Taxonomic revision of Bisifusarium (Nectriaceae)

© 2025 Westerdijk Fungal Biodiversity Institute You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at https://creativecommons.org/licenses/by-nc-nd/4.0/. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights. ISSN (Online) 1878-9080 https://doi.org/10.3114/persoonia.2025.54.06 Persoonia 54, 2025: 197–223 https://www.persoonia.org RESEARCH ARTICLE Taxonomic revision of Bisifusarium (Nectriaceae) K. Zhang1,2#*, M. Sandoval-Denis3#, H. Kandemir3, N. Yilmaz4, J.Z. Groenewald3, F. Roets5, M. de J. Yáñez-Morales6, M.J. Wingfield4, P.W. Crous3,4* 1Institute of Ecology and Biodiversity, School of Life Sciences, Shandong University, Qingdao, 266237, China 2Shandong Agriculture and Engineering University, Jinan, Shandong 250100, China 3Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands 4Department of Genetics, Biochemistry and Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0002, South Africa 5Department of Conservation Ecology and Entomology, Stellenbosch University, Private Bag X1, Matieland, 7602, South Africa 6Fitosanidad-Fitopatología, Colegio de Postgraduados, campus Montecillo, km 36.5 carretera México-Texcoco, Montecillo, Texcoco, Estado de México 56264, México #These authors contributed equally *Corresponding authors: K. Zhang, [email protected]; P.W. Crous, [email protected] Abstract: Species of Bisifusarium (previously the Fusarium dimerum species complex) have been associated with cheese fermentation and a wide range of opportunistic human infections, but they are generally regarded as saprotrophs. Bisifusarium spp. are also commonly isolated from soils and tissues of plants growing in arid climates. The genus is typically characterized by its distinct pionnotal growth in culture, and typically very short, 0–2(–3)-septate macroconidia, produced in sporodochia or on lateral phialidic hyphal pegs. Only 16 species of Bisifusarium have been described to date, and this study sought to re-evaluate these taxa by examining 116 Bisifusarium isolates from the culture collection of the Westerdijk Fungal Biodiversity Institute in Utrecht, The Netherlands. A multi-gene phylogenetic analysis using partial nucleotide sequences of the translation elongation factor 1-alpha (tef1), partial RNA polymerase II second largest subunit (rpb2), the 5.8S nrDNA with its flanking intergenic spacer regions (ITS), and partial β-tubulin (tub2) genes resolved 25 phylogenetic lineages. Further evaluation of culture and morphological characters, and host-substrates, confirmed eight of these clades as novel taxa that are formally described here. In addition, two putative novel species were identified but not described due to limited available data. We provide the morphological descriptions and photographic illustrations for B. hedylamarriae and B. lovelliae, which were formerly known only from their DNA data. This study significantly increases the number of species in Bisifusarium and provides a crucial foundation for future studies to elucidate the ecology and evolutionary relationships within this expanding genus. Citation: Zhang K, Sandoval-Denis M, Kandemir H, Yilmaz N, Groenewald JZ, Roets F, Yáñez-Morales M de J, Wingfield MJ, Crous PW (2025). Taxonomic revision of Bifusarium (Nectriaceae). Persoonia 54: 197–223. doi: 10.3114/persoonia.2025.54.06 Received: 1 April 2025; Accepted: 18 April 2025; Effectively published online: 14 May 2025 Corresponding editor: U. Braun Key words: cheese fermentation multi-locus new taxa opportunistic human infections systematics INTRODUCTION The Nectriaceae encompasses a vast number of fungal species including numerous important plant and human pathogens. The family currently accommodates 82 genera (Hyde et al. 2024), including Fusarium, which represents the fourth most cited genera in the literature (Bhunjun et al. 2024). Lombard et al. (2015) introduced a new nomenclature for the Nectriaceae, resulting in the description of several new genera based on morphological observations and a 10-gene phylogeny. A direct consequence of this new classification was that the Fusarium dimerum species complex was placed in the genus Bisifusarium. The name Bisifusarium refers to the macroconidia of species that mostly have 1–2-septa, in contrast to those of Fusarium s. str. where the macroconidia are multiseptate (Lombard et al. 2015). The genus Bisifusarium presently encompasses 16 species (Schroers et al. 2009, Lombard et al. 2015, Sun et al. 2017, Wang et al. 2022, 2024, Visagie et al. 2024), commonly found as saprotrophs, occurring in various habitats with very different intrinsic and extrinsic characteristics (Schroers et al. 2009, Lombard et al. 2015, Sun et al. 2017) (Fig. 1). For example, B. domesticum is of particular interest to food mycologists as it is used in the dairy industry for cheese production (Bachmann et al. 2003, 2005, Schroers et al. 2009, Ropars et al. 2012, Lombard et al. 2015). Bisifusarium biseptatum, B. delphinoides, B. nectrioides, B. penzigii and B. solicola are associated with soil (Schroers et al. 2009, Visagie et al. 2024) while B. tonghuanum was isolated from Atriplex centralasiatica and Suaeda salsa plants (Sun et al. 2017). Bisifusarium lunatum has mainly been isolated from Opuntia or Gymnocaycium cacti (Schroers et al. 2009), and could be a plant pathogen (Flores-Flores et al. 2013, Koch et al. 2015, Gryzenhout et Persoonia – Volume 54, 2025198 Fig. 1. Substrates associated with Bisifusarium spp. A. B. africanum from Encephalartos leaf. B. B. hedylamarriae on leaves of Aloe sp. C. B. cacticola on Opuntia ficus-indica. D. B. sechiicola on Sechium edule. E. B. colchici on Trachyandra sp. F. B. allantoides on Saint Nectaire cheese (photo credit: Monica Coton). G. B. solicola from soil adjacent to Agapanthus praecox. H. B. namibense from desert soil in Namibia. al. 2017). Bisifusarium delphinoides is also considered a possible plant pathogen, isolated from stem lesions of Hoodia gordonii and found in soil and other plant materials (Schroers et al. 2009, Kulkarni et al. 2013). Other taxa associated with plants include B. aseptatum from Orchidaceae (Wang et al. 2022), B. lovelliae from leaves of Epipremnum pinnatum (Tan & Shivas 2023a), and B. hedylamarriae from Sansevieria (Tan & Shivas 2023b). Savary et al. (2021) described B. allantoides and B. penicilloides associated