In May and June 2023, a field survey was carried out in three hazelnut orchards located in the Viterbo area (Central Italy), to investigate the presence of early NGN symptoms. Twenty five symptomatic hazelnut samples, collected from cultivar ‘Nocchione’ and ‘Tonda Romana’, were sealed in sterile plastic bags and brought to the Plant Pathology Laboratory of the Tuscia University in Viterbo. Briefly, hazelnut inner kernel sections were surface-sterilized with 3% sodium hypochlorite for 3 min, rinsed twice with sterile distilled water, and dried under laminar flow. One-centimeter sections from both healthy and symptomatic tissues were aseptically placed onto potato dextrose agar (PDA, Dinkelberg analytics, Gablingen, Germany) plates and incubated at 25 ± 1 °C for 5–7 days. The pure fungal cultures were obtained through serial transfer of emerging colonies onto fresh PDA plates and further used for morphological and molecular analyses. For morphological characterization, single hyphal tips were transferred to plates containing the standard PDA and oatmeal agar (OA, Condalab, Madrid, Spain), which were incubated at 25 °C in the dark. After 10 days, the morphology of the colony was observed. Morphology of the conidia was examined by growing the isolates on synthetic nutrient agar (SNA), prepared according to Nirenberg (1976). Plates were incubated at 25 °C in the dark at 100% relative humidity. Observations on the conidial morphology and size were carried out at 100x magnification through a microscope (Leitz).

In addition to those available from our previous study (Zimowska et al., 2024), four more endophytic isolates conforming to the FCCSC were collected from secondary branches of both hazelnut and small-leaved linden (Tilia cordata) in the Kraków area (Southwest Poland), following the procedure described in the mentioned reference. The details concerning the isolation sources and the GenBank accession numbers of the DNA barcode sequences used in this study are indicated in Table 1.

Genomic DNA (gDNA) was extracted from fresh, filtered mycelium obtained from pure colonies grown in 50 mL tubes containing potato dextrose broth (PDB) using the Plant Genomic DNA Extraction Mini Kit (Fisher Molecular Biology, Rome, Italy). Molecular characterization was carried out following published protocols (O'Donnell et al., 1998; Liu et al., 1999; Hofstetter et al., 2007), through amplification of the translation elongation factor 1-alpha (tef-1) and the second largest subunit of RNA polymerase II (rpb2) gene regions, using the primers listed in Table 2. For each PCR reaction, 5 ng of gDNA was used as a template in a final volume of 25 μL, containing 2 × PCRBIO HS Taq DNA Polymerase (PCR Biosystems, UK) and 0.5 μM of both forward and reverse primers. The thermal cycling program for tef-1, using primers EF-1 and EF-2, consisted of an initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s, with a final extension at 72 °C for 5 min. For rpb2 amplification, using primers fRPB2-5f and fRPB2-7cR, the program consisted of an initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 60 s, annealing at 57 °C for 75 s, and extension at 72 °C for 60 s, with a final extension at 72 °C for 10 min. Two μL of the amplified products were analyzed by 1.2% agarose gel electrophoresis, while the remaining products were sent to Macrogen Europe (Milan, Italy) for Sanger sequencing.

All the collected Fusarium isolates were submitted to a detailed phylogenetic analysis, along with 60 strains belonging to the FCCSC and the FTSC whose sequences are available in the GenBank database (Supplementary Table 1); F. lateritium NRRL 13622 was included as an outgroup. The original sequences were edited using UGENE v48.1 (Okonechnikov et al., 2012), concatenated, and aligned with MUSCLE v3.8.31 (Edgar, 2004). The resulting alignment file was used as input to construct a maximum likelihood (ML) phylogenetic tree with RAxML-HPC v8.2.12 (Stamatakis, 2014), employing the GTRGAMMAI substitution model and 1,000 bootstrap replicates. The tree was visualized with FigTree v1.4.4¹ and further edited with Inkscape v0.92.²

