Biscogniauxia rosacearum, first evidence in Germany and pest risk analysis for the potentially quarantine relevant charcoal canker fungus

Biscogniauxia rosacearum, first evidence in Germany and pest risk analysis for the potentially quarantine relevant charcoal canker fungus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Biscogniauxia rosacearum, first evidence in Germany and pest risk analysis for the potentially quarantine relevant charcoal canker fungus Gritta Schrader, Steffen Bien, Clovis Douanla-Meli, Björn Hoppe, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7752155/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 6 You are reading this latest preprint version Abstract Since Biscogniauxia rosacearum has been detected in Germany for the first time, a pest risk analysis (PRA) for this wood-decaying fungus with potential quarantine relevance is presented. This species which is known to be distributed throughout the Mediterranean region and presumably native to the Middle East, is usually found on Rosaceae and other deciduous trees. Two new host tree species were identified, Abies grandis and Pseudotsuga menziesii . Furthermore, this ascomycete was compared to Biscogniauxia mediterranea , which is prevalent in Germany, and was distinguished in a multigene phylogeny based on ITS, TUB , and ACT sequence alignment. In addition, a qPCR -assay using a previously published species-specific primer combination for the detection of B. mediterranea was tested on a selection of isolated B. mediterranea and B. rosacearum strains, and subsequently assigned to B. rosacearum . Graphostromataceae wood-decay fungus pest risk analysis multigene phylogeny tree pathogen Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The genus Biscogniauxia Kuntze comprises approximately 90 species names (as of September 2025; www.indexfungorum.org , www.mycobank.org ) and belongs to the family Graphostromataceae M.E. Barr, J.D. Rogers & Y.M. Ju, order Xylariales Nannf. within the phylum Ascomycota (Senanayake et al. 2015 ; Daranagama et al. 2018 ; Hyde et al. 2020 ). The family furthermore includes the genera Camillea Fr., Cryptostroma P.H. Greg. & S. Waller, Graphostroma Piroz., Obolarina Pouzar, and Vivantia J.D. Rogers, Y.M. Ju & Cand. (Hyde et al. 2020 ; Li et al. 2021 ) (Hyde et al. 2020 ; Li et al., 2021 ). The genus Biscogniauxia was named by Carl Ernst Otto Kuntze in 1891 to honour the Belgian botanist Célestin Alfred Cogniaux (1841–1916) (Kuntze 1891 ). Species of Biscogniauxia have a worldwide distribution and have been recorded on a wide range of woody host plants (Bahmani et al. 2021 ). Biscogniauxia species comprise pathogens and endophytes of angiosperms (Hyde et al. 2020 ; Tropf et al. 2025 ). However, they are also known as endophytes of the woody and foliar tissues of conifers. For instance, the type species B. nummularia (Bull.) Kuntze and B. mediterranea (De Not.) Kuntze have recently been found in association with Scots pine ( Pinus sylvestris L.) and giant sequoia ( Sequoiadendron giganteum (Lindl.) J. Buchholz) (Bußkamp et al. 2020 ; Langer et al. 2024 ). As plant pathogens, Biscogniauxia species are able to cause severe wood-decay and such events frequently occur in trees which are already significantly weakened for example by abiotic factors, such as intense drought (Ju et al. 1998 ). According to the phylogenetic analysis by Koukol et al. ( 2014 ), the anamorphic fungus Cryptostroma corticale (Ellis & Everh.) P. H. Greg. & H. Waller, which is the causal agent of Sooty bark disease (SBD), is closely related to B. bartholomei (Peck) Lar. N. Vassiljeva and G. platystomum (Schwein.) Piroz. According to Daranagama et al. ( 2018 ), the genus Biscogniauxia seems to be paraphyletic within the Graphostromataceae. As indicated by Ju et al. ( 1998 ), Biscogniauxia species are well adapted to dry habitats, or at least, to habitats that are dry for part of the year. The mycelial development occurs inside the bark of the host plants and the fructifications, both anamorphic and teleomorphic, are produced superficially on the twigs, branches, and trunks of angiosperm plants (Hyde et al. 2020 ). In addition to teleomorphs, several species exhibit a periconiella- or nodulisporium-like anamorph (Hyde et al. 2020 ). According to the USDA database (Farr and Rossman 2022 ), 13 Biscogniauxia species are known to occur in Europe. These include B. anceps (Sacc.) J.D. Rogers, Y.M. Ju & Cand. (Rogers et al. 1996 ), B. capnodes (Berk.) Y.M. Ju & J.D. Rogers. ( https://www.gbif.org/species/3461055 ), B. cinereolilacina (J.H. Mill.) Pouzar (Pouzar 1979 ; Pouzar 1986 ), B. communapertura Y.M. Ju & J.D. Rogers (Ju et al. 1998 ), B. dennisii (Pouzar) Pouzar (Pouzar 1979 ), B. granmoi Lar.N. Vassiljeva (Læssøe et al. 1999 ; Zíbarová and Kout 2017 ), B. marginata (Fr.) Pouzar (Pouzar 1979 ; Pouzar 1986 ; Chlebicki and Bujakiewicz 1994 ), B. mediterranea (Ju et al. 1998 ; Zíbarová and Kout 2017 ), B. nummularia (Pouzar 1979 ; Pouzar 1986 ; Læssøe et al. 1999 ), B. querna Pouzar (Pouzar 1986 ), B. repanda (Fr.) Kuntze (Pouzar 1979 ; Pouzar 1986 ; Chlebicki and Bujakiewicz 1994 ), B. rosacearum M.L. Raimondo & Carlucci, and B. simplicior Pouzar (Pouzar 1986 ). Until recently, only four species were known to occur in Germany, namely B. capnodes on a non-native Latania sp., (Ju et al. 1998 ), B. mediterranea on oak (Ju et al. 1998 ; Bußkamp et al. 2020 ; Langer and Bußkamp 2021 ; Langer and Bußkamp 2023 ; Bußkamp et al. 2024 ) and Scots pine (Bußkamp et al. 2020 ), B. nummularia on Fagus sylvatica (Ju et al. 1998 ; Læssøe et al. 1999 ; Tropf et al. 2025 )d repanda on the Betulaceae Alnus glutinosa (L.) Gaertn. and Betula pendula Roth (Schmid-Heckel 1988 ; Chlebicki and Bujakiewicz 1994 ). Biscogniauxia nummularia, B. mediterranea , and B. rosacearum can induce strip-cankers on their host trees, also referred to as charcoal canker or tarcrust (Hendry et al. 1998 ; Granata and Sidoti 2004 ; Nugent et al. 2005 ; Ragazzi et al. 2007 ; Henriques et al. 2015 ; Luchi et al. 2015 ; Raimondo et al. 2016 ; Yangui et al. 2019 ). First visible signs of infection or parasitic growth are often bark blisters on the host tree (Fig. 1 e), later on blackish, elongated bark lesions (strip cankers) on the trunks (Fig. 1 d) and branches appear and teleomorphs and/ or anamorphs were produced (Fig. 1 a-f). The parasitic growth of the three Biscogniauxia species lead to wood decay often followed by green wood and brittle fracture (Fig. 1 g). The occurrence of B. mediterranea has been reported in Africa, Europe, North and Central America (Ju et al. 1998 ). It is widely distributed in Europe, where it is associated with oak (Ju et al. 1998 ) and the causal agent of charcoal canker on Quercus suber L. (Fagaceae) in the Mediterranean basin (Hyde et al. 2020 ). It has been hypothesized that B. mediterranea enters the host tree initially through the trunk and leaves (Gharbi et al. 2020 ), where it can be recognised as an endophyte in apparently healthy plants (Tropf et al. 2025 ). However, it can switch to its pathogenic lifestyle in water-stressed host plants (Edwards et al. 2003 ). Biscogniauxia mediterranea has been detected frequently in various tree species ( Ju et al. 1998 , Langer and Bußkamp 2021 ; Bußkamp et al. 2024 ). The latent pathogen B. nummularia is considered to be one of the most frequently detected fungal endophytes in European beech ( Fagus sylvatica L., Fagaceae) (Chapela and Boddy 1988 ; Nugent et al. 2005 ; Langer et al. 2021 ; Langer and Bußkamp 2021 ; Tropf et al. 2025 ). When its host is stressed by drought and heat, the fungus can transit from an endophytic to a pathogenic lifestyle, causing various symptoms such as bark necrosis, bark blistering, bark ruptures, strip-cankers, and brittle fractures (Granata and Whalley 1994 ; Hendry et al. 1998 ; Nugent et al. 2005 ). Biscogniauxia nummularia is the causal agent of beech bark tar crust disease and charcoal canker in F. orientalis and F. sylvatica (Vujanovic et al. 2020 ; Patejuk et al. 2022 ; Zamani et al. 2024 ). Additionally, it is one of the key fungal agents in beech decline (Granata and Whalley 1994 ; Granata and Sidoti 2004 ) and vitality loss of beech (Langer and Bußkamp 2021 ; Langer and Bußkamp 2023 ). Biscogniauxia nummularia can form periconiella-like anamorphs in vitro and in planta, which produce powdery, light-coloured, globose conidia (Petrini and Petrini 1985 ; Langer 2024 ). Although B. nummularia has been detected in many different angiosperms and gymnosperms (Petrini and Petrini 1985 ), to date, fruiting bodies and damage to trees have only been documented in F. orientalis Lipsky and F. sylvatica (Petrini and Petrini 1985 ; Nugent et al. 2005 ; Langer and Bußkamp 2023 ; Zamani et al. 2024 ). Biscogniauxia rosacearum is presumably native to the Middle East, and has already been reported in the EU. For instance, it is the causal agent of the charcoal canker on species in the family Rosaceae Juss., including Pyrus communis L. (pear), Prunus domestica L. (plum), and Cydonia oblonga Mill. (quince), first observed in Italy (Raimondo et al. 2016 ). Despite the fact that B. rosacearum is of phytosanitary significance (EFSA 2022; JKI 2025 ), it is not currently listed as a quarantine pest or a regulated non-quarantine pest in the annexes to Regulation (EU) 2019/2072. Furthermore, it is not listed by the European and Mediterranean Plant Protection Organization (EPPO). Generally, B. rosacearum infects a broad range of host plants, including almond ( Prunus dulcis (Mill.) D.A.Webb, Rosaceae), grapevine ( Vitis vinifera L., Vitaceae), pear, plum, quince, strawberry tree ( Arbutus unedo L., Ericaceae) and various oak species in the Mediterranean region, Middle East, and Africa (Raimondo et al. 2016 ; Bahmani et al. 2021 ; Sohrabi et al. 2022 ; Bashiri et al. 2022 ; Yangui et al. 2024 ). Recently, B. rosacearum has been found for the first time in Germany on Abies grandis (Douglas ex D.Don) Lindl. (Pinaceae) and on Pseudotsuga menziesii (Mirbel) Franco (Pinaceae). Therefore, an express pest risk analysis (Express-PRA) on Biscogniauxia rosacearum was conducted and disseminated (JKI 2025 ). Both, B. rosacearum and the closely related species B. mediterranea are thought to have a high degree of intraspecific diversity (Henriques et al. 2016 ; Bashiri et al. 2022 ). Regarding B. mediterranea , it is assumed that the epidemiology of the disease caused by this species may have changed. Its high genetic variability, even in small populations, is attributed to its heterothallic mating system, its high sexual reproduction rate, and the production of large quantities of ascospores (Vannini et al. 2008 ; Henriques et al. 2016 ). The same likely applies to B. rosacearum . A previously unpublished phylogenetic analysis based on ITS sequences (JKI 2025 ) indicates that B. rosacearum has a much broader host range and a more extensive geographical distribution than is currently recognised. This is largely related to the fact that in the past, some sequences belonging to B. rosacearum have been incorrectly assigned to B. mediterranea and deposited in sequence databases as such, due to inadequate taxonomical identification. Therefore, comparison of current sequence data with older entries can easily lead to confusion. Therefore, the aims of this study were to 1) present additional information on B. mediterranea , B. nummularia , and B. rosacearum 2) identify B. rosacearum strains that may not have been identified initially and that were assigned to Biscogniauxia in the strain collection of the Northwest German Forest Research Institute (NW-FVA), 3) identify potential B. rosacearum entries in the NCBI GenBank database that may not have been identified previously, 4) test and evaluate the species-specific primers for B. mediterranea published by Luchi et al. (2005), 5) evaluate the host and distribution range of B. rosacearum so far as known, and 6) assess the phytosanitary risk of B. rosacearum by conducting a pest risk analysis (PRA) according to the standards of the International Plant Protection Convention (IPPC) and the European and Mediterranean Plant Protection Organization (EPPO). Material and Methods Cases of disease and fungal isolation The forest sites and cases of diseases associated with the incidences of B. rosacearum in Germany were studied in 2021 and 2024 as part of routine forest protection consulting and causal analysis of forest damage by the Northwest German Forest Research Institute (NW-FVA). Two specimens of B. rosacearum were isolated and identified according to the methods described in Langer and Bußkamp ( 2023 ) and Langer et al. ( 2024 ) and stored in the NW-FVA strain collection. The Biscogniauxia rosacearum -strain NW-FVA 6753 (= isolate 2021-69-SB-Z1-112, NCBI Genbank Accession No. PX239669) was isolated from a twig (Fig. 1 ) of a diseased, 48-year-old grand fir ( A. grandis ) sampled on 16 June 2021 in a private forest close to Kirchlinteln, Lower Saxony, Germany. The affected tree, suffering from Fir bark disease (Langer and Rohde 2018 ), was also infected among others with B. nummularia, Diplodia mutila (Fr.) Fr., D. sapinea (Fr.) P. Karst., Fusarium lateritium Nees, Nemania serpens (Pers.) Gray, Neonectria neomacrospora (C. Booth & Samuels) Mantiri & Samuels, Phomopsis sp., and Xylaria sp. Furthermore, Heterobasidion annosum (Fr.) Bref. and Armillaria root rot were detected. The Biscogniauxia rosacearum -strain NW-FVA 13169 = isolate 2024-73-Ü1–2, Accession No. PX239675) was retrieved from a shoot from Douglas fir ( P. menziesii ) aged 30 years, sampled on 27 August 2024 in a forest close to Genthin, Saxony-Anhalt, Germany. The studied tree suffered from Swiss needle cast (causal agent: Nothophaeocryptopus gaeumannii (T. Rohde) Videira, C. Nakash., U. Braun & Crous), H. annosum root rot and infestation with Cooley spruce gall adelgid ( Adelges cooleyi (Gillette)) and weevils of the Curculionidae family. Beside a few other fungi, B. nummularia and Peniophora cinerea (Pers.) Cooke were additionally isolated from the studied Douglas fir tissue. In order to compare the cultural characteristics of the German B. rosacearum strains with other similar species, strains of B. mediterranea (NW-FVA 9136 = Dgl-2022-2-Z2-68, Accession No. PX239670) isolated from P. menziesii sampled on 9 June 2022 in Baden-Wuerttemberg close to Iffezheim (Langer et al. 2025 ), and NW-FVA 11840 = 2023-110-1-Ü3-37b, Accession No. PX239672) isolated from Quercus petraea (Mattuschka) Liebl., sampled on 11 October 2023 in Hesse) were inoculated onto PDA agar, observed and documented with culture photos after 7, 14, 21, and 28 days (Fig. 2 ). The B. mediterranea teleomorph of the specimen 2025-72-3 (strain NW-FVA 13980, Accession No. PX239676, Fig. 1 ) was isolated from F. sylvatica sampled by J. Bußkamp on 21 May 2025 in Germany, in the Harz mountains close to Wernigerode. Molecular Identification and phylogenetic analysis Extraction of genomic DNA from all isolated strains was conducted following the method described in Tropf et al. ( 2025 ). The 5.8S nuclear ribosomal gene with the two flanking internal transcribed spacers ITS-1 and ITS-2 (ITS) was amplified using the primer pair ITS-1F + ITS4 (White et al. 1990 ). Additionally, partial sequences of the β-tubulin gene ( TUB ) and the actin gene ( ACT ) were generated using the primer pairs T1 + T2 (O’Donnell and Cigelnik 1997 ) and ACT-512F + ACT-783R (Carbone and Kohn 1999 ), respectively. The mixture for all PCR reactions consisted of 2.5 µl 10×PCR reaction buffer (with 20 mmol/l MgCl 2 , Carl Roth, Karlsruhe, Germany), 1 µl of each primer (10 mmol/l), 2.5 µl MgCl 2 (25 mmol/l), 0.1 µl Roti®-Pol Taq HY Taq polymerase (Carl Roth, Karlsruhe, Germany), 2.5 µl of 2 mmol/l dNTPs (Biozym Scientific GmbH, Hessisch Oldendorf, Germany), and 1 µl of extracted DNA solution. Each reaction was topped up to a volume of 20 µl by adding HPLC grade water (Carl Roth, Karlsruhe, Germany). A GeneExplorer 96 (Hangzhou BIOER Technology, Hangzhou, China) was used to carry out the DNA amplifications. PCR conditions for ITS and ACT followed Bien et al. ( 2020 ) and Raimondo et al. ( 2016 ), respectively. The PCR conditions for the TUB region were as follows: initial denaturation at 95°C for 5 min, followed by 35 cycles of 95°C for 30 s, 58°C for 30 s and 72°C for 60 s, with a 10 min extension at 72°C on the final cycle. PCR products were visualised in 1% agarose gel, and sent to Eurofins Scientific Laboratory (Ebersberg, Germany) for sequencing. Consensus sequences were generated from all the resulting sequences, which were then visually checked and edited as necessary using the BioEdit Sequence Alignment Editor (v. 7.2.5; Hall ( 1999 )). Sequences were submitted to GenBank (Sayers et al. 2022 ), see also Online Resource 1. For the phylogenetic analysis, two datasets were compiled. Dataset 1 consists of sequence data from three loci (ITS, TUB , ACT ; if available) from strains of Biscogniauxia isolated in this study and further sequences retrieved from the NCBI GenBank including those of ex-type strains, following the data composition used in the phylogenetic analyses of Raimondo et al. ( 2016 ) and Qiao et al. ( 2024 ). For dataset 2, ITS sequence of B. rosacearum ex-type strain CBS 141046 (GenBank Accession No. KT253493) was used for a Blastn search on NCBI GenBank on April 11, 2025. Of the higher-matching sequences identified, those with a percentage identity value > 98% were used together with ITS sequences from B. mediterranea and B. rosacearum strains isolated in this study and the ones reported in the study by Luchi et al. ( 2005 ) and Raimondo et al. ( 2016 ). Sequence data from both datasets and from all single loci were aligned automatically using MAFFT v. 7.490 (Katoh et al. 2002 ; Katoh and Standley 2013 ) as implemented in Geneious R11 (Kearse et al. 2012 ), and manually adjusted if necessary. The multilocus (ML analyses for dataset 1 (separately for all single loci and the concatenated dataset) and dataset 2 were performed by RAxML v. 8.2.11 (Stamatakis 2006 ; Stamatakis 2014 ) as implemented in Geneious Prime® 2025.0.3 (Kearse et al. 2012 ) using the GTRGAMMA model with the rapid bootstrapping and search for best scoring ML tree algorithm including 1000 bootstrap replicates. qPCR Analysis DNA extracts from B. rosacearum strains NW-FVA 6753 and NW-FVA 13169 as well as from B. mediterranea strains NW-FVA 6717, NW-FVA 9136, NW-FVA 10508, NW-FVA 11840, NW-FVA 12572, and NW-FVA 13121 were used for qPCR following Luchi et al. ( 2005 ) using the primer and probe combination specific for B. mediterranea presented therein. DNA extracts from all samples were brought to final DNA concentration of 80 ng/µl. qPCR was performed in a StepOnePlus™ PCR System (Applied Biosystems, Waltham, MA, USA). The reaction was performed in a final volume of 20 µl. qPCR mixture contained 300 nmol/l each forward primer and reverse primer, 200 nmol/l fluorogenic probe, 1x qPCR Blue Probe Mix and 50 µM ROX Additive (Biozym Scientific GmbH, Hessisch-Oldendorf, Germany), and 1 µl template DNA. Each DNA sample was assayed in four replicates. Four wells containing 1 µl HPLC grade water each instead of DNA sample as ‘no-template control’. The PCR protocol according to Luchi et al. ( 2005 ) was followed. Pest Risk Analysis (PRA) To identify the risk of introducing the fungus into new areas, i.e. the potential of its entry and establishment, as well as its risk of spread and the potential impacts, an Express-PRA for the PRA area, i.e. EU member states, in particular Germany, was conducted as described in Schrader et al. ( 2024 ). In case the outcome of the PRA indicates that the pest is a potential quarantine pest, phytosanitary measures have to be applied (Schrader et al. 2024 ). The PRA followed the steps according to the standards of the IPPC (IPPC 2001 ) and the European and Mediterranean Plant Protection Organization (EPPO) (EPPO 2024 ). PRA and the different aspects to be assessed are also regulated by the European Union (EU) (EU 2016 ), in line with IPPC and EPPO. The PRA describes the biology of the fungus, as well as its geographical distribution and areas infested. It further assesses its host plants and their presence in the PRA area, i.e. Germany and the EU. In addition, the PRA includes information about the symptoms of the pathogen, a discussion, whether climate in its area of distribution is comparable to the PRA area, and identifies potential pathways of introduction and spread. An important aspect is the assessment of known impacts in infested areas and in areas potentially being infested. Detection and diagnostics are briefly described as well. Finally, it is discussed, whether an infestation could easily be eradicated. Results Additional information on B. mediterranea , B. nummularia , and B. rosacearum Typical habitus of a Biscogniauxia teleomorph (Fig. 1 a-d) and anamorph (Fig. 1 e-f) and some of the typically caused symptoms (e.g. strip-canker Fig. 1 d), as well as the studied material of the first evidences from B. rosacearum are presented in Fig. 1 h-l. Stroma of B. nummularia (Fig. 1 e) were produced as a cushion-like fungal mass under the bark (Fig. 1 f), visible as bark blisters. The pressure from the stroma causes the bark to peel or strip off in patches or strips (Fig. 1 e-g). Wood-decay by Biscogniauxia species leads to brittle fracture (Fig. 1 g). Molecular Identification and phylogenetic analysis Except from two strains isolated and identified in the study, no other Biscogniauxia of the strain NW-FVA-collection could be assigned to B. rosacearum. The combined sequence dataset over all DNA loci analyzed (dataset 1) using ML consisted of 76 isolates including the outgroup and comprised 2139 characters. The gene boundaries in the concatenated multi-locus alignment were as follows: ITS: 1–878, TUB : 879–1829, ACT : 1830–2139. The analyses of all single loci datasets (not shown) as well as the concatenated dataset revealed phylogenetic trees of highly similar topology. The ML phylogeny of the multi locus dataset including ML bootstrap values is shown in Fig. 3 . Strains of B. mediterranea and B. rosacearum form well-supported, closely related but distinctly separate monophyletic clades, respectively. Six of the German Biscogniauxia isolates (NW-FVA 10508, NW-FVA 9136, NW-FVA 12572, NW-FVA 6717, NW-FVA 13121 and NW-FVA 11840) have been shown to cluster in the B. mediterranea clade, and two isolates (NW-FVA 6753 and NW-FVA 13169) cluster in the B. rosacearum clade. The sequence dataset 2 of ITS sequences primarily retrieved from GenBank after Blast search with B. rosacearum consisted of 129 isolates including the outgroup and comprised 517 characters. The ML phylogeny of dataset including ML bootstrap values is shown in Fig. 4 . Apart from the strains used as outgroup and strains used as reference strains of Biscogniauxia mediterranea all strains retrieved from GenBank cluster in a well-supported clade together with strains of B. rosacearum analyzed in depth in Raimondo et al. ( 2016 ) and Sohrabi et al. ( 2022 ) and strains isolated in this study. The majority of strains retrieved from GenBank which should be assigned to B. rosacearum according to our analysis were designated as B. mediterranea . qPCR Analysis qPCR revealed positive amplification curves for all tested B. rosacearum strains (NW-FVA 6753: Ct mean ± SD = 17,92 ± 0,06, ΔRn mean = 5,119; NW-FVA 13169: Ct mean ± SD = 17,34 ± 0,03, ΔRn mean = 5,222). No signal was observed for wells containing sample DNA of B. mediterranea strains and NTCs. An amplification plot is displayed in Fig. 5 . Pest Risk Analysis Based on the findings of this study and the literature review on Biscogniauxia , in accordance with the procedure described in Material and Methods, the following results have been obtained: Biology of the organism Fungi from the genus Biscogniauxia are generally considered opportunistic pathogens that occur endophytically in the sapwood and bark of healthy trees without causing any obvious disease symptoms. However, they can cause significant damage to severely weakened or damaged trees. Drought conditions in the Mediterranean region have been found to influence the aggressiveness of B. rosacearum and can lead to the outbreak of the disease (van Dyk et al. 2021 ). The fungus forms a hard mycelial mat (stroma) to multiply and spread to new host plants. Biscogniauxia rosacearum has a high reproduction rate and is heterothallic, i.e. the mycelia are self-incompatible or self-sterile. Initially, powdery, light-coloured, asexual spores (conidia) are produced. A specific vector is not mentioned in the literature. According to Raimondo et al. ( 2016 ), the fungus is primarily dispersed by wind or insects; occasionally also by other animals or by water. In a later stage, the fungus produces black sexual spores (ascospores), which are also transported by wind, water, or animals. While conidia are typically formed in spring or early summer, sexual spores are found in summer and fall. Both spore types can cause infections (Raimondo et al. 2016 ; Crocker et al. 2018 ). Geographical distribution of the fungus and areas infested Apart from the two findings in Germany, the fungus has been detected in Iran, Italy, Portugal, Spain, South Africa, and Tunisia (Raimondo et al. 2016 ; Pinna et al. 2019 ; Spies et al. 2020 ; Bahmani et al. 2021 ; van Dyk et al. 2021 ; Sohrabi et al. 2022 ; Bashiri et al. 2022 ; Yangui et al. 2024 ). However, the phylogenetic analysis based on ITS sequences retrieved from GenBank presented here suggests a much larger distribution area (North Africa, Middle East, Southern to Central Europe, China, USA, see Fig. 4 ). So far, occurrences in the EU have been confirmed only sporadically. Surveys would be necessary to determine the actual distribution status. Host plants and their presence in the PRA area To date, several host plants are known. Biscogniauxia rosacearum was found to be endophytic, parasitic, saprobic, and also associated with woody tissues damaged by other pathogens: In Italy, B. rosacearum was found on 13- to 20-year-old pear trees ( Pyrus communis ), plum ( Prunus domestica ), quince ( Cydonia oblonga ), and downy oak ( Quercus pubescens Willd.) in Apulia (Raimondo et al. 2016 ). Additionally, it has also been isolated from insect boreholes in oak trees in Sardinia (Pinna et al. 2019 ). In Iran, the fungus has been detected on Vitis vinifera (confirmed by greenhouse pathogenicity tests), as well as on Quercus castaneifolia C.A.Mey., Q. infectoria Oliv., Q. libani Oliv., Q. brantii Lindl., and Prunus dulcis (Bahmani et al. 2021 ; Sohrabi et al. 2022 ; Bashiri et al. 2022 ). Studies of oak dieback in the Iranian forests of the Zagros Mountains (Bashiri and Abdollahzadeh 2024 ) revealed that B. rosacearum was one of the most frequently detected fungi associated with oak dieback, alongside Obolarina persica Mirab., Y.M. Ju, H.M. Hsieh & J.D. Rogers (wrongly addressed as Biscogniauxia persica ), Cytospora hedjaroudei Bashiri & Abdollahz., C. zagrosensis Bashiri & Abdollahz., Neocosmospora metavorans (Al-Hatmi, S.A. Ahmed & de Hoog) Sand.-Den. & Crous, and Neocosmospora sp. In Tunisia, B. rosacearum was detected on Arbutus unedo in 2020 (Yangui et al. 2024 ) and on Myrtus communis L. in co-infection with Diaporthe foeniculina (Sacc.) Udayanga & Castl. (Khadraoui et al. 2025 ). In South Africa, B. rosacearum was associated with olive ( Olea europaea L.) trunk disease symptoms (Spies et al. 2020 ; van Dyk et al. 2021 ). Results of phylogenetic studies conducted by Raimondo et al. ( 2016 ) also included Platypus cylindrus (Fabricius, J.C., 1792), arthropods (Portugal), Holcus lanatus L., Quercus ilex L., and Pinus sylvestris (Spain) as hosts. The European Food Safety Authority (EFSA) (EFSA 2022) also mentions P. sylvestris as a host plant, but no further evidence is found for this. In Germany, B. rosacearum was detected in two diseased trees in two federal states, Lower Saxony and Saxony-Anhalt. In 2021, a strain of B. rosacearum was isolated from the shoot tissue of a 48-year-old grand fir ( Abies grandis ) suffering from fir bark necrosis, which was also infected with Diplodia sapinea , D. mutila , and Neonectria neomacrospora . In 2024, another strain was isolated together with a Coniochaeta sp. from dying shoots of a 30-year-old Douglas fir ( Pseudotsuga menziesii ). The affected Douglas fir also exhibited Swiss needle cast caused by the pathogen Nothophaeocryptopus gaeumannii , root rot caused by Heterobasidion annosum , shoot dieback, nicking, and mealybug infestation (this study). Fruiting bodies (teleomorphs) and charcoal cankers have so far only been recorded on the following plant species: pears, plums and quinces (Raimondo et al. 2016 ), almond trees (Sohrabi et al. 2022 ), grapevine (Bahmani et al. 2021 ), and oaks (Bashiri et al. 2022 ). Again, phylogenetic analysis of ITS Blast results suggests an even broader host range, including several additional Quercus ( Q. cerris L., Q. faginea Lam., Q. ilex , Q. robur L., Q. suber L.) and Pinus ( P. mugo Turra, P. nigra J.F.Arnold, P. pinaster Ait.) species, and further woody and herbaceous plants. Most of the host plants, including pear, plum, quince, grapevine, Douglas fir, grand fir and various oak and pine species are economically and ecologically important plants used for agriculture and forestry in Germany, as well as in the EU. Symptoms In pathogenicity tests on Vitis vinifera , disease symptoms such as yellowing and necrosis of the leaves were observed two weeks after inoculation under greenhouse conditions. Brown lesions spread on the stems above and below the inoculation site. Stem cross-sections showed wedge-shaped necrosis of the vascular tissue (Bahmani et al. 2021 ). Charcoal-like cankers in the bark were observed on stems of pears, plums, and quinces (Raimondo et al. 2016 ). Climate in distribution area and PRA area So far, the fungus has been reliably detected in Mediterranean regions and in South Africa, but also on two individual plants (grand fir and Douglas fir) in Lower Saxony and Saxony-Anhalt, showing that at least in Mediterranean EU member states, climate is matching, but possibly also in Central Europe due to the confirmed occurrence in Germany. In addition, analysis of the ITS sequence data of