,38,39,40,41,42]. Genes encoding the zearalenone and bikaverin pathways are also gaining more attention [41,43]. The high level of sequence divergence among mycotoxin biosynthetic genes (especially TRI and FUM genes) can be applied to distinguish the populations even on a sub-specific (or even host-specific) level [26], being often more valuable phylogenetic markers for the evaluation of the Fusarium species diversity.

The main aim of this study was to analyze the sequence divergences of the translation elongation factor 1 alpha (tef-1α) and FUM1 genes (encoding the essential enzyme of the fumonisin biosynthetic pathway—polyketide synthase) in two populations of F. proliferatum and F. verticillioides originating from several Polish pea varieties, compared to the collection strains of both species obtained from different host species. Moreover, the abilities of the selected strains to produce fumonisins in vitro were evaluated together with the contamination of the pea seed material with those mycotoxins.

Seeds of twelve cultivars of pea were screened for presence of the pathogenic fungi. Each genotype was grown in four replicates and two distinct localities in Central Poland: Radzików and Wiatrowo. Fungal species were identified morphologically using optical microscope and only samples containing Fusarium species were included in the study. Two cultivars appeared to contain fumonisin-producing Fusaria in more than one replicate and in both localities: EZOP and TURNIA. Furthermore, several other cultivars also contained the species of interest: EUREKA, SOKOLIK, TARCHALSKA and WIATO. F. proliferatum was predominantly occurring on cv. TURNIA and F. verticillioides on cv. EZOP, EUREKA and TURNIA (Table 1). Apart from both studied species, F. poae, F. equiseti, F. acuminatum, F. avenaceum, F. graminearum and F. sporotrichioides were identified occasionally in plant tissues (results not shown).

All isolates were re-identified molecularly on the basis of the translation elongation factor 1alpha (tef-1α) sequence analysis and aligned to the sequences of the collection strains from different host species to evaluate the in-population genetic variability (Figure 1). Additionally, a fragment of a FUM1 gene was partially sequenced using primers developed and validated during previous works [26,40]. Based on the multiple alignment of the sequences obtained, a dendrogram was calculated using the Maximum Parsimony approach (Figure 2). All strains under study fell firmly into the clades of F. proliferatum and F. verticillioides, discriminated on the basis of the collection strains sequences (Table 1). Moreover, a certain level of sub-specific polymorphism has been observed among the strains of F. proliferatum (Figure 1, Figure 2).

Having uncovered that pea seed samples quite frequently carried the dangerous Fusarium pathogens, a survey for fumonisin quantification has been performed for the respective seed samples of two cultivars EUREKA and TURNIA (four replicates from two localities each). Cultivars were chosen on the basis of fungal species present (Table 1). All of the samples tested contained low amounts of FBs (Table 2). However, no significant differences have been observed in FB levels between seeds containing F. proliferatum and F. verticillioides as the prevailing pathogens. A maximum amount of FBs detected in TURNIA IV from Radzików was 1.72 μg g⁻¹, and the lowest (for TURNIA I from Radzików) was 0.63 μg g⁻¹. FB₁ was dominating markedly, representing more than 90% of the total amount at all times (Table 2).

In order to evaluate the efficacy of the fumonisin B₁–B₃ biosynthesis by F. proliferatum and F. verticillioides isolates, the in vitro cultures on sterile rice grain were prepared [40]. Eighteen genotypes originating from TURNIA, EUREKA and SOKOLIK cultivars were analyzed. The amounts of FBs were quantified using a standardized high-performance liquid chromatography (HPLC) method (Section 4.5). Additionally, several collection strains of F. proliferatum and F. verticillioides originating from various host species [26] have been included to show the intraspecific variability of this trait (Table 3).

Seeds of only six out of twelve pea cultivars screened appeared to contain fumonisin-producing Fusarium species and in the seeds of EZOP and TURNIA the pathogens occurred frequently. F. proliferatum was predominantly occurring on cv. TURNIA and F. verticillioides on cv. EZOP (Table 1). Other Fusarium species were isolated only occasionally. Species identification was performed on the basis of the translation elongation factor 1alpha (tef-1α) sequence analysis. This gene is widely used in phylogenetic studies of fungi, successfully resolving most of the closely related Fusarium species [17,44,45]. However, in populations of some less polymorphic species, where the genotypes studied display a low level of genetic diversity, different genomic regions should be used to increase the polymorphism revealed [38,46].

