Introduction

Oilseed rape (Brassica napus L.) is one of the world’s most important oilseed crops. Stem canker also known as ‘blackleg’ of Brassica crops is the most damaging disease causing yield loss worldwide (Fitt et al. 2008). Stem canker is caused by two closely related fungal species sharing a similar life pattern: Plenodomus lingam (Tode) Desm. and Plenodomus biglobosus (Shoemaker & H. Brun) Gruyter, Aveskamp & Verkley (Dilmaghani et al. 2009). Their virulence differs significantly, with the greater yield loss being attributed to P. lingam (Williams and Fitt 1999). In the early 2000s, the species were distinguished by morphological differences of pseudothecia and classified in the L. genus (Shoemaker and Brun 2001), but later studies suggested that they rather belong to the P. genus (de Gruyter et al. 2012; Wijayawardene et al. 2014).

In Hungary, only P. lingam was previously reported as the causal agent of blackleg, but the presence of P. biglobosus was also confirmed a few years ago (Bagi et al. 2020). Despite some divergence, the pathogens cannot always be distinguished by morphological characteristics (Williams and Fitt 1999). DNA-based identification is required for reliable identification (Rouxel et al. 2004) and subclade differentiation. Plenodomus lingam can be divided into two subclades, whereas P. biglobosus includes seven distinct subclades (Mendes-Pereira et al. 2003; Vincenot et al. 2008; Zou et al. 2019). The subclade classification is based on geographical distribution, natural host range and phylogenetic analysis (Zou et al. 2019). The Plenodomus isolates previously reported from oilseed rape belong to P. lingam ‘brassicae’ (Mendes-Pereira et al. 2003), P. biglobosus ‘brassicae’ (Liu et al. 2014), ‘canadensis’ (Van de Wouw et al. 2008; Dilmaghani et al. 2009), ‘australensis’ (Plummer et al. 1994; Voigt et al. 2005) and ‘occiaustralensis’ (Vincenot et al. 2008; Dilmaghani et al. 2009) subclades. As pointed out by King and West (2022), the distribution patterns of P. biglobosus subclades need to be mapped because P. biglobosus is becoming an increasingly important pathogen of oilseed rape. The rDNA sequences, partial small subunit nrDNA (18S, SSU) and partial large subunit nrDNA (28S, LSU) are highly conserved and can discriminate at the levels of orders and kingdoms (Balesdent et al. 1998; de Gruyter et al. 2009). Molecular analysis of the ITS regions and the 5.8S rRNA gene has been used for many years to estimate diversity and classify Ascomycota fungi to species level (White et al. 1990; Capote et al. 2012). In the subclade related studies, ITS rDNA has been used for the phylogenetic analysis and reliable subclade identification of both species (Zou et al. 2019). Fragments of β-tubulin 2 gene sequences were also involved in the cluster analysis (Mendes-Pereira et al. 2003), but these sequences have not been published in international databases (NCBI, ENA). In order to distinguish closely related Plenodomus species, the RNA polymerase II second largest subunit (rpb2) region, which is more informative and variable than the ITS region, was also analyzed (Chen et al. 2015). This region can indicate the differences at species level (Drehmel et al. 2008).

We aimed to monitor and determine the distribution of the P. lingam and the newly reported P. biglobosus in Hungary, in order to estimate the arising importance of the fungi in Central Europe. To elucidate the genetic diversity within the Plenodomus population infecting oilseed rape in Hungary, our goal was to observe the phylogenetic relationship among the Plenodomus isolates by multi-locus sequence analysis of ITS1-5.8S-ITS2 region, partial sequences of the 28S nrDNA (LSU), the β-tubulin 2 gene (tub2) and the RNA polymerase second largest subunit (rpb2 region) and provide available sequence data in the NCBI.

Materials and methods

Collection of Plenodomus isolates

Leaf and stem tissues of oilseed rape with blackleg symptoms were collected for the survey in major producing counties across Hungary in commercial fields in 2017–2021. The fungus was induced to sporulate by incubating unsterilized, diseased tissues in humidity chambers during 2–3 days to allow cirri exudation from pycnidia. After formation of pycnidiospores, the cirrus was transferred with a sterile glass needle (Goh 1999) and suspended in sterile distilled water. Suspensions of conidia were transferred to PDA (potato dextrose agar, BioLab Zrt., Hungary) plates and incubated at 24 ± 1 °C temperature for 3–4 days. The cultures were purified by single-spore colony isolation. Growing hyphal tips from germinating conidia were transferred onto fresh PDA plates with the aid of a sterile dissection needle. Identification was initially based on morphological criteria, and cultures identified as Plenodomus spp. (Mitrovic et al. 2016) were grown on PDA plates and maintained at 4 °C.