Species delimitation analysis was performed employing the distance-based Automatic Barcode Gap Discovery (ABGD), the Generalized Mixed Yule-Coalescent (GMYC) model, and the Poisson Tree Processes method with multi-rate (mPTP) implementations, using RAxML output tree as input. In particular, the ABGD algorithm splits sequences based on break points in pairwise genetic distances (“barcode gaps,” Puillandre et al., 2012). The mPTP algorithm (Kapli et al., 2017) inspects a substitution-based, non-ultrametric phylogenetic tree to detect changes in branching rates. These rates, modeled as a Poisson process, occur randomly at a relatively constant rate within species and more slowly between species, allowing the algorithm to delineate species boundaries. The GMYC algorithm uses a time-calibrated (ultrametric) tree to find the point where the branching pattern shifts from splits between species to merge among individuals of the same species; thus, it is really sensitive to recent divergence when they tend to over-split. For the ABGD analysis, the parameters were set to test variability (P) between 0.001 (Pmin) and 0.1 (Pmax), standard for fungal ITS or protein-coding markers (Puillandre et al., 2012), with a minimum gap width of 0.1, employing the Kimura 2-parameter model and 50 screening steps. For the mPTP approach, 50 million generations were employed with MCM or ML algorithm, with sampling every 1,000 generations, using the minimum branch length calculated from each tree. For the GMYC analysis, an ultrametric tree was constructed on BEAST2 v2.7.7 (Bouckaert et al., 2014), setting the gamma site model with GTR + G + I as substitution, the Yule prior with a relaxed molecular clock, and Markov Chain Monte Carlo (MCMC) run for 50 million generations, sampled every 1,000 generations. Convergence and effective sample size (ESS) higher than 200 was checked using Tracer v.1.7.2 (Rambaut et al., 2018) and the maximum credibility clade tree was obtained with Treeannotator v2.7.7 (Bouckaert et al., 2014) applying the mean heights parameter and discarding the first 10% of the trees as burn-in period. The resulting tree was then imported into the R environment and the GMYC analysis was performed using the splits package using the single threshold approach (Fujisawa and Barraclough, 2013).

The hazelnut samples collected in orchards located in the Viterbo area from NGN symptomatic plants showed the presence of orange to light brown sporodochia on the fruit surface, indicating active fungal growth; these fruiting structures contained numerous hyaline, multiseptate, crescent-shaped macroconidia (Figure 1). The isolations done from the inner kernel tissues consistently yielded Fusarium from all the samples examined; a total of 17 isolates were recovered and stored in pure culture for the subsequent assessments. Four more Fusarium isolates exhibiting similar morphological features were recovered in the Kraków area, respectively two from hazelnut and two from linden tree, within the cooperative work in progress concerning endophytic associates of forest trees (Nicoletti and Zimowska, 2023). Notably, no symptoms referable to NGN were observed in the course of inspections carried out in Southern Poland in summer 2025.

When grown in axenic culture on PDA, the morphological characteristics of both pathogenic and endophytic isolates were comparable and consistent with the description previously reported for Polish isolates (Zimowska et al., 2024). On OA, radial growth was more pronounced; however, sporulation was generally reduced, and only minor differences in colony color and morphology were observed (Figure 2). On SNA, abundant macroconidia were produced from monophialidic conidiogenous cells. These conidia were hyaline, multicellular, typically with three to five septa, slightly curved and tapering toward both ends. The apical cells were more distinctly curved, whereas the basal cells exhibited a characteristic foot-shaped morphology (Figure 3). Throughout the 14-day incubation period, no pigmentation of the medium was detected, and microconidia were not produced. Chlamydospores were absent in the Italian isolates, whereas they were consistently observed in all the Polish isolates examined (Figure 4). The relevant morphological features as assessed in comparison with the descriptions of the reference FCCSC species, and reported for homogeneous groups of isolates, are resumed in Table 3.

The phylogenetic analysis based on the selected DNA markers (Figure 5) confirmed that all strains analyzed belong to the Fusarium citricola species complex (FCCSC), although they exhibit a certain degree of intra-clade variation. The 17 strains collected from the Viterbo area are positioned in the lower portion of the dendrogram and form four main clusters. The first cluster consists exclusively of isolate FUS 25; the second includes the type strain of F. aconidiale together with isolate PT, previously assigned to the FTSC (Turco et al., 2021); the third group includes F. celtidicola and the endophytic isolates from the Lublin area; and the fourth occupies an intermediate position, encompassing F. juglandicola and the isolates from the Kraków area. As a whole, the FCCSC and its related isolates are clearly separated from the rest of the strains, reinforcing their distinction from the FTSC.