Biscogniauxia strains provided in the NCBI GenBank also suggests a large distribution area based on unverified evidence from China and the USA (see ITS phylogram, Fig. 4 ). Pathways and natural spread Plants for planting are considered the main pathway. According to Raimondo et al. ( 2016 ), the fungus is naturally spread by wind or insects. Known impacts in infested areas Biscogniauxia species are primarily known as endophytes, secondary pathogens and facultative saprophytes. They can infect old and weakened host plants and persist as endophytes in the aboveground parts of oaks and other tree species. Furthermore, they may remain latent and only develop symptoms when the trees are stressed, for example by drought. Biscogniauxia species are able to rapidly colonize xylem and bark tissues, and can cause necrosis and canker formation, in severe cases even leading to tree death. Also younger and healthier trees are increasingly being infected (Henriques et al. 2016 ; Raimondo et al. 2016 ). In the case of B. rosacearum Bahmani et al. ( 2021 ) and Masi et al. ( 2021 ) have demonstrated that phytotoxins play a considerable role in the disease process. Expected (further) establishment and spread in Germany and the EU Based on the detection of B. rosacearum in Lower Saxony and Saxony-Anhalt, further establishment of the fungus in Germany is to be expected, also because of its high reproduction rate and spread potential by wind and insects. Since B. rosacearum occurs endophytically and has not been originally associated with damage in Germany (and thus does not attract attention), it may already be more widespread than previously thought. There is evidence (see ITS phylogram, Fig. 4 ) that B. rosacearum already occurs in some, primarily southern, EU Member States. Further establishment is expected as well in these areas, out of the reasons mentioned above. Expected impacts caused by the fungus in Germany and the EU In climatically suitable areas, significant damage to the host plants is to be expected, especially if they are already weakened by drought or infestation with other pests. However, B. rosacearum was probably not the cause of the observed damage in the two trees infected in Germany. Detection and diagnosis Biscogniauxia rosacearum can be unambiguously identified using the ITS DNA region. This result was confirmed by multi-locus sequence analysis (MLSA). In the past, especially before its initial description by Raimondo et al. ( 2016 ), the fungus was generally referred to as the closely related species B. mediterranea and deposited accordingly in databases. Therefore, confusion can easily arise when comparing new sequence data with existing ITS database entries. Until recently, findings on genetic diversity or host range, for example, were assigned erroneously to B. mediterranea based on incorrect species delimitation presumably due to these mentioned Blast inconsistencies (Yangui et al. 2022 ; Zamani et al. 2025 ). Furthermore, in addition to species assignment based on ITS database comparisons, rapid detection using qPCR and the specific primer/probe combination presented by Luchi et al. ( 2005 ) can currently lead to misinterpretation of test results. The latter authors developed the specific primer/probe combination based on ITS data from strains that, according to current knowledge, can be assigned to B. rosacearum . Accordingly, it was possible to demonstrate in this study that the specific primer/probe combination established by Luchi et al. ( 2005 ) does not amplify B. mediterranea but B. rosacearu m. Raimondo et al. ( 2016 ) provides specific identification guidelines but detection is difficult in asymptomatic but latently infected host plants. Figure 2 shows a synopsis of Biscogniauxia mediterranea and rosacearum pure cultures that were cultivated for 7, 14, 21, and 28 days on PDA (potato dextrose agar). Eradication of an infestation There is no known way to control the fungus by fungicides. To date, the only effective control method is felling and destroying (burning) the infected host plants. However, this would only be useful if B. rosacearum was not yet widespread. In principle, it would be necessary to identify infestation-free areas, including determining whether and to what extent the fungus occurs endophytically without symptoms. Depending on the results, preventative measures could then be taken to avoid or mitigate further spread of the fungus. If possible, in urban agricultural areas measures to improve tree health, such as reducing compaction, mulching, irrigation, and fertilization, should be taken. Visibly infected branches should be removed, both to reduce the amount of inoculum present and to prevent potentially dangerous tree breaks (Crocker et al. 2018 ). Since occurrences of B. rosacearum in the EU have so far only been confirmed sporadically, and there are only two isolated cases in Germany, further surveys would be necessary to determine the actual distribution status. Discussion Raimondo et al. ( 2016 ) were able to show that the two species B. mediterranea and B. rosacearum can be distinguished by their micromorphological characteristics. As described by the latter authors, the species differ in the size of their asci and ascospores as well as in their anamorph type. Biscogniauxia mediterranea forms a periconiella-like anamorph form, while B. rosacearum forms a nodulisporium-like form. If it is not possible to examine these micromorphological characteristics and only pure cultures are available, it will not be possible to distinguish between the two species on the basis of their culture-based characteristics alone. Therefore, the addition of molecular information (ITS sequence data according to this study is sufficient) is essential for an unambiguous identification. The multi-locus sequence analysis (MLSA) using three target genes in combination with morphological comparison of reference material allowed to designate the two newly isolated cultures as B. rosacearum and to identify false GenBank database entries that require an updated species annotation. Rapid advancements in (meta)barcoding of organisms in various environments not only require the application of modern molecular techniques, but also high-quality database entries containing reference sequence material to identify species accordingly. This of course has been under debate for various reasons and is not restricted to false entries due to lacking morphological identification but also due to duplicates, redundancies and errors in species determination algorithms (Chen et al. 2017 , Stein and Gailing 2025 ). Therefore, it is necessary to carefully interpret the obtained information and, preferably, verify the results with additional information and expertise beyond DNA sequences. Reliable species-level assignments require both, curated reference material and an accurate phylogeny based on molecular analyses, as described in this article. In conclusion, the study successfully achieved the aims outlined in the introduction. It was demonstrated that the fungal strains that could initially only be assigned to the Biscogniauxia genus in reference to the NW-FVA strain collection could be identified as B. rosacearum . It was shown that the species-specific primers for B. mediterranea published by Luchi et al. ( 2005 ) can lead to misinterpretation of test results, since the specific primer/probe combination used amplifies B. rosacearum instead of B. mediterranea . As a result of this, it is now possible to identify B. rosacearum entries in the NCBI GenBank database not having been identified previously. In the PRA, it could be shown that - with these new results for the identification and distinction of the two Biscogniauxia species – the distribution range of B. rosacearum is much larger than previously known. Furthermore, in the PRA it is assumed that B. rosacearum can establish in Germany due to suitable climatic conditions and the availability of potential host plants; (further) establishment in southern European and Mediterranean EU member states is to be expected. A more detailed assessment of climatic conditions required by the fungus to establish and spread, in combination with modelling its spread potential would therefore be useful to better estimate the risks for its host plants present in Europe. Biscogniauxia rosacearum poses a high damage potential for some Rosales and oaks, causing "charcoal canker". The development of "charcoal canker" in other host plants has not been observed to date, so that it would be important to investigate whether the Koch's postulates could be fulfilled for these species. In addition, insufficient information exists on the phylogenetic diversity and pathogenicity of different B. rosacearum strains towards different host species. In other tree species, the fungus seems not to cause any damage, but only occurs endophytically or opportunistically. The confusion regarding the two species B. rosacearum and B. mediterranea further complicates matters, as there may be errors with regard to the assignments of host plants and impacts of the two different species and their current distribution. Due to these uncertainties, it was not possible to conclusively assess the phytosanitary risk of B. rosacearum at this stage of the study. Therefore, there is an urgent need for further research, also to finally determine whether the fungus qualifies as a potential quarantine pest for the EU. Declarations Author contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Gitta Langer, Steffen Bien and Gritta Schrader. The molecular analyses were performed by Steffen Bien. The first draft of the manuscript was written by Gritta Schrader, Steffen Bien, and Gitta Langer and all authors commented on previous versions of the manuscript. Clovis Douanla-Meli and Björn Hoppe made substantial contributions to the manuscript. All authors read and approved the final manuscript. Funding acquisition: Gitta Langer. Funding The study was conducted as part of the TroWaK project (“Trockenheitsrisiken im Wald unter Klimawandel”, “Drought risks in forests under climate change”) which receives funding via the Waldklimafonds (“world climate fund”, WKF) funded by the German Federal Ministry of Agriculture, Food and Regional Identity (BMLEH), formerly Federal Ministry of Food and Agriculture (BMEL) and the Federal Ministry for the Environment, Climate Action, Nature Conservation and Nuclear Safety (BMUKN), formerly Federal Ministry for the Environment, Nature Conservation, Nuclear Safety and Consumer Protection (BMUV) administrated by the Agency for Renewable Resources (FNR) under grant agreement No 2220WK92D4. Additional funding was provided by the Julius Kuehn Institute (JKI) Federal Research Centre for Cultivated Plants, Institute for National and International Plant Health. Conflicts of interest The author(s) declare that they do not have any conflicts of interest. 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Czech Mycology 69:77–108. https://doi.org/10.33585/cmy.69106 Supplementary Files OnlineResource1Brosacearum.pdf Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Major revisions 21 Apr, 2026 Reviewers agreed at journal 07 Oct, 2025 Reviewers invited by journal 02 Oct, 2025 Editor invited by journal 02 Oct, 2025 Editor assigned by journal 01 Oct, 2025 First submitted to journal 30 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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16:01:00","extension":"png","order_by":31,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":392170,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7752155/v1/55151853e910cab0358dfd0a.png"},{"id":93609389,"identity":"80970f11-e767-413f-a2a9-6ea799f63b85","added_by":"auto","created_at":"2025-10-15 16:01:00","extension":"png","order_by":32,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":102427,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7752155/v1/7824653ae9e935657638261c.png"},{"id":93609385,"identity":"9c67ee5d-447f-4740-9b42-ad7ce190cf78","added_by":"auto","created_at":"2025-10-15 16:01:00","extension":"png","order_by":33,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":67959,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7752155/v1/70a2831a5d450bf1ea7439dc.png"},{"id":93608279,"identity":"ffa0847d-99d9-4f10-a731-aecc59d1857b","added_by":"auto","created_at":"2025-10-15 15:53:00","extension":"png","order_by":34,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":68107,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7752155/v1/e1929ebd5c71a1608eb5f2bf.png"},{"id":93609388,"identity":"a898dc01-a5f5-4c73-85b6-d9e53ea533d7","added_by":"auto","created_at":"2025-10-15 16:01:00","extension":"png","order_by":35,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":50071,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7752155/v1/9fafddd5041b96c3b03cc13d.png"},{"id":93608283,"identity":"5bf872d8-c175-4e4a-9063-fea0eef51877","added_by":"auto","created_at":"2025-10-15 15:53:00","extension":"xml","order_by":36,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":188581,"visible":true,"origin":"","legend":"","description":"","filename":"JPDPD25016250structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7752155/v1/6256adf810cfebd70a02bbb8.xml"},{"id":93608286,"identity":"971b523d-8a94-4cce-b385-6136643f297e","added_by":"auto","created_at":"2025-10-15 15:53:00","extension":"html","order_by":37,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":205397,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7752155/v1/699eb83574d7c6e607a33ed5.html"},{"id":93608246,"identity":"bb74ffb2-37a4-4fed-b81f-d86794a1aa51","added_by":"auto","created_at":"2025-10-15 15:52:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":9149371,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eBiscogniauxia\u003c/em\u003e spp. and infected host tissues. a-c) Fructification of \u003cem\u003eBiscogniauxia mediterranea\u003c/em\u003e d-g) Symptoms of damage due to \u003cem\u003eB. nummularia\u003c/em\u003e, h-l) the studied host tissue from which \u003cem\u003eBiscogniauxia rosacearum\u003c/em\u003e was isolated, h-j) \u003cem\u003eAbies grandis\u003c/em\u003e (2021-69), k-l) \u003cem\u003ePseudotsuga menziesii\u003c/em\u003e (2024-73). A comparison of the growth patterns exhibited by \u003cem\u003eB. mediterranea\u003c/em\u003e and \u003cem\u003eB. rosacearum\u003c/em\u003e on PDA medium did not reveal any discernible distinguishing characteristics with regard to their cultural characteristics (see Figure 2). Nevertheless, both species demonstrate a certain degree of variability in their cultural appearance\u003c/p\u003e","description":"","filename":"Fig.1Photoplate.png","url":"https://assets-eu.researchsquare.com/files/rs-7752155/v1/26e6e8b63043b73a50a718f2.png"},{"id":93608245,"identity":"748e7823-4883-4507-9e01-274481da3062","added_by":"auto","created_at":"2025-10-15 15:52:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4343081,"visible":true,"origin":"","legend":"\u003cp\u003eCultural characteristics of \u003cem\u003eB. rosacearum\u003c/em\u003e and \u003cem\u003eB. mediterranea\u003c/em\u003eisolated from different host species (\u003cem\u003eAbies grandis\u003c/em\u003e, \u003cem\u003ePseudotsuga menziesii\u003c/em\u003e, \u003cem\u003eQuercus petraea\u003c/em\u003e) on PDA medium\u003c/p\u003e","description":"","filename":"Fig.2cultureplate.png","url":"https://assets-eu.researchsquare.com/files/rs-7752155/v1/fc34de45f7ffdb4725289f17.png"},{"id":93608247,"identity":"ba8d58d2-a17f-4f73-9fab-47ac1bad9d35","added_by":"auto","created_at":"2025-10-15 15:52:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1868740,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogeny obtained by maximum likelihood analysis of the combined ITS, \u003cem\u003eTUB\u003c/em\u003e, and \u003cem\u003eACT\u003c/em\u003e sequence alignment of \u003cem\u003eBiscogniauxia\u003c/em\u003e strains (dataset 1). ML bootstrap support values above 70 % are shown at the nodes. \u003cem\u003eHypoxylon rubiginosum\u003c/em\u003e strain YMJ 24 is used as outgroup. Strain numbers of ex-type strains are emphasised with an asterisk (*). Strains analysed in this study are emphasised in bold. Branches that are crossed by diagonal lines are shortened by 50 %\u003c/p\u003e","description":"","filename":"Fig.3Multigenephylogeny.png","url":"https://assets-eu.researchsquare.com/files/rs-7752155/v1/81fd2d26978834806bb0cb0e.png"},{"id":93612241,"identity":"e1416ff3-4b66-4f59-af9c-a58511e77258","added_by":"auto","created_at":"2025-10-15 16:17:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2247195,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogeny obtained by maximum likelihood analysis of ITS sequence alignment of \u003cem\u003eBiscogniauxia\u003c/em\u003e strains (dataset 2). ML bootstrap support values above 70 % are shown at the nodes. Stains of \u003cem\u003eB. atropunctata \u003c/em\u003eand \u003cem\u003eB. latirima\u003c/em\u003e Y.M. Ju \u0026amp; J.D. Rogersare used as outgroup. For every strain clustered in the \u003cem\u003eB. rosacearum\u003c/em\u003e clade GenBank accession number, taxon designation as listed in GenBank (in bold), host or substrate (if available), country of isolation (if available), and strain number are given. Strain numbers of ex-type strains are emphasised with an asterisk (*). Branches that are crossed by diagonal lines are shortened by 50 %. Blue = strains of \u003cem\u003eB. rosacearum\u003c/em\u003e analysed intensively in Raimondo et al. (2016) and Sohrabi et al. (2022), and this study, respectively. Green = strains designated as \u003cem\u003eB. rosacearum\u003c/em\u003e in GenBank. hosNA = no host or substrate available, locNA = no country of isolation available\u003c/p\u003e","description":"","filename":"Fig.4ITSphylogenypart1.png","url":"https://assets-eu.researchsquare.com/files/rs-7752155/v1/67a7aeed06ad858cf8175581.png"},{"id":93608253,"identity":"85eb64bd-ba66-4155-bbb1-1915853647bd","added_by":"auto","created_at":"2025-10-15 15:53:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6648720,"visible":true,"origin":"","legend":"\u003cp\u003eAmplification plot from qPCR analysis for \u003cem\u003eBiscogniauxia mediterranea \u003c/em\u003ewith specific primer/probe combination. Analysis adapted from Luchi et al. (2005) including strains of \u0026nbsp;\u003cem\u003eB. mediterranea\u003c/em\u003eand \u003cem\u003eB. rosacearum\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.5qPCRanalysis.png","url":"https://assets-eu.researchsquare.com/files/rs-7752155/v1/0a6cba8620e367a03162e908.png"},{"id":93613234,"identity":"5c04d5dc-5d46-4792-999d-5205349019a4","added_by":"auto","created_at":"2025-10-15 16:25:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":25723400,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7752155/v1/ba431c61-eee7-46b4-b28f-5c91011470fd.pdf"},{"id":93608257,"identity":"0f2c79fc-bd21-4210-b02c-8d84bf6b958d","added_by":"auto","created_at":"2025-10-15 15:53:00","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":237696,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineResource1Brosacearum.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7752155/v1/feca286cd189b55cc7227f5f.pdf"}],"financialInterests":"","formattedTitle":"Biscogniauxia rosacearum, first evidence in Germany and pest risk analysis for the potentially quarantine relevant charcoal canker fungus","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe genus \u003cem\u003eBiscogniauxia\u003c/em\u003e Kuntze comprises approximately 90 species names (as of September 2025; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.indexfungorum.org\u003c/span\u003e\u003c/span\u003e, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.mycobank.org\u003c/span\u003e\u003c/span\u003e) and belongs to the family Graphostromataceae M.E. Barr, J.D. Rogers \u0026amp; Y.M. Ju, order Xylariales Nannf. within the phylum Ascomycota (Senanayake et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e; Daranagama et al. \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e; Hyde et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). The family furthermore includes the genera \u003cem\u003eCamillea\u003c/em\u003e Fr., \u003cem\u003eCryptostroma\u003c/em\u003e P.H. Greg. \u0026amp; S. Waller, \u003cem\u003eGraphostroma\u003c/em\u003e Piroz., \u003cem\u003eObolarina\u003c/em\u003e Pouzar, and \u003cem\u003eVivantia\u003c/em\u003e J.D. Rogers, Y.M. Ju \u0026amp; Cand. (Hyde et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e; Li et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e) (Hyde et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e; Li et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). The genus \u003cem\u003eBiscogniauxia\u003c/em\u003e was named by Carl Ernst Otto Kuntze in 1891 to honour the Belgian botanist C\u0026eacute;lestin Alfred Cogniaux (1841\u0026ndash;1916) (Kuntze \u003cspan class=\"CitationRef\"\u003e1891\u003c/span\u003e). Species of \u003cem\u003eBiscogniauxia\u003c/em\u003e have a worldwide distribution and have been recorded on a wide range of woody host plants (Bahmani et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). \u003cem\u003eBiscogniauxia\u003c/em\u003e species comprise pathogens and endophytes of angiosperms (Hyde et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e; Tropf et al. \u003cspan class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, they are also known as endophytes of the woody and foliar tissues of conifers. For instance, the type species \u003cem\u003eB. nummularia\u003c/em\u003e (Bull.) Kuntze and \u003cem\u003eB. mediterranea\u003c/em\u003e (De Not.) Kuntze have recently been found in association with Scots pine (\u003cem\u003ePinus sylvestris\u003c/em\u003e L.) and giant sequoia (\u003cem\u003eSequoiadendron giganteum\u003c/em\u003e (Lindl.) J. Buchholz) (Bu\u0026szlig;kamp et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e; Langer et al. \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e). As plant pathogens, \u003cem\u003eBiscogniauxia\u003c/em\u003e species are able to cause severe wood-decay and such events frequently occur in trees which are already significantly weakened for example by abiotic factors, such as intense drought (Ju et al. \u003cspan class=\"CitationRef\"\u003e1998\u003c/span\u003e). According to the phylogenetic analysis by Koukol et al. (\u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e), the anamorphic fungus \u003cem\u003eCryptostroma corticale\u003c/em\u003e (Ellis \u0026amp; Everh.) P. H. Greg. \u0026amp; H. Waller, which is the causal agent of Sooty bark disease (SBD), is closely related to \u003cem\u003eB. bartholomei\u003c/em\u003e (Peck) Lar. N. Vassiljeva and \u003cem\u003eG. platystomum\u003c/em\u003e (Schwein.) Piroz. According to Daranagama et al. (\u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e), the genus \u003cem\u003eBiscogniauxia\u003c/em\u003e seems to be paraphyletic within the Graphostromataceae. As indicated by Ju et al. (\u003cspan class=\"CitationRef\"\u003e1998\u003c/span\u003e), \u003cem\u003eBiscogniauxia\u003c/em\u003e species are well adapted to dry habitats, or at least, to habitats that are dry for part of the year. The mycelial development occurs inside the bark of the host plants and the fructifications, both anamorphic and teleomorphic, are produced superficially on the twigs, branches, and trunks of angiosperm plants (Hyde et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). In addition to teleomorphs, several species exhibit a periconiella- or nodulisporium-like anamorph (Hyde et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eAccording to the USDA database (Farr and Rossman \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e), 13 \u003cem\u003eBiscogniauxia\u003c/em\u003e species are known to occur in Europe. These include \u003cem\u003eB. anceps\u003c/em\u003e (Sacc.) J.D. Rogers, Y.M. Ju \u0026amp; Cand. (Rogers et al. \u003cspan class=\"CitationRef\"\u003e1996\u003c/span\u003e), B. \u003cem\u003ecapnodes\u003c/em\u003e (Berk.) Y.M. Ju \u0026amp; J.D. Rogers. (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.gbif.org/species/3461055\u003c/span\u003e\u003c/span\u003e), \u003cem\u003eB. cinereolilacina\u003c/em\u003e (J.H. Mill.) Pouzar (Pouzar \u003cspan class=\"CitationRef\"\u003e1979\u003c/span\u003e; Pouzar \u003cspan class=\"CitationRef\"\u003e1986\u003c/span\u003e), \u003cem\u003eB. communapertura\u003c/em\u003e Y.M. Ju \u0026amp; J.D. Rogers (Ju et al. \u003cspan class=\"CitationRef\"\u003e1998\u003c/span\u003e), B. \u003cem\u003edennisii\u003c/em\u003e (Pouzar) Pouzar (Pouzar \u003cspan class=\"CitationRef\"\u003e1979\u003c/span\u003e), \u003cem\u003eB. granmoi\u003c/em\u003e Lar.N. Vassiljeva (L\u0026aelig;ss\u0026oslash;e et al. \u003cspan class=\"CitationRef\"\u003e1999\u003c/span\u003e; Z\u0026iacute;barov\u0026aacute; and Kout \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e), \u003cem\u003eB. marginata\u003c/em\u003e (Fr.) Pouzar (Pouzar \u003cspan class=\"CitationRef\"\u003e1979\u003c/span\u003e; Pouzar \u003cspan class=\"CitationRef\"\u003e1986\u003c/span\u003e; Chlebicki and Bujakiewicz \u003cspan class=\"CitationRef\"\u003e1994\u003c/span\u003e), \u003cem\u003eB. mediterranea\u003c/em\u003e (Ju et al. \u003cspan class=\"CitationRef\"\u003e1998\u003c/span\u003e; Z\u0026iacute;barov\u0026aacute; and Kout \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e), \u003cem\u003eB. nummularia\u003c/em\u003e (Pouzar \u003cspan class=\"CitationRef\"\u003e1979\u003c/span\u003e; Pouzar \u003cspan class=\"CitationRef\"\u003e1986\u003c/span\u003e; L\u0026aelig;ss\u0026oslash;e et al. \u003cspan class=\"CitationRef\"\u003e1999\u003c/span\u003e), B. \u003cem\u003equerna\u003c/em\u003e Pouzar (Pouzar \u003cspan class=\"CitationRef\"\u003e1986\u003c/span\u003e), \u003cem\u003eB. repanda\u003c/em\u003e (Fr.) Kuntze (Pouzar \u003cspan class=\"CitationRef\"\u003e1979\u003c/span\u003e; Pouzar \u003cspan class=\"CitationRef\"\u003e1986\u003c/span\u003e; Chlebicki and Bujakiewicz \u003cspan class=\"CitationRef\"\u003e1994\u003c/span\u003e), \u003cem\u003eB. rosacearum\u003c/em\u003e M.L. Raimondo \u0026amp; Carlucci, and \u003cem\u003eB. simplicior\u003c/em\u003e Pouzar (Pouzar \u003cspan class=\"CitationRef\"\u003e1986\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eUntil recently, only four species were known to occur in Germany, namely \u003cem\u003eB. capnodes\u003c/em\u003e on a non-native \u003cem\u003eLatania\u003c/em\u003e sp., (Ju et al. \u003cspan class=\"CitationRef\"\u003e1998\u003c/span\u003e), B. \u003cem\u003emediterranea\u003c/em\u003e on oak (Ju et al. \u003cspan class=\"CitationRef\"\u003e1998\u003c/span\u003e; Bu\u0026szlig;kamp et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e; Langer and Bu\u0026szlig;kamp \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e; Langer and Bu\u0026szlig;kamp \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e; Bu\u0026szlig;kamp et al. \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e) and Scots pine (Bu\u0026szlig;kamp et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e), B. \u003cem\u003enummularia\u003c/em\u003e on \u003cem\u003eFagus sylvatica\u003c/em\u003e (Ju et al. \u003cspan class=\"CitationRef\"\u003e1998\u003c/span\u003e; L\u0026aelig;ss\u0026oslash;e et al. \u003cspan class=\"CitationRef\"\u003e1999\u003c/span\u003e; Tropf et al. \u003cspan class=\"CitationRef\"\u003e2025\u003c/span\u003e)d \u003cem\u003erepanda\u003c/em\u003e on the Betulaceae \u003cem\u003eAlnus glutinosa\u003c/em\u003e (L.) Gaertn. and \u003cem\u003eBetula pendula\u003c/em\u003e Roth (Schmid-Heckel \u003cspan class=\"CitationRef\"\u003e1988\u003c/span\u003e; Chlebicki and Bujakiewicz \u003cspan class=\"CitationRef\"\u003e1994\u003c/span\u003e). \u003cem\u003eBiscogniauxia nummularia, B. mediterranea\u003c/em\u003e, and \u003cem\u003eB. rosacearum\u003c/em\u003e can induce strip-cankers on their host trees, also referred to as charcoal canker or tarcrust (Hendry et al. \u003cspan class=\"CitationRef\"\u003e1998\u003c/span\u003e; Granata and Sidoti \u003cspan class=\"CitationRef\"\u003e2004\u003c/span\u003e; Nugent et al. \u003cspan class=\"CitationRef\"\u003e2005\u003c/span\u003e; Ragazzi et al. \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e; Henriques et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e; Luchi et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e; Raimondo et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e; Yangui et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). First visible signs of infection or parasitic growth are often bark blisters on the host tree (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ee), later on blackish, elongated bark lesions (strip cankers) on the trunks (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed) and branches appear and teleomorphs and/ or anamorphs were produced (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea-f). The parasitic growth of the three \u003cem\u003eBiscogniauxia\u003c/em\u003e species lead to wood decay often followed by green wood and brittle fracture (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eg).