Identification and selection of isolates for analysis

In five years, 308 Hungarian Plenodomus isolates (data not shown) were obtained and identified to species level by multiplex PCR with specific primers: LmacR, LmacF and LbigF (Liu et al. 2006). The rapid and simultaneous detection of species is performed by the length of specific bands of the target sequence: 331 bp for P. lingam isolates, and 444 bp for P. biglobosus isolates.

For the detailed phylogenetic analysis and comparison, 26 P. lingam and 17 P. biglobosus isolates were selected from different locations (Table 1). We aimed to include at least one P. lingam and one P. biglobosus isolate from each studied county. For two counties, this was not possible because we could not identify P. biglobosus at all. From these counties, two P. lingam isolates were chosen for the analysis.

Table 1 Plenodomus isolates used in this study with the accession numbers of the gene sequences
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DNA isolation, amplification and sequencing

Hyphae were collected from a PDA plate of each isolate. Then, genomic DNA was extracted using the cetyl-trimethyl-ammonium-bromide (CTAB) method (Maniatis et al. 1983), followed by chloroform/isoamyl alcohol (24:1, v/v) extraction and isopropanol precipitation. The concentration and the purity of the DNA were evaluated by NanoDrop™ Spectrophotometer.

The PCR-based assay was conducted by amplifying the 18S-28S rRNA region (a partial sequence of the 18S ribosomal gene, the ITS1 region, the 5.8S ribosomal gene, the ITS2 region and a partial sequence of the 28S ribosomal gene), other section of 28S nrDNA (LSU), part of the tub2 gene and partial rpb2 region. Amplifications of fragments were carried out in a total reaction volume of 50 μL containing 15 ng of genomic DNA, 0.2–0.2 µM forward and reverse primers, DreamTaq Green PCR Master Mix (2X) (Thermo Scientific™).

To amplify the 18S-28S region, PN3 and PN10 primers and PCR conditions were performed according Mitrovic et al. 2016. The length of the target sequence without primers was 507 bp for P. lingam and 535 bp at P. biglobosus. The LSU region was amplified with the primers LR0R and LR7 (Rehner and Samuels 1994) according to the protocol of Chen et al. 2015. The target sequence length without the primers was 1328 bp for P. lingam and 1327 bp for P. biglobosus. The part of the tub2 gene was amplified with the primer pair Btub2Fd and Btub4Rd (Woudenberg et al. 2009) by amplification conditions of Chen et al. 2015. The target sequence length without primers was 345 bp for P. lingam and 337 bp for P. biglobosus. To amplify partial region of rpb2, RPB2F (5′-AGGCTTGTGGTTTGGTCAAGA-3′) and the RPB2R (5′-ATCATAGCRGTCTCTTCCTCCT-3′), we designed primers based on sequences from the P. lingam rpb2 region (Accession Nos. DQ470894; KT389669; KY064047; XM_003841144) and P. biglobosus rpb2 region (Accession Nos. KY064037; MT683512). For rpb2 region, thermal cycling conditions consisted of denaturation at 95 °C for 3 min, followed by 35 cycles of the following steps: denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s and extension at 72 °C for 45 s, with a final extension step at 72 °C for 10 min. The target sequence length for both species was 492 bp without primers.

Amplification products were analyzed first by gel electrophoresis on 1% agarose gel (Sigma) stained with ECO Safe Nucleic Acid Staining Solution (Biocenter), then visualized under UV light. Amplicons were purified by High Pure PCR Product Purification Kit (Roche) according to the manufacturer’s protocol. Fragments of the 18S-28S were sequenced by PN10 primer, fragment of the LSU, fragment of the tub2 gene, fragment of the rpb2 region were sequenced in both directions using the primers for PCR, in an ABI Prism automatic sequencer (BaseClear B.V.). Sequences were manually checked, edited and compared to reference sequences deposited in the NCBI BLAST database (Altschul et al. 1999).

Phylogenetic analyses of the P. lingam and P. biglobosus isolates

Further phylogenetic analyses were carried out with the MEGA11 program package (Tamura et al. 2021). The trees were obtained by applying neighbor-joining algorithms to matrixes of pairwise distances estimated using the maximum composite likelihood (MCL) approach (Tamura et al. 2004). The tree is drawn to scale, with branch length measured by the number of substitutions per site. The clade stability was assessed with 1000 replicates of bootstrap values, and Leptosphaeria doliolum (CBS 505.75) was designated as the outgroup in all rooted trees.

Sequences of the ITS regions including the 5.8S gene of rDNA used for subclade identification (Mendes-Pereira et al. 2003) were compared to P. lingam strain CBS 275.63, P. biglobosus ‘brassicae’ UBIP01000001 genome, ITS1-5.8S-ITS2 sequences of the P. lingam subclades ‘brassicae’ (AJ550885; AJ550887), ‘lepidii’ (AJ550890) and the P. biglobosus subclades ‘thlaspi’ (AJ550891), ‘australensis’ (AJ550869; AJ550870), ‘erysimii’ (AJ550872), ‘canadensis’ (AJ550868; FJ172238; AJ550867), ‘occiaustralensis’ (AM410082), ‘americensis’ (MG321243) and ‘brassicae’ (DQ133890). For the multi-locus analysis (ITS1-5.8S-ITS2, partial LSU, partial tub2 gene, partial rpb2 region), the whole-genome isolates (P. lingam ‘brassicae’ CBS 275.63 and P. biglobosus ‘brassicae’ UBIP01000001) were included, because only these two isolates had published sequences from all these regions.

Results

Across all sites and surveillance years, 308 Hungarian Plenodomus isolates were identified: 158 isolates were P. lingam (51.3%), while P. biglobosus was detected in case of 150 isolates (48.7%) (Table 2). The newly reported Plenodomus biglobosus was identified from six new counties, so it can be concluded that the pathogen is widespread and common in Hungary.

Table 2 Number of locations with the isolate number from 2017 to 2021
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Phylogenetic analysis

For phylogenetic analyses, ITS1-5.8S-ITS2 sequences (468 bp for P. lingam and 496 bp for P. biglobosus), partial LSU sequences (877–881 bp for P. lingam and 874–881 bp for P. biglobosus), partial tub2 gene sequences (343–345 bp for P. lingam and 336–337 bp for P. biglobosus) and partial rpb2 region sequences (492–493 bp for P. lingam and 492–493 bp for P. biglobosus) of the examined 43 isolates were generated and deposited in GenBank.

Phylogenetic tree based on ITS1-5.8S-ITS2 region

The ITS1-5.8S-ITS2 sequences of Hungarian isolates were 100% identical in all 26 P. lingam isolates with the reference isolate from UK (JF740234 from CBS275.63 complete genome). Similarly, the ITS1-5.8S-ITS2 sequences of the 17 P. biglobosus isolates were also 100% identical with the reference sequence (UBIP01000001) (Fig. 1). Based on these results, it can be stated that all Plenodomus isolates investigated in the present study could be classified into P. lingam ‘brassicae’ and P. biglobosus ‘brassicae’ subclades. This region is highly conserved, as it has been determined by Mendes-Pereira et al. (2003).

Fig. 1
figure 1

Phylogenetic neighbor-joining tree based on ITS1-5.8S-ITS2 sequences of 26 isolates of Plenodomus lingam and 17 isolates of Plenodomus biglobosus from Hungary and isolates from NCBI database. The numbers are the percent bootstrap support for 1000 resampling and evolutionary analyses conducted in MEGA11. Leptosphaeria doliolum (CBS 505.75) was used as the outgroup

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Phylogenetic tree based on multi-locus phylogenetic analysis

Sequences of the P. lingam isolates: the partial tub2 gene sequences were 99.42–100%, the partial LSU sequences 99.43–100% and the partial rpb2 region sequences 98.37–100% identical with the reference strain sequences (CBS 275.63). Sequences of the P. biglobosus isolates: the partial tub2 gene sequences were 99.70–100%, the partial LSU sequences 99.09–100% and the partial rpb2 region sequences 100% identical with the reference strain sequences (UBIP01000001) (Fig. 2). Noteworthy, these are the first sequence data in relation to the genes and regions of interest of the P. lingam and P. biglobosus in Hungary.

Fig. 2
figure 2

Phylogenetic tree obtained from the combined partial tub2 gene, ITS1-5.8S-ITS2, partial LSU, partial rpb2 gene sequence alignment of 26 Plenodomus lingam and 17 Plenodomus biglobosus isolates under survey and reference strains. The tree was rooted using Leptosphaeria doliolum (CBS 505.75) as outgroup taxon. Bootstrap support values > 95% are indicated near the nodes

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The combined four‐locus data set consisted of 46 isolates with the whole-genome isolates of P. lingam ‘brassicae’ and P. biglobosus ‘brassicae’ and with Leptosphaeria doliolum as the outgroup taxon. In the tree (Fig. 2), the surveyed P. lingam and P. biglobosus isolates were clustered separately to their reference strains with 96% bootstrap support, respectively. Within species, we observed only parsimony-uninformative variability. The low genetic diversity among isolates in this investigation is not surprising, as the isolates belong to the same subclade (Fig. 1).

Discussion

Outbreak of a new pathogen or changes in genetic composition of a population could compromise the efficiency of established plant protection strategies. Plenodomus biglobosus was at first described in 2020 in Hungary (Bagi et al. 2020). The monitoring of new pathogens is of high importance as it can help to optimize breeding strategies and crop protection technologies (Huang et al. 2014). There is a lack of information about the distribution of Plenodomus species causing blackleg of brassicas in Central Europe; therefore, our goal was to identify and describe the local pathogens in oilseed rape cultivation. Based on the occurrence and frequency of P. lingam and P. biglobosus, it can be stated that P. biglobosus is more common and widespread in Hungary than previously thought.

Furthermore, we also tried to investigate the sources of variation in genetic diversity observed in the Hungarian P. lingam and P. biglobosus population. According to some views, in the UK additional genetic subclades may be responsible for the growing importance of P. biglobosus (King and West 2022). Molecular identification and characterization clearly identified the Hungarian P. lingam isolates as members of the P. lingam ‘brassicae’ subclade. This subclade is distributed worldwide and can infect several Brassica species (Mendes-Pereira et al. 2003), while P. lingam ‘lepidii’ subclade has been only isolated from Lepidium sp. from Canada (Mendes-Pereira et al. 2003). Similarly, the analysis of Hungarian P. biglobosus isolates showed that all isolates belong to the P. biglobosus ‘brassicae’ subclade, admittedly. The subclade ‘brassicae’ that infects Brassica species (Mendes-Pereira et al. 2003) is the most widely distributed subclade of P. biglobosus (Liu et al. 2014). Plenodomus biglobosus ‘canadensis’ is the most closely related subclade to P. biglobosus ‘brassicae’ and has been isolated from oilseed rape and Chinese mustard (Van de Wouw et al. 2008; Dilmaghani et al. 2009). Plenodomus biglobosus ‘australensis’ (Voigt et al. 2005), ‘occiaustralensis’ (Vincenot et al. 2008) and ‘americensis’ (Zou et al. 2019) subclades can also infect Brassica species, incl. oilseed rape, while other subclades (Mendes-Pereira et al. 2003) ‘thlaspi’ (obtained from Thlaspi arvense) and ‘erysimii’ (isolated from Erysimum sp.) have not been reported from brassicas yet.

Molecular methods have been used in taxonomic studies of Plenodomus to reveal phylogenetic relationship among the species and subclades (Zou et al. 2019). Combined DNA phylogenetic analysis based on ITS, 28S nrDNA (LSU) and β-tubulin, sequences are often used to reconstruct these relationships.

Hungarian P. lingam and P. biglobosus isolates, based on four DNA regions, resulted consistent phylogenetic trees and the sequences were extremely similar to each other. The low molecular diversity among the P. biglobosus isolates also suggests that the pathogen was introduced to Hungary much earlier than it was first identified. Most likely its emergence has remained hidden due to very similar symptoms. These fungi coexist in hosts and cause leaf lesions, in addition P. lingam is associated with basal stem canker, while P. biglobosus rather causes upper stem lesions (Eckert et al. 2010; Sprague et al. 2017). Low genetic diversity in a population is more likely to mean reduced adaptation potential, which may also provide important information on the risk of fungicide resistance development.

Worldwide large-scale monitoring and surveys are required to prevent the extreme economic losses. Our results indicate that for both pathogens only the ‘brassicae’ subclades are present in the Hungarian populations at the moment. As a result of globalization, there is a risk that additional subclades will emerge in Hungary on oilseed rape, but in the meantime, it can be concluded that the importance of blackleg pathogens will not change in the near future.