Polyketide synthase is the essential enzyme of the fumonisin biosynthetic pathway is encoded by FUM1 gene [47]. Using primers developed and validated during the previous works [26,40], FUM1 gene fragments were sequenced and comparatively analyzed using all the strains included in the study. Finally, a dendrogram was calculated using the Maximum Parsimony approach to show the divergences among the strains originating from different host species. All strains formed two separate and well-supported clades of F. proliferatum and F. verticillioides (Figure 2). Furthermore, a certain level of sub-specific polymorphism has been observed among the strains in the case of F. proliferatum genotypes (Figure 2). In the case of FUM genes this observation was already reported [26,40]; however, the analysis of the pea-derived strains is presented here for the first time. It seems that the pea-derived strains of F. proliferatum are highly uniform and show the highest similarity level to some genotypes originating from pineapple and date palm (Figure 2). On the contrary, F. verticillioides strains have shown virtually no difference among the populations from different hosts. It could implicate that F. proliferatum displays a higher evolutionary potential. In fact, this hypothesis seems to be fairly supported by the results of analyses performed during this and the previous studies [8,26,36,40].

Furthermore, the sequences of the biosynthetic genes from other mycotoxin pathways have been utilized in phylogenetic studies of Fusarium species [38,41,42], showing considerably higher polymorphism than the commonly used conserved genes from the primary metabolic pathways. Thus, markers for secondary metabolite biosynthetic genes can be sensitive tools for the prediction of the mycotoxin presence in plant samples. Here, pea seeds of the cultivars containing F. verticillioides have been analyzed. All samples tested contained low amounts of FBs (Table 1), though the levels of FBs were similar in the samples of seeds containing F. proliferatum and F. verticillioides, as the prevailing pathogens. FB₁ dominated markedly, representing more than 90% of the total amount at all times (Table 2). Moreover, there was no correlation between the frequency of the pathogen detection in particular pea cultivar samples coming from different locations and the observed fumonisin content. The efficacies of the fumonisin B₁–B₃ biosynthesis by eighteen genotypes of F. proliferatum and F. verticillioides strains originating from TURNIA, EUREKA and SOKOLIK cultivars, were evaluated on the basis of the in vitro cultures on sterile rice grain [40]. Concentrations of fumonisins produced on this substrate were lower than these observed on maize kernels [16,36], but still exceeded 1.5 mg g⁻¹ for some strains (Table 3). This difference may be related to the starch content of maize grain. Another possible reason is the crucial role of fumonisins during maize infestation, which has not been proven for other host-pathogen systems yet [48]. Generally, in rice cultures F. proliferatum genotypes produced higher amounts of FBs than F. verticillioides strains, however, the most efficient strain was F. verticillioides strain KF3661 from cultivar EUREKA (Table 3). Remarkably, the ratios between FB₁, FB₂ and FB₃ have shown higher variance than in the case of pea seed analyses. Some F. proliferatum genotypes accumulated FB₂ in amounts measuring as much as 1/3 of FB₁ level (e.g., KF 3357 and KF 3654). Conversely, few F. verticillioides genotypes (KF 3780 and KF 3781) synthesized virtually no FB₂ with simultaneous higher amounts of FB₃. Similar results were obtained for F. verticillioides strains originating from maize, though, FB₂ and FB₃ were almost absent there (e.g., KF 3644, KF 3488). In fact, for those incidences also FB₁ was produced in very low amounts. Moreover, for several medium-producing strains (e.g., F. proliferatum KF 3731 and KF 3737) the levels of FB₂ and FB₃ were similar and reached almost a half of the FB₁ amounts (Table 3).

It can be concluded that the pea-originating F. proliferatum and F. verticillioides isolates produced less fumonisins than the genotypes originating from different host species, like maize, garlic or asparagus [8,26,38]. Also, some pineapple-derived F. proliferatum strains were found to be very efficient FB-producers [33]. Comparing the genetic diversity of the two species, F. verticillioides appears as more uniform, but still, the strains differed remarkably in FBs synthesis. Taking into account the divergence of the FUM1 gene in relation to the variance observed in the amounts of FBs produced in vitro, it is the differential regulation pattern governing this variance, rather than the structural divergences of the essential fumonisin biosynthetic genes. This hypothesis, however, needs to be confirmed by conducting additional experiments, e.g., by analyzing the transcription levels of the essential FUM genes.