with cheese, while B. keratinophilum was noted for its ability to degrade keratin from chicken feathers (Wang et al. 2024). Some Bisifusarium species such as B. penzigii and B. lunatum are considered as possible human pathogens, because they have been isolated from a corneal ulcer and human sinus, respectively (Schroers et al. 2009, do Carmo et al. 2016). Likewise, B. delphinoides was reported from human eye and skin infections (Schroers et al. 2009, Collado et al. 2013, Salah et al. 2015, Hassan et al. 2016, Park et Zhang et al.: Taxonomic revision of Bisifusarium 199 al. 2019). Bisifusarium dimerum, initially isolated from citrus (Citrus medica; Schroers et al. 2009), is also considered a human pathogen causing several human mycoses, mainly keratomycosis, but also disseminated infections in immunocompromised patients, endocarditis, endogenous endophthalmitis and onychomycosis (Vismer et al. 2002, Schroers et al. 2009, Hassan et al. 2016, Simon et al. 2018, de Hoog et al. 2020). Given the limited number of described species and the ecological significance of the genus, the aim of this study was to re-evaluate 116 isolates identified as Bisifusarium spp., or erroneously classified as Fusarium spp. preserved in the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands. These isolates were collected from more than 35 hosts/substrates in 24 countries. All isolates were treated or identified based on their morphology and analyses of multi-gene DNA sequence data. MATERIALS AND METHODS Isolates Fungal isolates were obtained from the CBS culture collection and research collections housed at the Westerdijk Institute. A total of 116 isolates (including some ex-type strains of previously described species) were studied based on their morphology and phylogenetic inference of DNA sequence data. DNA extraction, PCR amplification and sequencing Genomic DNA was extracted from fungal colonies growing on oatmeal agar (OA; Crous et al. 2019) for 7–14 d at room temperature using the Wizard® Genomic DNA purification Kit (Promega Corporation, Madison, WI, USA), following the manufacturer’s protocol. Four loci were amplified: the 5.8S nrDNA with its flanking intergenic spacer regions (ITS) region was amplified using the primer pair ITS5 / ITS4 (White et al. 1990); the partial RNA polymerase II second largest subunit (rpb2), translation elongation factor 1-alpha (tef1) and β -tubulin (tub2) genes were amplified using the primer pairs RPB2-5F2 / RPB2-7CR (Liu et al. 1999, Sung et al. 2007), EF1 / EF2 (O’Donnell et al. 1998) and T1 / TUB4Rd (O’Donnell & Cigelnik 1997, Woudenberg et al. 2009), respectively. For sequencing, the same primer sets were used. The consensus sequences of each isolate were assembled from forward and reverse sequences using Geneious Prime v. 2022 (Biomatters Inc., New Zealand). Newly generated sequences and those retrieved from GenBank (http://www.ncbi.nlm.nih. gov) are shown in Table 1. Phylogenetic analyses Individual alignments for the four loci (ITS, rpb2, tef1, and tub2) were generated with MAFFT v. 7 using the default settings on the web server of the European Bioinformatics Institute (EMBL-EBI) (http://www.ebi.ac.uk/Tools/msa/ mafft/) (Katoh et al. 2019, Li et al. 2015). These alignments were manually edited in MEGA v. 7.0.21 when necessary (Tamura et al. 2013). Maximum-likelihood (ML) and Bayesian inference analyses (BI) were used for phylogenetic inferences of individual sequence alignments and the concatenated 4-gene dataset. The ML analyses were conducted using IQ-TREE v. 2.1.2 (Nguyen et al. 2015, Minh et al. 2020) with evolutionary models for each partition estimated using ModelFinder (Kalyaanamoorthy et al. 2017, Minh et al. 2020) as implemented in IQ-TREE. Estimation of branch support was carried out by 1000 replicates of ultrafast bootstrapping (UFBoot2; Hoang et al. 2018). Additional ML analyses were performed in raxmlGUI v. 2.0 (Edler et al. 2021), with evolutionary models calculated using ModelTest-NG (Darriba et al. 2020), followed by 1000 replicates of non-parametric bootstrapping. The BI analyses were run on the CIPRES Science Gateway portal (https://www.phylo.org/; Miller et al. 2012) using MrBayes v. 3.2.7a (Ronquist et al. 2012). Four simultaneous Markov chains were run for 10 M generations with the stopval argument on and set to an average standard deviation of split frequencies below 0.01. Trees were sampled every 1000 generations, and the initial 25 % of sampled trees were discarded as the burn-in fraction. The remaining trees were used to calculate the posterior probabilities (PP). The resulting trees for all analyses were plotted using FigTree v. 1.4.2 (http://tree.bio.ed.ac.uk/ software/figtree) and edited using Adobe Illustrator v. 28.2. Alignments and phylogenetic trees derived from this study were uploaded to figshare (www. figshare.com; DOI: 10.6084/m9.figshare.28816022). Morphology Fungal colonies were grown on oatmeal agar (OA), potato dextrose agar (PDA), synthetic nutrient-poor agar (SNA) and carnation leaf agar (CLA) (recipes in Crous et al. 2019). Colony diameters were measured after incubation in the dark for 7 d at 25 °C. Colony colours (upper surface and reverse) were described using the colour charts of Rayner (1970). Micromorphological characters were recorded from 7–14-d-old colonies on OA, SNA or CLA incubated under near-UV light at 24 °C, with structures mounted in sterile distilled water. Observations of micro-morphological characteristics were processed with a Nikon Eclipse 80i compound microscope with differential interference contrast (DIC) optics and a Nikon AZ100 dissecting microscope. Photomicrographs and measurements were made with a Nikon DS-Ri2 high-definition colour digital camera using the software NIS-elements D v. 4.50 (Nikon, Tokyo, Japan). All descriptions, illustrations and nomenclatural data were deposited in MycoBank (www. MycoBank.org; Crous et al. 2004). RESULTS Phylogenetic analyses The combined ITS, rpb2, tef1, and tub2 dataset included 403 sequences from 116 isolates, including two outgroup taxa (the ex-type strains of Rectifusarium robinianum CBS 430.91, and R. ventricosum CBS 748.79) selected according to previous studies (Crous et al. 2021, Visagie et al. 2024). The alignment included a total of 2516 sites, including alignment gaps (ITS 473, rpb2 763, tef1 775, and tub2 505) of which 1499 were invariable (ITS 344, rpb2 432, tef1 405, and tub2 318), 1181 Persoonia – Volume 54, 2025200 Table 1. Metadata and GenBank accession numbers of the the strains included in the phylogenetic analyses. Species Strain1Origin Substrate/host GenBank accession number ITS rpb2 tef1 tub2 Bisifusarium africanum CBS 153387 = CPC 34902 ex-type South Africa Encephalartos sp. leaf PV158417 PV167580 PV167641 PV167670 Bisifusarium allantoides CBS 110147 = BBA 70993 Germany Citrus sp. PV158418 PV167581 — — CBS 147587 = UBOCC-A-120036 ex-type France Cheese rind MW654548 MW811072 MW811087 MW811102 UBOCC-A-120037 France Cheese rind MW654549 MW811073 MW811088 MW811103 UBOCC-A-120035 Spain Cheese MW654536 MW811060 MW811075 MW811090 Bisifusarium aseptatum CGMCC 3.20816 ex-type China Orchidaceae sp. MW016389 MW474375 MW580429 MW533716 LC13608 China Orchidaceae sp. MW016391 MW474377 MW580431 MW533718 Bisifusarium australianum CBS 366.73 = ATCC 16553 = ATCC 24368 = IMI 117087b = NRRL 20712 ex-type Australia Poultry feed PV158419 PV167582 PV167642 — Bisifusarium biseptatum CBS 110138 = FRC E-0127 = NRRL 36158 South Africa Soil EU926251 PV167583 EU926318 EU926384 CBS 110144 = FRC E-0282 = NRRL 36164 South Africa Soil EU926253 PV167584 EU926320 EU926386 CBS 110146 = FRC E-0292 = NRRL 36166 South Africa Soil EU926254 PV167585 EU926321 EU926387 CBS 110306 = FRC E-0162 = NRRL 36179 South Africa Soil EU926255 — EU926322 EU926388 CBS 110311 = FRC E-0228 = NRRL 36184 ex-type South Africa Soil NR_137706 MW811071 EU926319 EU926385 Bisifusarium cacticola CBS 101615 ex-type Italy Opuntia ficus-indica PV158420 —PV167643 — CBS 110312 = FRC E-0306 = NRRL 34031 = NRRL 36185 USA Human sinus EU926223 PV167586 EU926290 EU926356 CBS 153382 = CPC 22100 South Africa Opuntia ficus-indica cv. Zastron PV158421 —PV167644 — CBS 153383 = CPC 22120 South Africa Opuntia ficus-indica cv. Malta PV158422 PV167587 PV167645 — CBS 153384 = CPC 22132 South Africa Opuntia ficus-indica cv. Morado PV158423 PV167588 PV167646 — Bisifusarium colchici CBS 153389 = CPC 45105 ex-type South Africa Colchicum sp. PV158431 PV167628 PV167655 — CBS 153390 = CPC 45109 South Africa Trachyandra sp. PV158432 PV167629 PV167656 — CBS 153391 = CPC 45111 South Africa Lachenalia sp. PV158433 PV167630 PV167657 — CBS 153392 = CPC 45112 South Africa Lachenalia sp. PV158434 PV167631 PV167658 — Bisifusarium delphinoides CBS 187.79 = NRRL 22108 Sudan Soil under Citrus sp. PV158424 PV167589 PV167647 — CBS 101047 Netherlands Pasteurization container PV158425 PV167590 PV167648 — CBS 109831 = MRC 7418 = NRRL 36152 South Africa Human cornea EU926232 — EU926299 EU926365 CBS 110140 = FRC E-0073 = NRRL 36160 USA Human eye EU926235 HM347219 EU926302 EU926368 CBS 110190 = FRC E-0074 USA Human eye PV158426 —PV167649 — CBS 110192 = FRC E-0065 = NRRL 37068 Australia Soil EU926238 PV167591 EU926305 EU926371 CBS 110304 = FRC E-0123 = NRRL 36177 = NRRL 37070 South Africa Soil debris EU926236 PV167592 EU926303 EU926369 CBS 110305 = FRC E-0152 = NRRL 36178 China Soil, bamboo grove clay EU926233 PV167593 EU926300 EU926366 Zhang et al.: Taxonomic revision of Bisifusarium 201 Table 1. (Continued). Species Strain1Origin Substrate/host GenBank accession number ITS rpb2 tef1 tub2 CBS 110309 = FRC E-0211 = NRRL 36182 South Africa Debris from corn soil EU926239 — EU926306 EU926372 CBS 110310 = FRC E-216 = NRRL 36183 South Africa Debris from corn soil EU926240 — EU926307 EU926373 CBS 110313 = FRC E-0309 = NRRL 36186 India Bryophyllum sp. EU926243 PV167594 EU926310 EU926376 CBS 110315 = FRC E-0063 = NRRL 36188 Australia Soil EU926234 PV167595 EU926301 EU926367 CBS 110316 = FRC E-0085 = NRRL 36189 USA Roots of citrus and soil EU926237 PV167596 EU926304 EU926370 CBS 115321 = NRRL 37072 Netherlands Water in pasteurisation equipment EU926244 PV167597 EU926311 EU926377 CBS 116510 = ATCC 24584 = NRRL 20715 Argentina Human cornea EU926231 PV167598 EU926298 EU926364 CBS 116513 = NRRL 22260 China Soil EU926241 — EU926308 EU926374 CBS 116522 = NRRL 34027 = UTHSC 95-2373 = UTHSC 97-2326 USA Human peritoneal fluid EU926242 PV167599 EU926309 EU926375 CBS 120714 = NRRL 53286 = CPC 13037 South Africa Hoodia sp. EU926225 PV167600 EU926292 EU926358 CBS 120715 = CPC 13038 South Africa Hoodia sp. EU926226 — EU926293 EU926359 CBS 120716 = CPC 13039 = NRRL 53288 South Africa Hoodia gordonii EU926227 PV167601 EU926294 EU926360 CBS 120717 = CPC 13040 = NRRL 53289 South Africa Hoodia sp. EU926228 PV167602 EU926295 EU926361 CBS 120718 = CPC 13041 = NRRL 53290 ex-type South Africa Hoodia gordonii EU926229 — EU926296 EU926362 CBS 120719 = CPC 13042 = NRRL 53291 South Africa Hoodia sp. EU926230 PV167603 EU926297 EU926363 CBS 139355 India Human cornea KU296244 PV167604 — — CBS 153386 = CPC 34708 Netherlands Echeveria sp. PV158427 PV167605 PV167650 PV167671 Bisifusarium dimerum CBS 175.31 = NRRL 36282 Germany Unknown EU926285 PV167606 EU926352 EU926418 CBS 254.50 = NRRL 36384 Unknown Human sputum MH856608 PV167607 EU926346 EU926412 CBS 489.81 = NRRL 20691 Unknown Mouldy soap EU926282 JX171592 EU926349 EU926415 CBS 102613 = NRRL 36130 Sweden Human wound EU926266 PV167608 EU926333 EU926399 CBS 108944 = NRRL 36140 ex-epitype Netherlands Human blood JQ434586 KM232363 KR673912 EU926400 CBS 109184 Netherlands Human peritoneal dialysate EU926268 PV167609 EU926335 EU926401 CBS 109187 = NRRL 36146 Canada Human peritoneal dialysate EU926269 PV167610 PV167651 EU926402 CBS 110141 = FRC E-0305 = NRRL 36161 USA Human blood EU926271 PV167611 EU926338 EU926404 CBS 110143 = FRC E-0115 Chile Human toe nail EU926272 PV167612 EU926339 EU926405 CBS 110314 = FRC E-0307 = NRRL 36187 USA Human eye EU926275 PV167613 EU926342 EU926408 CBS 110317 = FRC E-0302 = NRRL 36190 USA Human toe EU926276 PV167614 EU926343 EU926409 CBS 110319 = FRC E-0284 = NRRL 36192 USA Human EU926278 — EU926345 EU926411 CBS 110320 = FRC E-0116 = NRRL 37071 Chile Human toe nail EU926273 PV167615 EU926340 EU926406 CBS 115623 Unknown Unknown PV158428 PV167616 PV167652 — Persoonia – Volume 54, 2025202 Table 1. (Continued). Species Strain1Origin Substrate/host GenBank accession number ITS rpb2 tef1 tub2 CBS 116518 = NRRL 32173 Switzerland Unknown EU926261 PV167617 EU926328 EU926394 CBS 116519 = NRRL 34024 = UTHSC 99-1164 USA Human wound EU926262 — EU926329 EU926395 CBS 116520 = NRRL 34025 = UTHSC 97-2326 USA Human blood EU926281 — EU926348 EU926414 CBS 116521 = NRRL 34026 = UTHSC 97-1942 USA Human leg EU926259 PV167618 EU926326 EU926392 CBS 116523 = NRRL 34028 = UTHSC 93-2332 USA Human vitreous fluid EU926263 PV167619 EU926330 EU926396 CBS 116524 = NRRL 34029 = UTHSC 93-2606 USA Human eye EU926264 PV167620 EU926331 EU926397 CBS 116526 = NRRL 36945 USA Hospital sink spigot EU926280 PV167621 EU926347 EU926413 CBS 116527 = NRRL 36950 USA Human scalp EU926284 PV167622 EU926351 EU926417 CBS 116637 = NRRL 34030 = UTHSC-93-2286 USA Human vitreous fluid EU926265 PV167623 EU926332 EU926398 CBS 116638 = NRRL 36946 USA Hospital sink drain EU926283 PV167624 EU926350 EU926416 Bisifusarium domesticum CBS 244.82 France Cheese EU926220 PV167625 EU926287 EU926354 CBS 434.34 = ATCC 13417 = MUCL 9826 exneotype Belgium Cheese NR_145050 — — JQ434532 CBS 102407 = NRRL 37582 Belgium Cheese EU926221 PV167626 EU926288 EU926355 CBS 116517 = NRRL 29976 Switzerland Cheese JQ434584 HQ897694 EU926286 EU926353 Bisifusarium hedylamarriae CBS 150895 = BRIP 52699a ex-type Australia Sansevieria sp. — OQ626864 OR269443 — CBS 153385 = CPC 25710 South Africa Lederbouria floribunda PV158429 PV167627 PV167653 — CBS 153388 = CPC 39039 South Africa Aloe dyeri PV158430 —PV167654 — CGMCC 3.25519 = ZY 22.066 ex-type of B. sinense China Soil OR680545 OR842960 OR858939 OR843243 ZY 22.067 China Soil OR680546 OR842961 OR858940 OR843244 ZY 22.068 China Soil OR680547 OR842962 OR858941 OR843245 Bisifusarium keratinophilum CGMCC 3.23621 ex-type China Soil OP693473 OR168079 — OR168085 GZUIFR 22.371 China Soil OP693474 OR168080 — OR168086 GZUIFR 22.372 China Soil OP693475 OR168081 — OR168087 Bisifusarium lovelliae CBS 110145 = FRC E-0167 Thailand Banana grove EU926247 — EU926314 EU926380 CBS 110307 = FRC E-0173 = NRRL 36180 Thailand Banana grove EU926248 PV167632 EU926315 EU926381 CBS 110308 = FRC E-0177 = NRRL 36181 Thailand Soil in banana grove EU926249 PV167633 EU926316 EU926382 CBS 150777 = BRIP 75047a ex-type Australia Epipremnum pinnatum OQ629340 OQ626864 OQ626865 — Bisifusarium lunatum CBS 632.76 = BBA 63199 = NRRL 20690 = NRRL 36168 = NRRL 37067 ex-neotype Germany Gymnocalycium damsii JQ434583 JX171648 EU926291 EU926357 CBS 110148 Germany Citrus sp. PV158435 PV167634 PV167659 — Bisifusarium namibense CBS 153393 = DTO 484-E6 = CN005E1 ex-type Namibia Soil PV158437 —PV167661 — CBS 153394 = DTO 484-E8 = CN005E3 Namibia Soil PV158439 —PV167663 PV167673 Zhang et al.: Taxonomic revision of Bisifusarium 203 Table 1. (Continued). Species Strain1Origin Substrate/host GenBank accession number ITS rpb2 tef1 tub2 CBS 153395 = DTO 484-E9 = CN005H2 Namibia Soil PV158440 —PV167664 PV167674 CBS 153537 = DTO 484-E7 = CN005E2 Namibia Soil PV158438 —PV167662 — DTO 484-E5 = CN005D8 Namibia Soil PV158436 —PV167660 PV167672 Bisifusarium nectrioides CBS 176.31 = NRRL 20689 ex-lectotype Honduras Soil in banana plantation EU926245 JX171591 EU926312 EU926378 Bisifusarium penicilloides UBOCC-A-120034 France Cheese MW654541 MW811065 MW811080 MW811095 CBS 147586 = UBOCC-A-120021 ex-type France Cheese MW654542 MW811066 MW811081 MW811096 VTT-D-041022 Scotland Outer surface of sandstone building MW654535 MW811059 MW811074 MW811089 Bisifusarium penzigii CBS 116508 = ATCC 15621 = NRRL 20711 Sri Lanka Human eye EU926256 — EU926323 EU926389 CBS 317.34 = NRRL 22109 ex-type England Fagus sylvatica NR_137707 KM232362 EU926324 EU926390 CBS 318.34 = NRRL 36436 England Fagus sylvatica EU926258 PV167635 EU926325 EU926391 Bisifusarium phloginum CBS 110137 = BBA 64304 ex-type Germany Phlox drummondii PV158441 PV167636 PV167665 PV167675 CBS 110142 = BBA 71614 Germany Phaseolus vulgaris PV158442 —PV167666 — Bisifusarium salinasense CBS 110139 = FRC E-0010 ex-type USA Air — PV167637 PV167667 — CBS 119875 = BBA 62195 = FRC E-0312 = MRC 1652 South Africa Human cornea EU926222 HQ897698 EU926289 EU926419 Bisifusarium sechiicola CBS 153536 = CPC 16016 ex-type Mexico Sechium edule PV158443 PV167638 PV167668 PV167676 Bisifusarium solicola CBS 151317 = CPC 47701 ex-type South Africa Soil adjacent to Agapanthus praecox PP590160 PP620557 PP620565 PP620571 CBS 151318 = CPC 47715 South Africa Soil adjacent to Agapanthus praecox PP590161 PP620558 PP620566 PP620572 Bisifusarium sp. 1 CBS 110318 = FRC E-0300 = NRRL 36191 Australia Cultivated soil EU926246 — EU926313 EU926379 Bisifusarium sp. 2 CBS 135686 India Human cornea PV158444 PV167639 — — Bisifusarium tonghuanum CBS 110195 = FRC E-0278 South Africa Soil PV158445 PV167640 PV167669 — CGMCC3.17369 ex-type China Atriplex centralasiatica KX790413 KY964339 KX790418 KX790417 CGMCC3.17370 China Suaeda salsa KX790415 KY964340 KX790420 KX790419 Rectifusarium robinianum CBS 430.91 = NRRL 25729 ex-type Germany Robinia pseudoacacia PP336536 JX171633 KM231923 — Rectifusarium ventricosum CBS 748.79 = BBA 62452 = NRRL 20846 = NRRL 22113 ex-epitype Germany Soil on wheat field NR_172389 JX171597 KM231924 — 1 ATCC: American Type Culture Collection, Manassas, VA, USA; BBA: Collection of the Julius Kühn Institute – Federal Research Centre for Cultivated Plants (former Biologische Bundesanstalt für Landund Forstwirtschaft) housed at the Institute for Epidemiology and Pathogen Diagnostics, Braunschweig, Germany; BRIP: Plant Pathology Herbarium, Department of Primary Industries, Queensland, Australia; CBS: Westerdijk Fungal Biodiverity Institute (WI), Utrecht, The Netherlands; CGMCC: China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China; CPC: Collection of P.W. Crous, held at WI; FRC: Fusarium Research Center, Pennsylvannia State University, PA, USA; IMI: CABI Bioscience, Eggham, UK; LC: Working collection of Lei Cai, State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, P.R. China; MRC: PROMEC, Medical Research Council, Tygerberg, South Africa; MUCL: Mycothèque de l´Université Catholique de Louvain, Louvain-la-Neuve, Belgium; NRRL: Agricultural Research Service Culture Collection, National Center for Agricultural Utilization Research, USDA, Peoria, IL, USA; UBOCC: Culture Collection of University of Western Brittany, LUBEM Plouzane, France; UTHSC: Fungus Testing Laboratory at the University of Texas Health Science Center, San Antonio, TX, USA; VTT: VTT Culture Collection, VTT Technical Research Center of Finland, Finland. Sequences newly generated in this study are shown in bold. Persoonia – Volume 54, 2025204 Fig. 2. Phylogenetic tree inferred from a Maximum Likelihood (RAxML) analysis based on aligned, concatenated ITS, rpb2, tef1 and tub2 sequences of 116 strains representing Bisifusarium and outgroups. Numbers at branches indicate support values (RAxML-BS / IQ-TREE-BS / BI-PP) above 50 % / 90 % / 0.9. New species are printed in bold font. “T” indicates ex-type, “NT” indicates neotype, “ET” indicates epitype and “LT” indicates lectotype strains, all of which are labelled in bold. The tree is rooted with two species of Rectifusarium. Scale bar represents expected number of changes per site. B. nectrioides B. lovelliae Bisifusarum sp. 1 B. colchici B. penzigii B. biseptatum B. solicola B. dimerum B. hedylamarriae B. tonghuanum B. aseptatum B. penicilloides B. allantoides B. keratinophilum B. lunatum B. domesticum outgroup B. africanum Bisifusarium sp. 2 B. namibense B. australianum B. phloginum B. salinasense B. sechiicola B. cacticola B. delphinoides R. robinianum R. ventricosum 0.08 CBS 151317T soil, South Africa CBS 116521 human leg, USA GZUIFR 22.371 soil, China GZUIFR 22.372 soil, China CBS 109831 human cornea, South Africa CBS 110308 soil, Thailand CBS 244.82 cheese, France CBS 153390 Trachyandra sp., South Africa CGMCC 3.17369 T Atriplex centralasiatica, China CBS 153391 Lachenalia sp., South Africa CBS 110314 human eye, USA CBS 102613 human wound, Sweden CBS 150777T Epipremnum pinnatum, Australia CBS 116527 human scalp, USA CBS 110141 human blood, USA CBS 110190 human eye, USA CBS 115623 unknown, unknown CBS 110318 soil, Australia CBS 110309 soil debris, South Africa CBS 110147 Citrus sp., Germany CBS 110313 Bryophyllum sp., India CBS 110140 human eye, USA CBS 175.31 unknown, Germany CBS 110307 banana grove, Thailand CBS 153392 Lachenalia sp., South Africa CBS 187.79 soil, Sudan CBS 116523 human vitreous fluid, USA VTT D 041022 sandstone, Scotland CGMCC 3.20816T Orchidaceae sp., China CBS153386 Echeveria sp., Netherlands CBS 110138 soil, South Africa CBS 110142 Phaseolus vulgaris, Germany CBS 110306 soil, South Africa CBS 110311T soil, South Africa CBS 116519 human wound, USA CBS 110146 soil, South Africa CBS 110320 human toe nail, Chile ZY 22.068 soil, China CBS 110144 soil, South Africa CBS 120715 Hoodia sp., South Africa CBS 110310 soil debris, South Africa CBS 116520 human blood, USA CBS 120719 Hoodia sp., South Africa CBS 101047 pasteurization container, Netherlands CBS 366.73T poultry field, Australia CBS 110319 human, USA CBS 116526 hospital sink spigot, USA CBS 110145 banana grove, Thailand UBOCC A 120034 cheese, France CBS 109184 human peritoneal dialysate, Netherlands CBS 116638 hospital sink drain, USA CBS 102407 cheese, Belgium CBS 120716 Hoodia gordonii, South Africa CBS 109187 human peritoneal dialysate, USA CBS 116637 human vitreous fluid, USA UBOCC-A-120037 cheese, France CBS 153383 Opuntia ficus-indica, South Africa CBS 115321 water in pasteurization equipment, Netherlands CBS 120717 Hoodia sp., South Africa CBS 135686 human cornea, India CBS 110148 Citrus sp., Germany CBS 153537 soil, Namibia CBS 110304 soil debris, South Africa CBS 110192 soil, Australia CGMCC 3.17370 Suaeda salsa, China CBS 318.34 Fagus sylvatica, England CBS 116513 soil, China CBS 116518 unknown, Switzerland LC13608 Orchidaceae sp., China UBOCC-A-120035 cheese, Spain CBS 150895T Sansevieria sp., Australia CGMCC 3.23621T soil, China CBS 116508 human eye, Sri Lanka CBS 116524 human eye, USA CBS 147587T cheese, France CBS 748.79ET soil, Germany CBS 110312 human sinus, USA CBS 151318 soil, South Africa CBS 153385 Lederbouria floribunda, South Africa CBS 254.50 human sputum, unknown CBS 153388 Aloe dyeri, South Africa CBS 110195 soil, South Africa CBS 489.81 mouldy soap, unknown CBS 176.31LT soil, Honduras CBS 120714 Hoodia sp., South Africa CBS 108944ET human blood, Netherlands CBS 110143 human toe nail, Chile CBS 110137T Phlox drummondii, Germany CBS 153389T Colchicum sp., South Africa CBS 153536T Sechium edule, Mexico CBS 101615T Opuntia ficus-indica, Italy CBS 116522 human peritoneal fluid, USA CBS 139355 human cornea, India CBS 147586T cheese, France CBS 153393T soil, Namibia CBS 430.91T Robinia pseudoacacia, Germany CBS 110315 soil, Australia CBS 153387T Encephalartos sp., South Africa CBS 116517 cheese, Switzerland CBS 110316 roots of Citrus sp. and soil, USA CBS 317.34T Fagus sylvatica, England CBS 153395 soil, Namibia CBS 110317 human toe, USA CBS 632.76NT Gymnocalycium damsii, Germany CBS 116510 human cornea, Argentina ZY 22.067 soil, China CGMCC 3.25519T soil, China (ex-type of B. sinensis) CBS 434.34NT cheese, Belgium CBS 119875 human cornea, South Africa CBS 153384 Opuntia ficus-indica, South Africa CBS 110305 soil, China CBS 153382 Opuntia ficus-indica, South Africa CBS 110139T air, USA CBS 153394 soil, Namibia CBS 120718T Hoodia gordonii, South Africa DTO 484-E5 soil, Namibia 99/99/1 98/88/1 -/72/0.98 100/100/1 100/100/1 100/100/1 100/100/1 100/100/1 100/100/1 -/-/0.99 95/85/1 100/97/1 -/82/0.99 -/70/1 100/100/1 100/100/1 97/84/0.96 100/100/1 100/100/1 100/100/1 100/100/1 96/98/0.96 100/100/1 100/93/0.99 100/100/1 100/99/1 -/-/0.98 99/98/0.98 -/75/1 100/100/1 100/100/1 100/100/1 100/99/1 100/98/1 100/100/1 98/100/1 100/100/1 100/100/1 100/100/1 99/100/1 99/94/0.99 100/100/1 97/81/1 97/72/- 97/-/- 100/100/1 100/100/1 100/99/1 Zhang et al.: Taxonomic revision of Bisifusarium 205 were unique site patterns (ITS 142, rpb2 343, tef1 473, and tub2 223), and 893 were phylogenetically informative (ITS 104, rpb2 299, tef1 319, and tub2 171). Best-fit evolutionary model selection based on ModelFinder in IQ-TREE determined TIM2e+R2 for ITS, TIM2e+I+G4 for tef1, and TIM3e+G4 for rpb2 and tub2; whereas TIM2+I+G4 was applied to all partitions in raxmlGUI. Additionally, for BI, GTR+I+G was selected for ITS, and SYM+I+G for rpb2, tef1 and tub2. The IQ-TREE-ML, raxmlGUI-ML and MrBayes-BI phylogenies were identical in the distribution of terminal clades and resolved 23 partiallyto fully-supported phylogenetic species within the ingroup taxa (Fig. 2). Two isolates (CBS 110318 and CBS 135686) clustered as unsupported lineages in both the combined and single locus phylogenies (not shown, single-gene phylogenies available in figshare (www. figshare.com; DOI: 10.6084/m9.figshare.28816022). In addition, a clade including the ex-type and two additional strains of the recently described B. sinense (isolates CGMCC 3.25519, ZY 22.068 and ZY 22.067), clustered within the phylogenetic variation of B. hedylamarriae. Taxonomy Among the 116 isolates considered in this study, 10 novel lineages were recognized, eight of which represent the new species described below. Given their uncertain phylogenetic position and due to lack of phylogenetic support, the single lineages representing Bisifusarium sp. 1 (CBS 110318) and Bisifusarium sp. 2 (CBS 135686) most likely represent insufficiently sampled and novel species. We have chosen not to provide names for them at this time, even though morphological descriptions are included. Bisifusarium L. Lombard et al., Stud. Mycol. 80: 223. 2015. Sexual morph unknown. Conidiophores simple (aerial conidiophores) or grouped on sporodochia; aerial conidiophores simple, unbranched or irregularly branched, mostly reduced to terminal or single lateral conidiogenous cells. Conidiogenous cells often formed as (i) lateral phialidic pegs arising from superficial or submerged intercalary hyphal cells or, (ii) cylindrical and slightly tapering towards apex or ampulliform, smoothand thin-walled monophialides, rarely polyphialides, with inconspicuous or absent periclinal thickening, solitary or aggregated to represent a poorly developed pionnotal sporodochial-like structure, producing microand macroconidia. Microconidia hyaline, thinwalled, 0–1-septate, ellipsoidal, allantoid, broadly lunate to reniform, straight or curved, tapering towards both ends. Sporodochial macroconidia falcate, (0–)1–2(–3)-septate, thick-walled, curved to lunate, with a blunt to hooked apical cell and obtuse to poorly developed, foot-shaped basal cell, typically formed on sporodochia. Sporodochia pale yellow to orange; sporodochial conidiophores verticillately branched and densely packed, consisting of short, smoothand thinwalled stipes bearing an apical whorl of 2–3 monophialides; sporodochial conidiogenous cells monophialidic, cylindrical to subulate, smoothand thin-walled, with reduced or flared collarette. Chlamydospores, if present, globose to subglobose to ellipsoidal, solitary or in chains, sometimes aggregated in sclerotia (Lombard et al. 2015, Crous et al. 2021). Type species: Bisifusarium dimerum (Penz.) L. Lombard & Crous Notes: Bisifusarium was established to accommodate fusarioid species characterized by pionnotal culture growth, their short, chiefly 1–2-septate sporodochial macroconidia and the formation of lateral phialidic pegs arising from hyphae (Gerlach & Nirenberg 1982, Schroers et al. 2009, Lombard et al. 2015). Bisifusarium forms a well-supported clade (BS = 100 %, PP = 1.0) closely related but separate from Fusarium s. str. (Crous et al. 2021). Bisifusarium africanum K. Zhang & Crous, sp. nov. MB 858737. Fig. 3. Etymology: Name refers to Africa, the continent where it was collected. Sporodochia orange, formed abundantly on SNA and CLA. Sporodochial conidiophores verticillately branched and densely packed, giving rise to 1–6 conidiogenous cells, 10– 35 × 3–5 µm; sporodochial phialides monophialidic, subulate to subcylindrical, smoothand thin-walled, 2–4 µm diam. Sporodochial macroconidia slender, falcate, slightly curved with almost parallel sides tapering slightly towards both ends, with a papillate to hooked, curved apical cell and a blunt to poorly developed foot-like basal cell, 2-septate, with septa a third up from hilum and down from apex, not median, hyaline, smoothand thin-walled, (27.2–)28.0–29.0(–30.4) × (3.2–)3.5–3.9(–4.1) µm. Microconidia not observed. Chlamydospores intercalary, solitary or in short chains, ellipsoid to cylindrical, 4–8 µm diam. Culture characteristics: Colonies in the dark for 7 d at 25 °C: on OA reaching 7–7.5 cm diam., raised, aerial mycelia dense, colony margin entire, surface and reverse white. Colonies on PDA reaching 7.5–8 cm diam., raised, aerial mycelium dense, colony margin erose, surface and reverse white. Colonies on SNA reaching 7–7.5 cm diam., flat, aerial mycelium scant, colony margin erose, surface and reverse white; pigment and odour absent. Typus: South Africa, Gauteng Province, from Encephalartos sp. leaf, Mar. 2018, P.W. Crous (holotype CBS H-25455, culture ex-type CBS 153387 = CPC 34902); ibid., culture CPC 34903. Notes: Bisifusarium africanum clusters within a fully supported clade containing B. biseptatum, B. colchici, B. penzigii and B. solicola; all phylogenetically well-separated species. Bisifusarium africanum differs from B. biseptatum by 36 bp in the combined alignment (ITS 9, rpb2 5, tef1 12 and tub2 10 bp), from B. colchici by 15 bp (ITS 1, rpb2 6 and tef1 8 bp, while no tub2 sequences are available B. colchici), from B. penzigii by 24 bp (ITS 2, rpb2 6, tef1 10, and tub2 6 bp), and from B. solicola by 17 bp (ITS 0, rpb2 2, tef1 11, and tub2 4 bp). Morphologically, B. africanum is similar to B. biseptatum, but can be distinguished from that species based on macroconidial septation (2-septate in B. africanum vs 0–1-septate in B. biseptatum), macroconidial shape (macroconidial ends equally hooked in B. biseptatum in contrast to more strongly apically hooked in B. africanum), Persoonia – Volume 54, 2025212 Fig. 7. Bisifusarium hedylamarriae (ex-type CBS 150895). A. Colony surface on OA after 14 d at 25 °C. B. Colony on PDA. C. Colony on SNA. D, E. Sporodochia on carnation leaf. F, G. Sporodochial conidiophores and conidiogenous cells. H–J. Aerial conidiophores and conidiogenous cells. K. Microconidia. L. Sporodochial macroconidia. Scale bars = 10 µm. Zhang et al.: Taxonomic revision of Bisifusarium 213 Fig. 8. Bisifusarium lovelliae (ex-type CBS 150777). A. Colony surface on OA after 14 d at 25 °C. B. Colony on PDA. C. Colony on SNA. D, E. Sporodochia on carnation leaf. F, G. Sporodochial conidiophores and conidiogenous cells. H, I. Aerial conidiophores and conidiogenous cells. J. Microconidia. K, L. Chlamydospores. M. Sporodochial macroconidia. Scale bars: G, L = 5 µm; H = 20 µm; all others = 10 µm. Persoonia – Volume 54, 2025214 Fig. 9. Bisifusarium namibense (ex-type CBS 153393). A. Surface of colony on OA after 7 d at 25 °C. B. Colony on PDA. C. Colony on SNA. D. Sporodochia on carnation leaf. E, F. Sporodochial conidiophores and conidiogenous cells. G, H. Aerial conidiophores and conidiogenous cells. I. Microconidia. J. Chlamydospores. K. Sporodochial macroconidia. Scale bars: J = 5 µm; all others = 10 µm. Zhang et al.: Taxonomic revision of Bisifusarium 215 Fig. 10. Bisifusarium phloginum (ex-type CBS 110137). A. Surface of colony on OA after 7 d at 25 °C. B. Colony on PDA. C. Colony on SNA. D, E. Sporodochia on carnation leaf. F–H. Sporodochial conidiophores and conidiogenous cells. I, J. Chlamydospores. K. Sporodochial macroconidia. Scale bars = 10 µm. Persoonia – Volume 54, 2025216 Fig. 11. Bisifusarium salinasense (ex-type CBS 110139). A. Surface of colony on OA after 7 d at 25 °C. B. Colony on PDA. C. Colony on SNA. D, E. Sporodochia on carnation leaf. F, G. Sporodochial conidiophores and conidiogenous cells. H. Sporodochial macroconidia. Scale bars = 10 µm. positions (Tan & Shivas 2023a). The species is described and illustrated here based on the ex-type strain (CBS 150777, Fig. 8). Phylogenetically, B. lovelliae is resolved within a fully supported clade that also includes B. delphinoides, B. nectrioides and the undescribed lineage Bisifusarium sp. 1; all these taxa are characterized by predominantly 2-septate macroconidia. Apart from their chlamydospores and aerial phialides having different sizes, B. lovelliae can be distinguished from all its sibling species by the presence of distinct, regularly curved macroconidia. This is in comparison to asymmetrical macroconidia of B. delphinoides, B. nectrioides and Bisifusarium sp. 1, which are more pronouncedly curved toward the apex, resulting in slightly hooked distal ends (Schroer et al. 2009). Bisifusarium namibense K. Zhang, Yilmaz & Crous, sp. nov. MB 858741. Fig. 9. Etymology: Name refers to the fact that it was isolated from soil collected in the Namib desert. Sporodochia formed on SNA and on CLA, giving rise to a creamy conidial mass. Sporodochial conidiophores apically branched, consisting of primary and secondary branches Zhang et al.: Taxonomic revision of Bisifusarium 217 giving rise to 1–2 conidiogenous cells, 20–50 × 3–4 µm. Sporodochial phialides monophialidic, ampulliform to ellipsoid, 10–15 × 3.5–4 µm, with a tubular collarette, 1–3 µm tall, 2 µm diam. Sporodochial macroconidia falcate, gently dorsiventrally curved, with blunt apical cell and blunt basal cell, or poorly developed foot-like basal cell which is rarely present; medianly 1-septate, (14–)15–17(–18) × 3(–3.5) µm. Microconidia mostly 0(–1)-septate, typically ellipsoidal, straight or curved, formed on PDA substrate or in aerial hyphae, 7.5–9.5 × 3–3.5 µm. Chlamydospores intercalary, solitary or in short chains, globose to ellipsoid, 5–10 µm diam. Culture characteristics: Colonies in the dark for 7 d at 25 °C: on OA reaching 7–8 cm diam., flat, aerial mycelium sparse, colony margin entire, surface and reverse pale luteous. Colonies on PDA 6–7 cm diam., raised, aerial mycelium dense, colony margin erose, surface and reverse dirty white. Colonies on SNA reaching 3–4 cm diam., flat, aerial mycelium scant, colony margin erose, surface and reverse cream; odour absent. Typus: Namibia, Gobabeb, desert soil, Nov. 2019, N. Yilmaz (holotype CBS H-25454, culture ex-type CBS 153393 = DTO 484-E6). Fig. 12. Bisifusarium sechiicola (ex-type CBS 153536). A. Surface of colony on OA after 7 d at 25 °C. B. Colony on PDA. C. Colony on SNA. D, E. Sporodochia on carnation leaf. F. Sporodochial conidiophores and conidiogenous cells. G. Sporodochial macroconidia. Scale bars: F = 10 µm; G = 5 µm. Persoonia – Volume 54, 2025218 Additional materials examined: Namibia, Gobabeb, desert soil, Nov. 2019, N. Yilmaz, cultures CBS 153394 = DTO 484E8, CBS 153395 = DTO 484-E9, CBS 153537 = DTO 484-E7. Note: Bisifusarium namibense is morphologically similar to B. lunatum (0–1-septate macroconidia, (11.5–)18–22.5(– 26.5) × (2.5–)3–3.5(–4) µm; Shroers et al. 2009), but can be distinguished based on its smaller macroconidia, and DNA sequence data. Bisifusarium phloginum K. Zhang & Crous, sp. nov. MB 858742. Fig. 10. Etymology: Name refers to Phlox, the host genus from which it was isolated. Sporodochia greyish orange, formed abundantly on CLA. Sporodochial conidiophores verticillately branched and densely packed, subulate to subcylindrical, septate, 13–17 × 2–4 µm, smoothand thin-walled. Sporodochial macroconidia slender, falcate, slightly curved with almost parallel sides, tapering slightly towards both ends, with a papillate to hooked, curved apical cell and a blunt to poorly developed foot-like basal cell, 1-septate, hyaline, smoothand thinwalled, (13.6–)15.5–18.0(–20.9) × (2.0–)2.3–2.8(–3.0) µm. Chlamydospores intercalary, globose to ellipsoid, solitary or in chains. Culture characteristics: Colonies in the dark for 7 d at 25 °C: on OA reaching 3–3.5 cm diam., raised, aerial mycelia dense, colony margin entire, surface and reverse pale orange. Colonies on PDA reaching 3–3.5 cm diam., raised, aerial mycelium dense, colony margin erose, surface and reverse white, orange at the centre. Colonies on SNA reaching 3–4 cm diam., flat, aerial mycelium scant, colony margin erose, surface and reverse white; odour absent. Typus: Germany, from Phlox drummondii, collection date and collector unknown (holotype CBS H-25448, culture extype CBS 110137 = BBA 64304). Additional material examined: Germany, from Phaseolus vulgaris, collection date and collector unknown, culture CBS 110142 = BBA 71614. Notes: Bisifusarium phloginum is phylogenetically closely related to B. penicilloides and B. salinasense, from which it differs by 68 bp (ITS 2, rpb2 17, tef1 28, and tub2 21 bp) and 47 bp (ITS 2, rpb2 8, tef1 27, and tub2 10 bp), respectively. Morphologically, B. phloginum is distinguished by its slender, falcate, slightly curved macroconidia with almost parallel sides, a papillate to hooked apical cell, and a blunt to poorly developed foot-like basal cell. This is in contrast to those of B. penicilloides that are ellipsoidal, straight or curved, lunate or reniform with distinct foot-like basal cells (Savary et al. 2021), or the more robust macroconidia of B. salinense. Bisifusarium salinasense K. Zhang & Crous, sp. nov. MB 858743. Fig. 11. Etymology: Name refers to Salinas, California (USA), where it was collected. Sporodochia greyish orange, formed abundantly on carnation leaves. Sporodochial conidiophores verticillately branched and densely packed; sporodochial phialides subulate to subcylindrical, 10–20 × 3–4 µm, smoothand thin-walled. Sporodochial macroconidia slender, falcate, slightly curved with almost parallel sides tapering slightly towards both ends, with a papillate to hooked, curved apical cell and a blunt to foot-like basal cell, 1-septate, rarely 2-septate, hyaline, smoothand thin-walled, (16.2–)17.5–19.5(–22.2) × (3.0– )3.5–4.0(–4.3) µm. Aerial conidiophores often reduced to single monophialides. Chlamydospores intercalary, solitary or in short chains, globose to ellipsoid. Culture characteristics: Colonies in the dark for 7 d at 25 °C: on OA reaching 4–4.5 cm diam., raised, aerial mycelia dense, colony margin entire, surface and reverse white. Colonies on PDA reaching 2.5–3 cm diam., raised, aerial mycelium dense, colony margin erose, surface and reverse peach. Colonies on SNA reaching 3–3.5 cm diam., flat, aerial mycelium scant, colony margin erose, surface and reverse white; odour absent. Typus: USA, California, Salinas, from air sampling, collection date and collector unknown (holotype CBS H-25447, culture ex-type CBS 110139 = FRC E-0010). Additional material examined: Germany, on rotting fruit of Solanum lycopersicum (≡ Lycopersicon esculentum), 1955, R. Schneider, culture CBS 119875 = BBA 62195 = MRC 1652. Notes: Schroers et al. (2009) treated B. salinasense as Fusarium sp. 1. This species is phylogenetically similar but clearly differentiated from B. penicilloides and B. phloginum, differing by 61 bp (ITS 2, rpb2 17, tef1 16, and tub2 26 bp) and 47 bp (ITS 2, rpb2 8, tef1 27. and tub2 10 bp), respectively, in the combined four locus dataset. Compared to other Bisifusarium species, both B. salinasense and B. penicilloides are characterized by relatively slow growth on PDA (Schroers et al. 2009, Savary et al. 2021 and this study). Bisifusarium salinasense and B. penicilloides differ in their macroconidial size and septation, averaging 18.4 × 3.7 µm and 1-septate in B. salinasense vs av. 6.5 × 2.7 µm and aseptate in B. penicilloides (Savary et al. 2021). Bisifusarium salinasense is distinguished from B. phloginum, by its slightly longer and wider macroconidia [(16.2–)17.5–19.5(–22.2) × (3.0–)3.5–4.0(–4.3) µm vs (13.6–)15.5–18.0(–20.9) × (2.0– )2.3–2.8(–3.0) µm in B. phloginum], and by its often white colonies, compared to pale orange in B. phloginum. Bisifusarium sechiicola K. Zhang & Crous, sp. nov. MB 858744. Fig. 12. Etymology: Name refers to Sechium, the host genus from which it was isolated. Sporodochia greyish orange, formed abundantly on carnation leaves. Sporodochial conidiophores verticillately branched and densely packed; sporodochial phialides subulate to subcylindrical, 13–17 × 3–4 µm, smoothand thin-walled. Sporodochial macroconidia slender, falcate, slightly curved with almost parallel sides tapering slightly towards both ends, Zhang et al.: Taxonomic revision of Bisifusarium 219 Fig. 13. Bisifusarium sp. 1 (CBS 110318). A. Surface of colony on OA after 7 d at 25 °C. B. Colony on PDA. C. Colony on SNA. D. Sporodochia on carnation leaf. E. Sporodochial conidiophores and conidiogenous cells. F. Chlamydospores. G. Sporodochial macroconidia. Scale bars: E = 20 µm; all others = 10 µm. with a papillate to hooked, curved apical cell and a blunt to poorly developed foot-like basal cell, aseptate, hyaline, smoothand thin-walled, (7.3–)8.0–11.0(–13.0) × (1.8– )2.0–2.5(–3.0) µm; aerial microconidia forming small false heads on tips of monoand polyphialides, hyaline, ellipsoid to falcate, partially club-shaped, smoothand thin-walled, aseptate, (4.4–)5.0–6.5(–7.0) × (1.5–)1.8–2.0(–2.3) µm. Chlamydospores not observed. Culture characteristics: Colonies in the dark for 7 d at 25 °C: on OA reaching 4–4.5 cm diam., raised, aerial mycelia dense, colony margin entire, surface and reverse white. Colonies on PDA reaching 3.5–4 cm diam., raised, aerial mycelium dense, colony margin erose, surface and reverse white. Colonies on SNA reaching 3.5–4 cm diam., flat, aerial mycelium scant, colony margin erose, surface and reverse white; odour absent. Typus: Mexico, Colima, from Sechium edule, 31 Oct. 2008, M. de J. Yáñez-Morales (holotype CBS H-25449, culture extype CBS 153536 = CPC 16016). Notes: Bisifusarium sechiicola is morphologically and phylogenetically close to B. allantoides (macroconidia 9.5– 23.6 × 2.72–4.1 µm, av. 12.41 × 3.2 µm; Savaray et al. 2021), from which it differs by its smaller macroconidia, and white Persoonia – Volume 54, 2025220 Fig. 14. Bisifusarium sp. 2 (CBS 135686). A. Surface of colony on OA after 7 d at 25 °C. B. Colony on PDA. C. Colony on SNA. D. Sporodochia on carnation leaf. E, F. Sporodochial conidiophores and conidiogenous cells. G. Sporodochial macroconidia. Scale bars = 10 µm. colonies on PDA, whereas those of B. allantoides are pale orange. Additionally, both species differ by 58 bp in the four loci dataset (ITS 3, rpb2 22, tef1 24, and tub2 9 bp). Bisifusarium sp. 1. Fig. 13. Sporodochia greyish orange, formed abundantly on SNA and CLA. Sporodochial conidiophores verticillately branched and densely packed, giving rise to 1–2 conidiogenous cells, 10–30 × 2–3 µm; sporodochial phialides subulate to subcylindrical, smoothand thin-walled, 2–4 µm diam. Sporodochial macroconidia slender, falcate, slightly curved with almost parallel sides tapering slightly towards both ends, with a papillate to hooked, curved apical cell and well-developed foot-like basal cell, 2-septate, with septa a third up from hilum and down from apex, not median, hyaline, smoothand thinwalled, (14.5–)15.0–17.0(–18.9) × (2.3–)2.5–3.0(–3.5) µm. Microconidia not observed. Chlamydospores intercalary, solitary or in short chains, ellipsoid to cylindrical, 6–10 µm diam. Culture characteristics: Colonies in the dark for 7 d at 25 °C: on OA reaching 5.5–6 cm diam., raised, aerial mycelia dense, colony margin entire, surface and reverse white. Colonies on Zhang et al.: Taxonomic revision of Bisifusarium 221 PDA reaching 7–8 cm diam., raised, aerial mycelia dense, colony margin erose, surface and reverse white. Colonies on SNA reaching 6–6.4 cm diam., flat, aerial mycelia scant, colony margin erose, surface and reverse white; pigment and odour absent. Material examined: Australia, cultivated soil, collection date and collector unknown, isol. L. Burgess, F 7749, culture CBS 110318 = FRC E-0300 = NRRL 36191. Notes: Bisifusarium sp. 1 was originally assigned to Fusarium sp. 3 by Schroers et al. (2009) and it is closely related to B. lovelliae (Tan & