Phylogenetic analysis of Plenodomus lingam and Plenodomus biglobosus isolates in Hungary

Blackleg (stem canker) of crucifers is a globally important disease caused by multiple genetic subclades of the fungi Plenodomuslingam (syn.: Leptosphaeria maculans) and Plenodomus biglobosus (syn.: Leptosphaeria biglobosa). In our study, we monitored the geographical distribution of these two pathogen species from rapeseed growing areas in Hungary. Multiplex PCR identified 48.7% of the isolates as Plenodomus biglobosus, which indicates the non-recent introduction of the pathogen into Hungary. In addition, multi-locus analysis revealed low genetic diversity within the species, as all isolates were clustered to the Plenodomuslingam ‘brassicae’ and Plenodomus biglobosus ‘brassicae’ subclades. The low genetic diversity of a population generally means reduced adaptation potential, which is essential information in breeding and in developing more effective management strategies.


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.

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.

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 Nan-oDrop™ 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 ) by amplification conditions of Chen et al. 2015. The target sequence length without primers was 1 3 345 bp for P. lingam and 337 bp for P. biglobosus. To amplify partial region of rpb2, RPB2F (5′-AGG CTT GTG GTT TGG TCA AGA-3′) and the RPB2R (5′-ATC ATA GCR GTC TCT TCC TCCT-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 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.

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.

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).
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 parsimonyuninformative 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.
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.