\u003c/p\u003e\n\u003cp\u003eThe occurrence of \u003cem\u003eB. mediterranea\u003c/em\u003e has been reported in Africa, Europe, North and Central America (Ju et al. \u003cspan class=\"CitationRef\"\u003e1998\u003c/span\u003e). It is widely distributed in Europe, where it is associated with oak (Ju et al. \u003cspan class=\"CitationRef\"\u003e1998\u003c/span\u003e) and the causal agent of charcoal canker on \u003cem\u003eQuercus suber\u003c/em\u003e L. (Fagaceae) in the Mediterranean basin (Hyde et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). It has been hypothesized that \u003cem\u003eB. mediterranea\u003c/em\u003e enters the host tree initially through the trunk and leaves (Gharbi et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e), where it can be recognised as an endophyte in apparently healthy plants (Tropf et al. \u003cspan class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, it can switch to its pathogenic lifestyle in water-stressed host plants (Edwards et al. \u003cspan class=\"CitationRef\"\u003e2003\u003c/span\u003e). \u003cem\u003eBiscogniauxia mediterranea\u003c/em\u003e has been detected frequently in various tree species ( Ju et al. \u003cspan class=\"CitationRef\"\u003e1998\u003c/span\u003e, Langer and Bu\u0026szlig;kamp \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e; Bu\u0026szlig;kamp et al. \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe latent pathogen \u003cem\u003eB. nummularia\u003c/em\u003e is considered to be one of the most frequently detected fungal endophytes in European beech (\u003cem\u003eFagus sylvatica\u003c/em\u003e L., Fagaceae) (Chapela and Boddy \u003cspan class=\"CitationRef\"\u003e1988\u003c/span\u003e; Nugent et al. \u003cspan class=\"CitationRef\"\u003e2005\u003c/span\u003e; Langer et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e; Langer and Bu\u0026szlig;kamp \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e; Tropf et al. \u003cspan class=\"CitationRef\"\u003e2025\u003c/span\u003e). When its host is stressed by drought and heat, the fungus can transit from an endophytic to a pathogenic lifestyle, causing various symptoms such as bark necrosis, bark blistering, bark ruptures, strip-cankers, and brittle fractures (Granata and Whalley \u003cspan class=\"CitationRef\"\u003e1994\u003c/span\u003e; Hendry et al. \u003cspan class=\"CitationRef\"\u003e1998\u003c/span\u003e; Nugent et al. \u003cspan class=\"CitationRef\"\u003e2005\u003c/span\u003e). \u003cem\u003eBiscogniauxia nummularia\u003c/em\u003e is the causal agent of beech bark tar crust disease and charcoal canker in \u003cem\u003eF. orientalis\u003c/em\u003e and \u003cem\u003eF. sylvatica\u003c/em\u003e (Vujanovic et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e; Patejuk et al. \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zamani et al. \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e). Additionally, it is one of the key fungal agents in beech decline (Granata and Whalley \u003cspan class=\"CitationRef\"\u003e1994\u003c/span\u003e; Granata and Sidoti \u003cspan class=\"CitationRef\"\u003e2004\u003c/span\u003e) and vitality loss of beech (Langer and Bu\u0026szlig;kamp \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e; Langer and Bu\u0026szlig;kamp \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e). \u003cem\u003eBiscogniauxia nummularia\u003c/em\u003e can form periconiella-like anamorphs in vitro and in planta, which produce powdery, light-coloured, globose conidia (Petrini and Petrini \u003cspan class=\"CitationRef\"\u003e1985\u003c/span\u003e; Langer \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e). Although \u003cem\u003eB. nummularia\u003c/em\u003e has been detected in many different angiosperms and gymnosperms (Petrini and Petrini \u003cspan class=\"CitationRef\"\u003e1985\u003c/span\u003e), to date, fruiting bodies and damage to trees have only been documented in \u003cem\u003eF. orientalis\u003c/em\u003e Lipsky and \u003cem\u003eF. sylvatica\u003c/em\u003e (Petrini and Petrini \u003cspan class=\"CitationRef\"\u003e1985\u003c/span\u003e; Nugent et al. \u003cspan class=\"CitationRef\"\u003e2005\u003c/span\u003e; Langer and Bu\u0026szlig;kamp \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zamani et al. \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBiscogniauxia rosacearum\u003c/em\u003e is presumably native to the Middle East, and has already been reported in the EU. For instance, it is the causal agent of the charcoal canker on species in the family Rosaceae Juss., including \u003cem\u003ePyrus communis\u003c/em\u003e L. (pear), \u003cem\u003ePrunus domestica\u003c/em\u003e L. (plum), and \u003cem\u003eCydonia oblonga\u003c/em\u003e Mill. (quince), first observed in Italy (Raimondo et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). Despite the fact that \u003cem\u003eB. rosacearum\u003c/em\u003e is of phytosanitary significance (EFSA 2022; JKI \u003cspan class=\"CitationRef\"\u003e2025\u003c/span\u003e), it is not currently listed as a quarantine pest or a regulated non-quarantine pest in the annexes to Regulation (EU) 2019/2072. Furthermore, it is not listed by the European and Mediterranean Plant Protection Organization (EPPO). Generally, \u003cem\u003eB. rosacearum\u003c/em\u003e infects a broad range of host plants, including almond (\u003cem\u003ePrunus dulcis\u003c/em\u003e (Mill.) D.A.Webb, Rosaceae), grapevine (\u003cem\u003eVitis vinifera\u003c/em\u003e L., Vitaceae), pear, plum, quince, strawberry tree (\u003cem\u003eArbutus unedo\u003c/em\u003e L., Ericaceae) and various oak species in the Mediterranean region, Middle East, and Africa (Raimondo et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e; Bahmani et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e; Sohrabi et al. \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e; Bashiri et al. \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e; Yangui et al. \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e). Recently, \u003cem\u003eB. rosacearum\u003c/em\u003e has been found for the first time in Germany on \u003cem\u003eAbies grandis\u003c/em\u003e (Douglas ex D.Don) Lindl. (Pinaceae) and on \u003cem\u003ePseudotsuga menziesii\u003c/em\u003e (Mirbel) Franco (Pinaceae). Therefore, an express pest risk analysis (Express-PRA) on \u003cem\u003eBiscogniauxia rosacearum\u003c/em\u003e was conducted and disseminated (JKI \u003cspan class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eBoth, \u003cem\u003eB. rosacearum\u003c/em\u003e and the closely related species \u003cem\u003eB. mediterranea\u003c/em\u003e are thought to have a high degree of intraspecific diversity (Henriques et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e; Bashiri et al. \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). Regarding \u003cem\u003eB. mediterranea\u003c/em\u003e, it is assumed that the epidemiology of the disease caused by this species may have changed. Its high genetic variability, even in small populations, is attributed to its heterothallic mating system, its high sexual reproduction rate, and the production of large quantities of ascospores (Vannini et al. \u003cspan class=\"CitationRef\"\u003e2008\u003c/span\u003e; Henriques et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). The same likely applies to \u003cem\u003eB. rosacearum\u003c/em\u003e. A previously unpublished phylogenetic analysis based on ITS sequences (JKI \u003cspan class=\"CitationRef\"\u003e2025\u003c/span\u003e) indicates that \u003cem\u003eB. rosacearum\u003c/em\u003e has a much broader host range and a more extensive geographical distribution than is currently recognised. This is largely related to the fact that in the past, some sequences belonging to \u003cem\u003eB. rosacearum\u003c/em\u003e have been incorrectly assigned to \u003cem\u003eB. mediterranea\u003c/em\u003e and deposited in sequence databases as such, due to inadequate taxonomical identification. Therefore, comparison of current sequence data with older entries can easily lead to confusion.\u003c/p\u003e\n\u003cp\u003eTherefore, the aims of this study were to\u003c/p\u003e\n\u003cp\u003e1) present additional information on \u003cem\u003eB. mediterranea\u003c/em\u003e,\u003cem\u003e\u0026nbsp;B. nummularia\u003c/em\u003e,\u003cem\u003e\u0026nbsp;\u003c/em\u003eand \u003cem\u003eB. rosacearum\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2) identify \u003cem\u003eB. rosacearum\u003c/em\u003e strains that may not have been identified initially and that were assigned to \u003cem\u003eBiscogniauxia\u003c/em\u003e in the strain collection of the Northwest German Forest Research Institute (NW-FVA),\u003c/p\u003e\n\u003cp\u003e3) identify potential\u0026nbsp;\u003cem\u003eB. rosacearum\u003c/em\u003e entries in the NCBI GenBank database that may not have been identified previously,\u003c/p\u003e\n\u003cp\u003e4) test and evaluate the species-specific primers for\u0026nbsp;\u003cem\u003eB. mediterranea\u0026nbsp;\u003c/em\u003epublished by\u003cem\u003e\u0026nbsp;\u003c/em\u003eLuchi et al. (2005),\u003c/p\u003e\n\u003cp\u003e5) \u0026nbsp; evaluate the host and distribution range of \u003cem\u003eB. rosacearum\u0026nbsp;\u003c/em\u003eso far as known,\u003cem\u003e\u0026nbsp;and\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e6) assess the phytosanitary risk of \u003cem\u003eB. rosacearum\u0026nbsp;\u003c/em\u003eby conducting a pest risk analysis (PRA) according to the standards of the International Plant Protection Convention (IPPC) and the European and Mediterranean Plant Protection Organization (EPPO).\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCases of disease and fungal isolation\u003c/h2\u003e\u003cp\u003eThe forest sites and cases of diseases associated with the incidences of \u003cem\u003eB. rosacearum\u003c/em\u003e in Germany were studied in 2021 and 2024 as part of routine forest protection consulting and causal analysis of forest damage by the Northwest German Forest Research Institute (NW-FVA). Two specimens of \u003cem\u003eB. rosacearum\u003c/em\u003e were isolated and identified according to the methods described in Langer and Bu\u0026szlig;kamp (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and Langer et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and stored in the NW-FVA strain collection.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eBiscogniauxia rosacearum\u003c/em\u003e-strain NW-FVA 6753 (=\u0026thinsp;isolate 2021-69-SB-Z1-112, NCBI Genbank Accession No. PX239669) was isolated from a twig (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) of a diseased, 48-year-old grand fir (\u003cem\u003eA. grandis\u003c/em\u003e) sampled on 16 June 2021 in a private forest close to Kirchlinteln, Lower Saxony, Germany. The affected tree, suffering from Fir bark disease (Langer and Rohde \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), was also infected among others with \u003cem\u003eB. nummularia, Diplodia mutila\u003c/em\u003e (Fr.) Fr., \u003cem\u003eD. sapinea\u003c/em\u003e (Fr.) P. Karst., \u003cem\u003eFusarium lateritium\u003c/em\u003e Nees, \u003cem\u003eNemania serpens\u003c/em\u003e (Pers.) Gray, \u003cem\u003eNeonectria neomacrospora\u003c/em\u003e (C. Booth \u0026amp; Samuels) Mantiri \u0026amp; Samuels, \u003cem\u003ePhomopsis\u003c/em\u003e sp., and \u003cem\u003eXylaria\u003c/em\u003e sp. Furthermore, \u003cem\u003eHeterobasidion annosum\u003c/em\u003e (Fr.) Bref. and \u003cem\u003eArmillaria\u003c/em\u003e root rot were detected.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eBiscogniauxia rosacearum\u003c/em\u003e-strain NW-FVA 13169\u0026thinsp;=\u0026thinsp;isolate 2024-73-\u0026Uuml;1\u0026ndash;2, Accession No. PX239675) was retrieved from a shoot from Douglas fir (\u003cem\u003eP. menziesii\u003c/em\u003e) aged 30 years, sampled on 27 August 2024 in a forest close to Genthin, Saxony-Anhalt, Germany. The studied tree suffered from Swiss needle cast (causal agent: \u003cem\u003eNothophaeocryptopus gaeumannii\u003c/em\u003e (T. Rohde) Videira, C. Nakash., U. Braun \u0026amp; Crous), \u003cem\u003eH. annosum\u003c/em\u003e root rot and infestation with Cooley spruce gall adelgid (\u003cem\u003eAdelges cooleyi\u003c/em\u003e (Gillette)) and weevils of the Curculionidae family. Beside a few other fungi, \u003cem\u003eB. nummularia\u003c/em\u003e and \u003cem\u003ePeniophora cinerea\u003c/em\u003e (Pers.) Cooke were additionally isolated from the studied Douglas fir tissue.\u003c/p\u003e\u003cp\u003eIn order to compare the cultural characteristics of the German \u003cem\u003eB. rosacearum\u003c/em\u003e strains with other similar species, strains of \u003cem\u003eB. mediterranea\u003c/em\u003e (NW-FVA 9136\u0026thinsp;=\u0026thinsp;Dgl-2022-2-Z2-68, Accession No. PX239670) isolated from \u003cem\u003eP. menziesii\u003c/em\u003e sampled on 9 June 2022 in Baden-Wuerttemberg close to Iffezheim (Langer et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), and NW-FVA 11840\u0026thinsp;=\u0026thinsp;2023-110-1-\u0026Uuml;3-37b, Accession No. PX239672) isolated from \u003cem\u003eQuercus petraea\u003c/em\u003e (Mattuschka) Liebl., sampled on 11 October 2023 in Hesse) were inoculated onto PDA agar, observed and documented with culture photos after 7, 14, 21, and 28 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The \u003cem\u003eB. mediterranea\u003c/em\u003e teleomorph of the specimen 2025-72-3 (strain NW-FVA 13980, Accession No. PX239676, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) was isolated from \u003cem\u003eF. sylvatica\u003c/em\u003e sampled by J. Bu\u0026szlig;kamp on 21 May 2025 in Germany, in the Harz mountains close to Wernigerode.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMolecular Identification and phylogenetic analysis\u003c/h3\u003e\n\u003cp\u003eExtraction of genomic DNA from all isolated strains was conducted following the method described in Tropf et al. (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe 5.8S nuclear ribosomal gene with the two flanking internal transcribed spacers ITS-1 and ITS-2 (ITS) was amplified using the primer pair ITS-1F\u0026thinsp;+\u0026thinsp;ITS4 (White et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). Additionally, partial sequences of the β-tubulin gene (\u003cem\u003eTUB\u003c/em\u003e) and the actin gene (\u003cem\u003eACT\u003c/em\u003e) were generated using the primer pairs T1\u0026thinsp;+\u0026thinsp;T2 (O\u0026rsquo;Donnell and Cigelnik \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1997\u003c/span\u003e) and ACT-512F\u0026thinsp;+\u0026thinsp;ACT-783R (Carbone and Kohn \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), respectively. The mixture for all PCR reactions consisted of 2.5 \u0026micro;l 10\u0026times;PCR reaction buffer (with 20 mmol/l MgCl\u003csub\u003e2\u003c/sub\u003e, Carl Roth, Karlsruhe, Germany), 1 \u0026micro;l of each primer (10 mmol/l), 2.5 \u0026micro;l MgCl\u003csub\u003e2\u003c/sub\u003e (25 mmol/l), 0.1 \u0026micro;l Roti\u0026reg;-Pol Taq HY Taq polymerase (Carl Roth, Karlsruhe, Germany), 2.5 \u0026micro;l of 2 mmol/l dNTPs (Biozym Scientific GmbH, Hessisch Oldendorf, Germany), and 1 \u0026micro;l of extracted DNA solution. Each reaction was topped up to a volume of 20 \u0026micro;l by adding HPLC grade water (Carl Roth, Karlsruhe, Germany). A GeneExplorer 96 (Hangzhou BIOER Technology, Hangzhou, China) was used to carry out the DNA amplifications. PCR conditions for ITS and \u003cem\u003eACT\u003c/em\u003e followed Bien et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and Raimondo et al. (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), respectively. The PCR conditions for the \u003cem\u003eTUB\u003c/em\u003e region were as follows: initial denaturation at 95\u0026deg;C for 5 min, followed by 35 cycles of 95\u0026deg;C for 30 s, 58\u0026deg;C for 30 s and 72\u0026deg;C for 60 s, with a 10 min extension at 72\u0026deg;C on the final cycle. PCR products were visualised in 1% agarose gel, and sent to Eurofins Scientific Laboratory (Ebersberg, Germany) for sequencing. Consensus sequences were generated from all the resulting sequences, which were then visually checked and edited as necessary using the BioEdit Sequence Alignment Editor (v. 7.2.5; Hall (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1999\u003c/span\u003e)). Sequences were submitted to GenBank (Sayers et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), see also Online Resource 1.