This separation is consistently supported by all the three independent species-delimitation algorithms (Figure 5). In fact, both mPTP and ABGD assigned all the 21 isolates to a single species-level unit, whereas GMYC identified three distinct entities. The first GMYC group comprised a single NGN isolate (FUS 25), which forms an independent lineage congruent with its unique placement in the phylogenetic tree. The second group included eight Italian NGN isolates, all the Polish isolates, and the strains identified as F. celtidicola and F. juglandicola. The third cluster encompassed the remaining NGN isolates, including isolate PT, isolate IHEM 28077, and the type strain of F. aconidiale. All the three delimitation methods consistently recognized F. citricola and F. salinense as distinct species, but the placement of isolate ZLVG982 was discordant. In the ABGD analysis this isolate grouped with F. salinense, whereas in both mPTP and GMYC it formed an independent lineage, supporting our earlier inference that it may represent a separate, as-yet undescribed species (Zimowska et al., 2024).

Within the FTSC, the three algorithms are generally concordant, consistently recovering well-supported clades corresponding to F. acuminatum, F. tricinctum, F. torulosum, F. reticulatum, F. avenaceum, and F. gamsii. Minor discrepancies concern the discrimination among F. iranicum, F. flocciferum and FTSC 24, which were grouped as a single taxon by all three delimitation methods, as well as isolate FTSC 21, which clustered together with F. avenaceum into a single operational unit in the GMYC analysis. Also noteworthy was the case of FTSC 25, represented by a single strain, which was placed in an intermediate position at a seemingly equal phylogenetic distance from both the FCCSC and the other FTSC lineages. This finding, consistent with our previous phylogenetic results (Becchimanzi et al., 2025), deserves further examination if additional isolates belonging to this provisional taxon become available.

Pairwise Kimura 2-parameter (K2P) distance matrices derived from the ABGD analysis confirmed a clear discontinuity between the FCCSC and FTSC species complexes (Supplementary Figure 1). Inter-complex distances ranged from 0.0614 to 0.0955, with a median of 0.0767, delineating a clear barcode gap that separates the two complexes (Supplementary Table 2). By contrast, intra-complex distances were much smaller: within the FTSC, distances were higher (Q3 = 0.0406, i.e., the 75th percentile of the dataset, meaning that 75% of all pairwise distances are smaller than this value and 25% are larger; max = 0.0560), reflecting greater internal structuring associated to several distinct, recognized species-level entities. Within the FCCSC, values remained very low, with a median of 0.0036, Q3 of 0.0229 and max distance of 0.0279 (Supplementary Figure 2; Supplementary Table 2). These results confirm a strong genetic separation between the two complexes, with no overlap between their intra- and inter-group distance distributions. This absence of overlap represents a clear “barcode gap”, quantitatively reinforcing the distinct evolutionary identity of the FCCSC and its separation from the FTSC.

When going deeper into the FCCSC, the K2P distance matrix revealed a cohesive but internally structured lineage composed of four genetic groups: F. citricola, F. salinense, Fusarium sp. ZLVG982, and a larger assemblage including F. aconidiale, F. celtidicola, F. juglandicola, and the Italian and Polish isolates (referred to as “the FCCSC comprehensive taxon, or FCCSC-CT”), in line with our previous phylogenetic consideration. Pairwise distances within these groups were uniformly low, with no variation observed in F. citricola (all distances = 0, indicating that these five isolates are genetically identical for the loci analyzed), and slight variability observed in F. salinense (median = 0.0048; max = 0.0048) and in FCCSC-CT isolates (median = 0.0024; Q3 = 0.0036, max = 0.0060). Between-group comparison within the FCCSC also yielded very low divergence, with median values ranging from 0.016 to 0.023 K2P (Supplementary Table 2). The smallest inter-group distance was recorded between ZLVG982 and F. salinense (median = 0.0144), whereas the largest occurred between F. citricola and F. salinense (median = 0.0230). The FCCSC-CT consistently showed low differentiation from all the other FCCSC subgroups (median = 0.0168-0.0229). Their internal distances (median = 0.0024) fall well within the expected intraspecific range, while their inter-group divergences (0.017-0.023) suggest incipient speciation rather than established separation. Collectively, these data indicate that the FCCSC-CT constitutes a single, genetically cohesive lineage distinct from, but closely related to, F. citricola, F. salinense and ZLVG982; together, they form a compact species complex characterized by shallow internal divergence and strong inter-complex separation. Regarding FTSC 25, which is intermediate between FTSC and FCCSC, the distance indicates that this provisional taxon is genetically closer to the former complex. Specifically, the distances from the FTSC were: min distance = 0.0333, max distance = 0.0560, and median = 0.0430; while higher values could be determined toward the FCCSC: min distance = 0.0674, max distance = 0.0739, and median = 0.0700.