\u003c/p\u003e\u003cp\u003eFor the phylogenetic analysis, two datasets were compiled. Dataset 1 consists of sequence data from three loci (ITS, \u003cem\u003eTUB\u003c/em\u003e, \u003cem\u003eACT\u003c/em\u003e; if available) from strains of \u003cem\u003eBiscogniauxia\u003c/em\u003e isolated in this study and further sequences retrieved from the NCBI GenBank including those of ex-type strains, following the data composition used in the phylogenetic analyses of Raimondo et al. (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and Qiao et al. (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). For dataset 2, ITS sequence of \u003cem\u003eB. rosacearum\u003c/em\u003e ex-type strain CBS 141046 (GenBank Accession No. KT253493) was used for a Blastn search on NCBI GenBank on April 11, 2025. Of the higher-matching sequences identified, those with a percentage identity value\u0026thinsp;\u0026gt;\u0026thinsp;98% were used together with ITS sequences from \u003cem\u003eB. mediterranea\u003c/em\u003e and \u003cem\u003eB. rosacearum\u003c/em\u003e strains isolated in this study and the ones reported in the study by Luchi et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) and Raimondo et al. (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Sequence data from both datasets and from all single loci were aligned automatically using MAFFT v. 7.490 (Katoh et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Katoh and Standley \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) as implemented in Geneious R11 (Kearse et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), and manually adjusted if necessary.\u003c/p\u003e\u003cp\u003eThe multilocus (ML analyses for dataset 1 (separately for all single loci and the concatenated dataset) and dataset 2 were performed by RAxML v. 8.2.11 (Stamatakis \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Stamatakis \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) as implemented in Geneious Prime\u0026reg; 2025.0.3 (Kearse et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) using the GTRGAMMA model with the rapid bootstrapping and search for best scoring ML tree algorithm including 1000 bootstrap replicates.\u003c/p\u003e\n\u003ch3\u003eqPCR Analysis\u003c/h3\u003e\n\u003cp\u003eDNA extracts from \u003cem\u003eB. rosacearum\u003c/em\u003e strains NW-FVA 6753 and NW-FVA 13169 as well as from \u003cem\u003eB. mediterranea\u003c/em\u003e strains NW-FVA 6717, NW-FVA 9136, NW-FVA 10508, NW-FVA 11840, NW-FVA 12572, and NW-FVA 13121 were used for qPCR following Luchi et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) using the primer and probe combination specific for \u003cem\u003eB. mediterranea\u003c/em\u003e presented therein. DNA extracts from all samples were brought to final DNA concentration of 80 ng/\u0026micro;l. qPCR was performed in a StepOnePlus\u0026trade; PCR System (Applied Biosystems, Waltham, MA, USA). The reaction was performed in a final volume of 20 \u0026micro;l. qPCR mixture contained 300 nmol/l each forward primer and reverse primer, 200 nmol/l fluorogenic probe, 1x qPCR Blue Probe Mix and 50 \u0026micro;M ROX Additive (Biozym Scientific GmbH, Hessisch-Oldendorf, Germany), and 1 \u0026micro;l template DNA. Each DNA sample was assayed in four replicates. Four wells containing 1 \u0026micro;l HPLC grade water each instead of DNA sample as \u0026lsquo;no-template control\u0026rsquo;. The PCR protocol according to Luchi et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) was followed.\u003c/p\u003e\n\u003ch3\u003ePest Risk Analysis (PRA)\u003c/h3\u003e\n\u003cp\u003eTo identify the risk of introducing the fungus into new areas, \u003cem\u003ei.e.\u003c/em\u003e the potential of its entry and establishment, as well as its risk of spread and the potential impacts, an Express-PRA for the PRA area, \u003cem\u003ei.e.\u003c/em\u003e EU member states, in particular Germany, was conducted as described in Schrader et al. (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In case the outcome of the PRA indicates that the pest is a potential quarantine pest, phytosanitary measures have to be applied (Schrader et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The PRA followed the steps according to the standards of the IPPC (IPPC \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) and the European and Mediterranean Plant Protection Organization (EPPO) (EPPO \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). PRA and the different aspects to be assessed are also regulated by the European Union (EU) (EU \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), in line with IPPC and EPPO. The PRA describes the biology of the fungus, as well as its geographical distribution and areas infested. It further assesses its host plants and their presence in the PRA area, \u003cem\u003ei.e.\u003c/em\u003e Germany and the EU. In addition, the PRA includes information about the symptoms of the pathogen, a discussion, whether climate in its area of distribution is comparable to the PRA area, and identifies potential pathways of introduction and spread. An important aspect is the assessment of known impacts in infested areas and in areas potentially being infested. Detection and diagnostics are briefly described as well. Finally, it is discussed, whether an infestation could easily be eradicated.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eAdditional information on B. mediterranea\u003c/b\u003e, \u003cb\u003eB. nummularia\u003c/b\u003e, \u003cb\u003eand B. rosacearum\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTypical habitus of a \u003cem\u003eBiscogniauxia\u003c/em\u003e teleomorph (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-d) and anamorph (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee-f) and some of the typically caused symptoms (e.g. strip-canker Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed), as well as the studied material of the first evidences from \u003cem\u003eB. rosacearum\u003c/em\u003e are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh-l. Stroma of \u003cem\u003eB. nummularia\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee) were produced as a cushion-like fungal mass under the bark (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef), visible as bark blisters. The pressure from the stroma causes the bark to peel or strip off in patches or strips (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee-g). Wood-decay by \u003cem\u003eBiscogniauxia\u003c/em\u003e species leads to brittle fracture (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eMolecular Identification and phylogenetic analysis\u003c/h2\u003e\u003cp\u003eExcept from two strains isolated and identified in the study, no other \u003cem\u003eBiscogniauxia\u003c/em\u003e of the strain NW-FVA-collection could be assigned to \u003cem\u003eB. rosacearum.\u003c/em\u003e The combined sequence dataset over all DNA loci analyzed (dataset 1) using ML consisted of 76 isolates including the outgroup and comprised 2139 characters. The gene boundaries in the concatenated multi-locus alignment were as follows: ITS: 1\u0026ndash;878, \u003cem\u003eTUB\u003c/em\u003e: 879\u0026ndash;1829, \u003cem\u003eACT\u003c/em\u003e: 1830\u0026ndash;2139. The analyses of all single loci datasets (not shown) as well as the concatenated dataset revealed phylogenetic trees of highly similar topology. The ML phylogeny of the multi locus dataset including ML bootstrap values is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Strains of \u003cem\u003eB. mediterranea\u003c/em\u003e and \u003cem\u003eB. rosacearum\u003c/em\u003e form well-supported, closely related but distinctly separate monophyletic clades, respectively. Six of the German \u003cem\u003eBiscogniauxia\u003c/em\u003e isolates (NW-FVA 10508, NW-FVA 9136, NW-FVA 12572, NW-FVA 6717, NW-FVA 13121 and NW-FVA 11840) have been shown to cluster in the \u003cem\u003eB. mediterranea\u003c/em\u003e clade, and two isolates (NW-FVA 6753 and NW-FVA 13169) cluster in the \u003cem\u003eB. rosacearum\u003c/em\u003e clade.\u003c/p\u003e\u003cp\u003eThe sequence dataset 2 of ITS sequences primarily retrieved from GenBank after Blast search with \u003cem\u003eB. rosacearum\u003c/em\u003e consisted of 129 isolates including the outgroup and comprised 517 characters. The ML phylogeny of dataset including ML bootstrap values is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Apart from the strains used as outgroup and strains used as reference strains of \u003cem\u003eBiscogniauxia mediterranea\u003c/em\u003e all strains retrieved from GenBank cluster in a well-supported clade together with strains of \u003cem\u003eB. rosacearum\u003c/em\u003e analyzed in depth in Raimondo et al. (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and Sohrabi et al. (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and strains isolated in this study. The majority of strains retrieved from GenBank which should be assigned to \u003cem\u003eB. rosacearum\u003c/em\u003e according to our analysis were designated as \u003cem\u003eB. mediterranea\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eqPCR Analysis\u003c/h3\u003e\n\u003cp\u003eqPCR revealed positive amplification curves for all tested \u003cem\u003eB. rosacearum\u003c/em\u003e strains (NW-FVA 6753: Ct mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u0026thinsp;=\u0026thinsp;17,92\u0026thinsp;\u0026plusmn;\u0026thinsp;0,06, ΔRn mean\u0026thinsp;=\u0026thinsp;5,119; NW-FVA 13169: Ct mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u0026thinsp;=\u0026thinsp;17,34\u0026thinsp;\u0026plusmn;\u0026thinsp;0,03, ΔRn mean\u0026thinsp;=\u0026thinsp;5,222). No signal was observed for wells containing sample DNA of \u003cem\u003eB. mediterranea\u003c/em\u003e strains and NTCs. An amplification plot is displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003ePest Risk Analysis\u003c/h3\u003e\n\u003cp\u003eBased on the findings of this study and the literature review on \u003cem\u003eBiscogniauxia\u003c/em\u003e, in accordance with the procedure described in Material and Methods, the following results have been obtained:\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eBiology of the organism\u003c/h2\u003e\u003cp\u003eFungi from the genus \u003cem\u003eBiscogniauxia\u003c/em\u003e are generally considered opportunistic pathogens that occur endophytically in the sapwood and bark of healthy trees without causing any obvious disease symptoms. However, they can cause significant damage to severely weakened or damaged trees. Drought conditions in the Mediterranean region have been found to influence the aggressiveness of \u003cem\u003eB. rosacearum\u003c/em\u003e and can lead to the outbreak of the disease (van Dyk et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The fungus forms a hard mycelial mat (stroma) to multiply and spread to new host plants. \u003cem\u003eBiscogniauxia rosacearum\u003c/em\u003e has a high reproduction rate and is heterothallic, \u003cem\u003ei.e.\u003c/em\u003e the mycelia are self-incompatible or self-sterile. Initially, powdery, light-coloured, asexual spores (conidia) are produced. A specific vector is not mentioned in the literature. According to Raimondo et al. (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), the fungus is primarily dispersed by wind or insects; occasionally also by other animals or by water. In a later stage, the fungus produces black sexual spores (ascospores), which are also transported by wind, water, or animals. While conidia are typically formed in spring or early summer, sexual spores are found in summer and fall. Both spore types can cause infections (Raimondo et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Crocker et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eGeographical distribution of the fungus and areas infested\u003c/h2\u003e\u003cp\u003eApart from the two findings in Germany, the fungus has been detected in Iran, Italy, Portugal, Spain, South Africa, and Tunisia (Raimondo et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Pinna et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Spies et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Bahmani et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; van Dyk et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Sohrabi et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Bashiri et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Yangui et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, the phylogenetic analysis based on ITS sequences retrieved from GenBank presented here suggests a much larger distribution area (North Africa, Middle East, Southern to Central Europe, China, USA, see Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). So far, occurrences in the EU have been confirmed only sporadically. Surveys would be necessary to determine the actual distribution status.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eHost plants and their presence in the PRA area\u003c/h2\u003e\u003cp\u003eTo date, several host plants are known. \u003cem\u003eBiscogniauxia rosacearum\u003c/em\u003e was found to be endophytic, parasitic, saprobic, and also associated with woody tissues damaged by other pathogens:\u003c/p\u003e\u003cp\u003eIn Italy, \u003cem\u003eB. rosacearum\u003c/em\u003e was found on 13- to 20-year-old pear trees (\u003cem\u003ePyrus communis\u003c/em\u003e), plum (\u003cem\u003ePrunus domestica\u003c/em\u003e), quince (\u003cem\u003eCydonia oblonga\u003c/em\u003e), and downy oak (\u003cem\u003eQuercus pubescens\u003c/em\u003e Willd.) in Apulia (Raimondo et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Additionally, it has also been isolated from insect boreholes in oak trees in Sardinia (Pinna et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In Iran, the fungus has been detected on \u003cem\u003eVitis vinifera\u003c/em\u003e (confirmed by greenhouse pathogenicity tests), as well as on \u003cem\u003eQuercus castaneifolia\u003c/em\u003e C.A.Mey., \u003cem\u003eQ. infectoria\u003c/em\u003e Oliv., \u003cem\u003eQ. libani\u003c/em\u003e Oliv., \u003cem\u003eQ. brantii\u003c/em\u003e Lindl., and \u003cem\u003ePrunus dulcis\u003c/em\u003e (Bahmani et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Sohrabi et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Bashiri et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Studies of oak dieback in the Iranian forests of the Zagros Mountains (Bashiri and Abdollahzadeh \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) revealed that \u003cem\u003eB. rosacearum\u003c/em\u003e was one of the most frequently detected fungi associated with oak dieback, alongside \u003cem\u003eObolarina persica\u003c/em\u003e Mirab., Y.M. Ju, H.M. Hsieh \u0026amp; J.D. Rogers (wrongly addressed as \u003cem\u003eBiscogniauxia persica\u003c/em\u003e), \u003cem\u003eCytospora hedjaroudei\u003c/em\u003e Bashiri \u0026amp; Abdollahz., \u003cem\u003eC. zagrosensis\u003c/em\u003e Bashiri \u0026amp; Abdollahz., \u003cem\u003eNeocosmospora metavorans\u003c/em\u003e (Al-Hatmi, S.A. Ahmed \u0026amp; de Hoog) Sand.-Den. \u0026amp; Crous, and \u003cem\u003eNeocosmospora\u003c/em\u003e sp. In Tunisia, \u003cem\u003eB. rosacearum\u003c/em\u003e was detected on \u003cem\u003eArbutus unedo\u003c/em\u003e in 2020 (Yangui et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and on \u003cem\u003eMyrtus communis\u003c/em\u003e L. in co-infection with \u003cem\u003eDiaporthe foeniculina\u003c/em\u003e (Sacc.) Udayanga \u0026amp; Castl. (Khadraoui et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In South Africa, \u003cem\u003eB. rosacearum\u003c/em\u003e was associated with olive (\u003cem\u003eOlea europaea\u003c/em\u003e L.) trunk disease symptoms (Spies et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; van Dyk et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eResults of phylogenetic studies conducted by Raimondo et al. (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) also included \u003cem\u003ePlatypus cylindrus\u003c/em\u003e (Fabricius, J.C., 1792), arthropods (Portugal), \u003cem\u003eHolcus lanatus\u003c/em\u003e L., \u003cem\u003eQuercus ilex\u003c/em\u003e L., and \u003cem\u003ePinus sylvestris\u003c/em\u003e (Spain) as hosts. The European Food Safety Authority (EFSA) (EFSA 2022) also mentions \u003cem\u003eP. sylvestris\u003c/em\u003e as a host plant, but no further evidence is found for this.\u003c/p\u003e\u003cp\u003eIn Germany, \u003cem\u003eB. rosacearum\u003c/em\u003e was detected in two diseased trees in two federal states, Lower Saxony and Saxony-Anhalt. In 2021, a strain of \u003cem\u003eB. rosacearum\u003c/em\u003e was isolated from the shoot tissue of a 48-year-old grand fir (\u003cem\u003eAbies grandis\u003c/em\u003e) suffering from fir bark necrosis, which was also infected with \u003cem\u003eDiplodia sapinea\u003c/em\u003e, \u003cem\u003eD. mutila\u003c/em\u003e, and \u003cem\u003eNeonectria neomacrospora\u003c/em\u003e. In 2024, another strain was isolated together with a \u003cem\u003eConiochaeta\u003c/em\u003e sp. from dying shoots of a 30-year-old Douglas fir (\u003cem\u003ePseudotsuga menziesii\u003c/em\u003e). The affected Douglas fir also exhibited Swiss needle cast caused by the pathogen \u003cem\u003eNothophaeocryptopus gaeumannii\u003c/em\u003e, root rot caused by \u003cem\u003eHeterobasidion annosum\u003c/em\u003e, shoot dieback, nicking, and mealybug infestation (this study).\u003c/p\u003e\u003cp\u003eFruiting bodies (teleomorphs) and charcoal cankers have so far only been recorded on the following plant species: pears, plums and quinces (Raimondo et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), almond trees (Sohrabi et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), grapevine (Bahmani et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and oaks (Bashiri et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAgain, phylogenetic analysis of ITS Blast results suggests an even broader host range, including several additional \u003cem\u003eQuercus\u003c/em\u003e (\u003cem\u003eQ. cerris\u003c/em\u003e L., \u003cem\u003eQ. faginea\u003c/em\u003e Lam., \u003cem\u003eQ. ilex\u003c/em\u003e, \u003cem\u003eQ. robur\u003c/em\u003e L., \u003cem\u003eQ. suber\u003c/em\u003e L.) and \u003cem\u003ePinus\u003c/em\u003e (\u003cem\u003eP. mugo\u003c/em\u003e Turra, \u003cem\u003eP. nigra\u003c/em\u003e J.F.Arnold, \u003cem\u003eP. pinaster\u003c/em\u003e Ait.) species, and further woody and herbaceous plants. Most of the host plants, including pear, plum, quince, grapevine, Douglas fir, grand fir and various oak and pine species are economically and ecologically important plants used for agriculture and forestry in Germany, as well as in the EU.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eSymptoms\u003c/h2\u003e\u003cp\u003eIn pathogenicity tests on \u003cem\u003eVitis vinifera\u003c/em\u003e, disease symptoms such as yellowing and necrosis of the leaves were observed two weeks after inoculation under greenhouse conditions. Brown lesions spread on the stems above and below the inoculation site. Stem cross-sections showed wedge-shaped necrosis of the vascular tissue (Bahmani et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Charcoal-like cankers in the bark were observed on stems of pears, plums, and quinces (Raimondo et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eClimate in distribution area and PRA area\u003c/h2\u003e\u003cp\u003eSo far, the fungus has been reliably detected in Mediterranean regions and in South Africa, but also on two individual plants (grand fir and Douglas fir) in Lower Saxony and Saxony-Anhalt, showing that at least in Mediterranean EU member states, climate is matching, but possibly also in Central Europe due to the confirmed occurrence in Germany. In addition, analysis of the ITS sequence data of \u003cem\u003eBiscogniauxia\u003c/em\u003e strains provided in the NCBI GenBank also suggests a large distribution area based on unverified evidence from China and the USA (see ITS phylogram, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003ePathways and natural spread\u003c/h2\u003e\u003cp\u003ePlants for planting are considered the main pathway. According to Raimondo et al. (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), the fungus is naturally spread by wind or insects.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eKnown impacts in infested areas\u003c/h2\u003e\u003cp\u003e\u003cem\u003eBiscogniauxia\u003c/em\u003e species are primarily known as endophytes, secondary pathogens and facultative saprophytes. They can infect old and weakened host plants and persist as endophytes in the aboveground parts of oaks and other tree species. Furthermore, they may remain latent and only develop symptoms when the trees are stressed, for example by drought. \u003cem\u003eBiscogniauxia\u003c/em\u003e species are able to rapidly colonize xylem and bark tissues, and can cause necrosis and canker formation, in severe cases even leading to tree death. Also younger and healthier trees are increasingly being infected (Henriques et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Raimondo et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In the case of \u003cem\u003eB. rosacearum\u003c/em\u003e Bahmani et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and Masi et al. (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) have demonstrated that phytotoxins play a considerable role in the disease process.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eExpected (further) establishment and spread in Germany and the EU\u003c/h2\u003e\u003cp\u003eBased on the detection of \u003cem\u003eB. rosacearum\u003c/em\u003e in Lower Saxony and Saxony-Anhalt, further establishment of the fungus in Germany is to be expected, also because of its high reproduction rate and spread potential by wind and insects. Since \u003cem\u003eB. rosacearum\u003c/em\u003e occurs endophytically and has not been originally associated with damage in Germany (and thus does not attract attention), it may already be more widespread than previously thought.\u003c/p\u003e\u003cp\u003eThere is evidence (see ITS phylogram, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) that \u003cem\u003eB. rosacearum\u003c/em\u003e already occurs in some, primarily southern, EU Member States. Further establishment is expected as well in these areas, out of the reasons mentioned above.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eExpected impacts caused by the fungus in Germany and the EU\u003c/h2\u003e\u003cp\u003eIn climatically suitable areas, significant damage to the host plants is to be expected, especially if they are already weakened by drought or infestation with other pests. However, \u003cem\u003eB. rosacearum\u003c/em\u003e was probably not the cause of the observed damage in the two trees infected in Germany.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eDetection and diagnosis\u003c/h2\u003e\u003cp\u003e\u003cem\u003eBiscogniauxia rosacearum\u003c/em\u003e can be unambiguously identified using the ITS DNA region. This result was confirmed by multi-locus sequence analysis (MLSA). In the past, especially before its initial description by Raimondo et al. (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), the fungus was generally referred to as the closely related species \u003cem\u003eB. mediterranea\u003c/em\u003e and deposited accordingly in databases. Therefore, confusion can easily arise when comparing new sequence data with existing ITS database entries. Until recently, findings on genetic diversity or host range, for example, were assigned erroneously to \u003cem\u003eB. mediterranea\u003c/em\u003e based on incorrect species delimitation presumably due to these mentioned Blast inconsistencies (Yangui et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zamani et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Furthermore, in addition to species assignment based on ITS database comparisons, rapid detection using qPCR and the specific primer/probe combination presented by Luchi et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) can currently lead to misinterpretation of test results. The latter authors developed the specific primer/probe combination based on ITS data from strains that, according to current knowledge, can be assigned to \u003cem\u003eB. rosacearum\u003c/em\u003e. Accordingly, it was possible to demonstrate in this study that the specific primer/probe combination established by Luchi et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) does not amplify \u003cem\u003eB. mediterranea\u003c/em\u003e but \u003cem\u003eB. rosacearu\u003c/em\u003em. Raimondo et al. (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) provides specific identification guidelines but detection is difficult in asymptomatic but latently infected host plants. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows a synopsis of \u003cem\u003eBiscogniauxia mediterranea\u003c/em\u003e and \u003cem\u003erosacearum\u003c/em\u003e pure cultures that were cultivated for 7, 14, 21, and 28 days on PDA (potato dextrose agar).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eEradication of an infestation\u003c/h2\u003e\u003cp\u003eThere is no known way to control the fungus by fungicides. To date, the only effective control method is felling and destroying (burning) the infected host plants. However, this would only be useful if \u003cem\u003eB. rosacearum\u003c/em\u003e was not yet widespread. In principle, it would be necessary to identify infestation-free areas, including determining whether and to what extent the fungus occurs endophytically without symptoms. Depending on the results, preventative measures could then be taken to avoid or mitigate further spread of the fungus. If possible, in urban agricultural areas measures to improve tree health, such as reducing compaction, mulching, irrigation, and fertilization, should be taken. Visibly infected branches should be removed, both to reduce the amount of inoculum present and to prevent potentially dangerous tree breaks (Crocker et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSince occurrences of \u003cem\u003eB. rosacearum\u003c/em\u003e in the EU have so far only been confirmed sporadically, and there are only two isolated cases in Germany, further surveys would be necessary to determine the actual distribution status.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eRaimondo et al. (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) were able to show that the two species \u003cem\u003eB. mediterranea\u003c/em\u003e and \u003cem\u003eB. rosacearum\u003c/em\u003e can be distinguished by their micromorphological characteristics. As described by the latter authors, the species differ in the size of their asci and ascospores as well as in their anamorph type. \u003cem\u003eBiscogniauxia mediterranea\u003c/em\u003e forms a periconiella-like anamorph form, while \u003cem\u003eB. rosacearum\u003c/em\u003e forms a nodulisporium-like form. If it is not possible to examine these micromorphological characteristics and only pure cultures are available, it will not be possible to distinguish between the two species on the basis of their culture-based characteristics alone. Therefore, the addition of molecular information (ITS sequence data according to this study is sufficient) is essential for an unambiguous identification.\u003c/p\u003e\u003cp\u003eThe multi-locus sequence analysis (MLSA) using three target genes in combination with morphological comparison of reference material allowed to designate the two newly isolated cultures as \u003cem\u003eB. rosacearum\u003c/em\u003e and to identify false GenBank database entries that require an updated species annotation. Rapid advancements in (meta)barcoding of organisms in various environments not only require the application of modern molecular techniques, but also high-quality database entries containing reference sequence material to identify species accordingly. This of course has been under debate for various reasons and is not restricted to false entries due to lacking morphological identification but also due to duplicates, redundancies and errors in species determination algorithms (Chen et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Stein and Gailing \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Therefore, it is necessary to carefully interpret the obtained information and, preferably, verify the results with additional information and expertise beyond DNA sequences. Reliable species-level assignments require both, curated reference material and an accurate phylogeny based on molecular analyses, as described in this article.\u003c/p\u003e\u003cp\u003eIn conclusion, the study successfully achieved the aims outlined in the introduction. It was demonstrated that the fungal strains that could initially only be assigned to the \u003cem\u003eBiscogniauxia\u003c/em\u003e genus in reference to the NW-FVA strain collection could be identified as \u003cem\u003eB. rosacearum\u003c/em\u003e. It was shown that the species-specific primers for \u003cem\u003eB. mediterranea\u003c/em\u003e published by Luchi et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) can lead to misinterpretation of test results, since the specific primer/probe combination used amplifies \u003cem\u003eB. rosacearum\u003c/em\u003e instead of \u003cem\u003eB. mediterranea\u003c/em\u003e. As a result of this, it is now possible to identify \u003cem\u003eB. rosacearum\u003c/em\u003e entries in the NCBI GenBank database not having been identified previously.\u003c/p\u003e\u003cp\u003eIn the PRA, it could be shown that - with these new results for the identification and distinction of the two \u003cem\u003eBiscogniauxia\u003c/em\u003e species \u0026ndash; the distribution range of \u003cem\u003eB. rosacearum\u003c/em\u003e is much larger than previously known. Furthermore, in the PRA it is assumed that \u003cem\u003eB. rosacearum\u003c/em\u003e can establish in Germany due to suitable climatic conditions and the availability of potential host plants; (further) establishment in southern European and Mediterranean EU member states is to be expected. A more detailed assessment of climatic conditions required by the fungus to establish and spread, in combination with modelling its spread potential would therefore be useful to better estimate the risks for its host plants present in Europe.\u003c/p\u003e\u003cp\u003e\u003cem\u003eBiscogniauxia rosacearum\u003c/em\u003e poses a high damage potential for some Rosales and oaks, causing \"charcoal canker\". The development of \"charcoal canker\" in other host plants has not been observed to date, so that it would be important to investigate whether the Koch's postulates could be fulfilled for these species. In addition, insufficient information exists on the phylogenetic diversity and pathogenicity of different \u003cem\u003eB. rosacearum\u003c/em\u003e strains towards different host species. In other tree species, the fungus seems not to cause any damage, but only occurs endophytically or opportunistically. The confusion regarding the two species \u003cem\u003eB. rosacearum\u003c/em\u003e and \u003cem\u003eB. mediterranea\u003c/em\u003e further complicates matters, as there may be errors with regard to the assignments of host plants and impacts of the two different species and their current distribution. Due to these uncertainties, it was not possible to conclusively assess the phytosanitary risk of \u003cem\u003eB. rosacearum\u003c/em\u003e at this stage of the study. Therefore, there is an urgent need for further research, also to finally determine whether the fungus qualifies as a potential quarantine pest for the EU.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Gitta Langer, Steffen Bien and Gritta Schrader. The molecular analyses were performed by Steffen Bien. The first draft of the manuscript was written by Gritta Schrader, Steffen Bien, and Gitta Langer and all authors commented on previous versions of the manuscript. Clovis Douanla-Meli and\u0026nbsp;Bj\u0026ouml;rn Hoppe\u003csup\u003e\u0026nbsp;\u003c/sup\u003emade substantial contributions to the manuscript. All authors read and approved the final manuscript. Funding acquisition: Gitta Langer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conducted as part of the TroWaK project (\u0026ldquo;Trockenheitsrisiken im Wald unter Klimawandel\u0026rdquo;, \u0026ldquo;Drought risks in forests under climate change\u0026rdquo;) which receives funding via the Waldklimafonds (\u0026ldquo;world climate fund\u0026rdquo;, WKF) funded by the German Federal Ministry of Agriculture, Food and Regional Identity (BMLEH), formerly Federal Ministry of Food and Agriculture (BMEL) and the Federal Ministry for the Environment, Climate Action, Nature Conservation and Nuclear Safety (BMUKN), formerly Federal Ministry for the Environment, Nature Conservation, Nuclear Safety and Consumer Protection (BMUV) administrated by the Agency for Renewable Resources (FNR) under grant agreement No 2220WK92D4. Additional funding was provided by the Julius Kuehn Institute (JKI) Federal Research Centre for Cultivated Plants, Institute for National and International Plant Health.\u003c/p\u003e\u003cp\u003eConflicts of interest\u003c/p\u003e\n\u003cp\u003eThe author(s) declare that they do not have any conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBahmani Z, Abdollahzadeh J, Amini J, Evidente A (2021) \u003cem\u003eBiscogniauxia rosacearum\u003c/em\u003e the charcoal canker agent as a pathogen associated with grapevine trunk diseases in Zagros region of Iran. Scientific Reports :14098. https://doi.org/10.1038/s41598-021-93630-w\u003c/li\u003e\n\u003cli\u003eBashiri S, Abdollahzadeh J (2024) Taxonomy and pathogenicity of fungi associated with oak decline in northern and central Zagros forests of Iran with emphasis on coelomycetous species. Front Plant Sci 15:20yay. https://doi.org/10.3389/fpls.2024.1377441\u003c/li\u003e\n\u003cli\u003eBashiri S, Abdollahzadeh J, Evidente A (2022) Diagnosing and pathogenicity of \u003cem\u003eBiscogniauxia\u003c/em\u003e species, the causal agents of oak charcoal canker and decline in Zagros forests of Iran. J Plant Pathol 104(3):1011\u0026ndash;1025. https://doi.org/10.1007/s42161-022-01124-z\u003c/li\u003e\n\u003cli\u003eBien S, Kraus C, Damm U (2020) Novel collophorina-like genera and species from \u003cem\u003ePrunus\u003c/em\u003e trees and vineyards in Germany. Persoonia 45(1):46\u0026ndash;67. https://doi.org/10.3767/persoonia.2020.45.02\u003c/li\u003e\n\u003cli\u003eBu\u0026szlig;kamp J, Bien S, Neumann L, Blumenstein K, Terhonen E, Langer GJ (2024) Endophytic community in juvenile \u003cem\u003eAcer pseudoplatanus\u003c/em\u003e and pathogenicity of \u003cem\u003eCryptostroma corticale\u003c/em\u003e and other associated fungi under controlled conditions. J Plant Pathol 106(2):565\u0026ndash;577. https://doi.org/10.1007/s42161-023-01575-y\u003c/li\u003e\n\u003cli\u003eBu\u0026szlig;kamp J, Langer GJ, Langer EJ (2020) \u003cem\u003eSphaeropsis sapinea\u003c/em\u003e and fungal endophyte diversity in twigs of Scots pine (\u003cem\u003ePinus sylvestris\u003c/em\u003e) in Germany. Mycol Progress 19(9):985\u0026ndash;999. https://doi.org/10.1007/s11557-020-01617-0\u003c/li\u003e\n\u003cli\u003eCarbone I, Kohn LM (1999) A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 91(3):553\u0026ndash;556. https://doi.org/10.1080/00275514.1999.12061051\u003c/li\u003e\n\u003cli\u003eChapela IH, Boddy L (1988) Fungal colonization of attached beech branches II. Spatial and temporal organization of communities arising from latent invaders in bark and functional sapwood, under different moisture regimes. New Phytologist 110(1):47\u0026ndash;57. https://doi.org/10.1111/j.1469-8137.1988.tb00236.x\u003c/li\u003e\n\u003cli\u003eChen Q, Zobel J, Verspoor K (2017) Duplicates, redundancies and inconsistencies in the primary nucleotide databases: a descriptive study. Database (Oxford) 2017:baw163. https://doi.org/10.1093/database/baw163\u003c/li\u003e\n\u003cli\u003eChlebicki A, Bujakiewicz A (1994) \u003cem\u003eBiscogniauxia repanda\u003c/em\u003e, \u003cem\u003eB. marginata\u003c/em\u003e and \u003cem\u003eCamarops polysperma\u003c/em\u003e (Pyrenomycetes) in Poland and Lithuania. Acta Mycologica 29:53\u0026ndash;58. https://doi.org/10.5586/am.1994.006\u003c/li\u003e\n\u003cli\u003eCrocker E, Bordas A, Coyle D (2018) Biology, Ecology, and Management of \u003cem\u003eBiscogniauxia\u003c/em\u003e (\u003cem\u003eHypoxylon\u003c/em\u003e) Canker in the Southeastern U.S. SREF Factsheet, SREF-FH- 009. Forest Health Research and Education Center, Department of Forestry and Natural Resources, University of Kentucky, Lexington, USA\u003c/li\u003e\n\u003cli\u003eDaranagama D, Hyde K, Sir E, Thambugala K, Tian Q, Samarakoon MC, Mckenzie E, Jayasiri S, Tibpromma S, Bhat DJ, Liu X, Stadler M (2018) Towards a natural classification and backbone tree for Graphostromataceae, Hypoxylaceae, Lopadostomataceae and Xylariaceae. Fungal Diversity 88:1\u0026ndash;165. https://doi.org/10.1007/s13225-017-0388-y\u003c/li\u003e\n\u003cli\u003eEdwards, Jonglaekha, Kshirsagar, Maitland, Mekkamol, Nugent, Phosri C, Rodtong S, Ruchikachorn, Sangvichien E, George S, Sihanonth P, Suwannasai N, Thienhirun, Whalley A, Whalley MA (2003) The Xylariaceae as phytopathogens. Recent Research Developments in Plant Sciences 1:1\u0026ndash;19\u003c/li\u003e\n\u003cli\u003eEFSA (European Food Safety) (2022) Plant Health Newsletter on Horizon Scanning EN-7282. EFSA Supporting Publications 19(4, March 2022):31. https://doi.org/10.2903/sp.efsa.2022.EN-7282\u003c/li\u003e\n\u003cli\u003eEPPO (2024) EPPO Standards \u0026ndash; PM 5 Pest Risk Analysis\u003c/li\u003e\n\u003cli\u003eEU (2016) Regulation (EU) 2016/2031 of the European Parliament of the Council of 26 October 2016 on protective measures against pests of plants, amending Regulations (EU) No 228/2013, (EU) No 652/2014 and (EU) No 1143/2014 of the European Parliament and of the Council and repealing Council Directives 69/464/EEC, 74/647/EEC, 93/85/EEC, 98/57/EC, 2000/29/EC, 2006/91/EC and 2007/33/EC.\u003c/li\u003e\n\u003cli\u003eFarr DF, Rossman AY (2022) Fungal databases - fungus-host distributions. In: U.S. National Fungus Collections Fungal Database. https://nt.ars-grin.gov/fungaldatabases/fungushost/fungushost.cfm. 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Zust\u0026auml;ndige Mitarbeiter: Dr. Gritta Schrader, Dr. Bj\u0026ouml;rn Hoppe, Dr. Clovis Douanla-Meli; unter Mitwirkung von Dr. Steffen Bien und Dr. Gitta Langer, Nordwestdeutsche Forstliche Versuchsanstalt, G\u0026ouml;ttingen\u003c/li\u003e\n\u003cli\u003eJu Y, Rogers J, Mart\u0026iacute;n F, Granmo A (1998) The genus \u003cem\u003eBiscogniauxia\u003c/em\u003e. Mycotaxon (66):1\u0026ndash;98\u003c/li\u003e\n\u003cli\u003eKatoh K, Misawa K, Kuma K, Miyata T (2002) MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res 30(14):3059\u0026ndash;3066. https://doi.org/10.1093/nar/gkf436\u003c/li\u003e\n\u003cli\u003eKatoh K, Standley DM (2013) MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. 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Aufl. 1990. Nationalparkverwaltung, Berchtesgaden\u003c/li\u003e\n\u003cli\u003eSchrader G, Kehlenbeck H, Wilstermann A, Kaminski K (2024) Pest Risk Analysis in Germany over the course of time. Themenheft: Pflanzengesundheit 76(02):15\u0026ndash;23. https://doi.org/10.5073/JfK.2024.02.03\u003c/li\u003e\n\u003cli\u003eSenanayake I, Maharachchikumbura S, Hyde K, Bhat DJ, Jones E, Mckenzie E, Dai D-Q, Daranagama D, Dayarathne M, Goonasekara I, Konta S, Li W, Shang Q-J, Stadler M, Wijayawardene N, Xiao Y-P, Norphanphoun C, Li Q, Liu X, Erio C (2015) Towards unraveling relationships in Xylariomycetidae (Sordariomycetes). Fungal diversity 73:73-144. https://doi.org/10.1007/s13225-015-0340-y\u003c/li\u003e\n\u003cli\u003eSohrabi M, Mohammadi H, Armengol Fort\u0026iacute; J, Le\u0026oacute;n Santana M (2022) New report of \u003cem\u003eBiscogniauxia rosacearum\u003c/em\u003e as a pathogen on almond trees in Iran. 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PCR protocols: a guide to methods and applications 18(1):315\u0026ndash;322\u003c/li\u003e\n\u003cli\u003eYangui I, Hlaiem S, Ben Jam\u0026acirc;a ML, Vannini A, Vettraino AM, Messaoud C (2022) Difference in pathogenicity and genetic variability among isolates of \u003cem\u003eBiscogniauxia mediterranea\u003c/em\u003e from \u003cem\u003eQuercus suber\u003c/em\u003e in Tunisia. Biologia 77(7):1713\u0026ndash;1721. https://doi.org/10.1007/s11756-021-01003-5\u003c/li\u003e\n\u003cli\u003eYangui I, Hlaiem S, Khadraoui H, Messaoud C, Ben Jam\u0026acirc;a ML, Ezzine O (2024) \u003cem\u003eBiscogniauxia rosacearum\u003c/em\u003e: A newly identified pathogen of strawberry tree (\u003cem\u003eArbutus unedo\u003c/em\u003e L.) in North Africa. 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Trees 39(1):17. https://doi.org/10.1007/s00468-024-02585-8\u003c/li\u003e\n\u003cli\u003eZamani SM, Sepasi N, Afarin S, Ahangaran Y, Gholami Ghavam Abad R, Askary H (2024) Report of \u003cem\u003eBiscogniauxia nummularia\u003c/em\u003e as pathogenic agent of charcoal canker disease of beech (\u003cem\u003eFagus orientalis\u003c/em\u003e) from Iran. Iranian Journal of Forest and Range Protection Research 21(2):350\u0026ndash;358. https://doi.org/10.22092/ijfrpr.2024.364708.1611\u003c/li\u003e\n\u003cli\u003eZ\u0026iacute;barov\u0026aacute; L, Kout J (2017) Xylariaceous pyrenomycetes from Bohemia: Species of \u003cem\u003eBiscogniauxia\u003c/em\u003e and \u003cem\u003eHypoxylon\u003c/em\u003e new to the Czech Republic, and notes on other rare species. Czech Mycology 69:77\u0026ndash;108. https://doi.org/10.33585/cmy.69106\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-plant-diseases-and-protection","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpdp","sideBox":"Learn more about [Journal of Plant Diseases and Protection](https://www.springer.com/journal/41348)","snPcode":"41348","submissionUrl":"https://www.editorialmanager.com/jpdp","title":"Journal of Plant Diseases and Protection","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Graphostromataceae, wood-decay fungus, pest risk analysis, multigene phylogeny, tree pathogen","lastPublishedDoi":"10.21203/rs.3.rs-7752155/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7752155/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSince \u003cem\u003eBiscogniauxia rosacearum\u003c/em\u003e has been detected in Germany for the first time, a pest risk analysis (PRA) for this wood-decaying fungus with potential quarantine relevance is presented. This species which is known to be distributed throughout the Mediterranean region and presumably native to the Middle East, is usually found on Rosaceae and other deciduous trees. Two new host tree species were identified, \u003cem\u003eAbies grandis\u003c/em\u003e and \u003cem\u003ePseudotsuga menziesii\u003c/em\u003e. Furthermore, this ascomycete was compared to \u003cem\u003eBiscogniauxia mediterranea\u003c/em\u003e, which is prevalent in Germany, and was distinguished in a multigene phylogeny based on ITS, \u003cem\u003eTUB\u003c/em\u003e, and \u003cem\u003eACT\u003c/em\u003e sequence alignment. In addition, a qPCR -assay using a previously published species-specific primer combination for the detection of \u003cem\u003eB. mediterranea\u003c/em\u003e was tested on a selection of isolated \u003cem\u003eB. mediterranea\u003c/em\u003e and \u003cem\u003eB. rosacearum\u003c/em\u003e strains, and subsequently assigned to \u003cem\u003eB. rosacearum\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Biscogniauxia rosacearum, first evidence in Germany and pest risk analysis for the potentially quarantine relevant charcoal canker fungus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-15 15:52:55","doi":"10.21203/rs.3.rs-7752155/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2026-04-22T02:07:59+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-10-07T12:53:05+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-02T19:35:38+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Journal of Plant Diseases and Protection","date":"2025-10-02T11:28:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-01T19:26:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Plant Diseases and Protection","date":"2025-09-30T09:46:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-plant-diseases-and-protection","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpdp","sideBox":"Learn more about [Journal of Plant Diseases and Protection](https://www.springer.com/journal/41348)","snPcode":"41348","submissionUrl":"https://www.editorialmanager.com/jpdp","title":"Journal of Plant Diseases and Protection","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b4df54d2-edb1-4f5c-91d1-42122fcf72f1","owner":[],"postedDate":"October 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-04-22T06:13:31+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-15 15:52:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7752155","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7752155","identity":"rs-7752155","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}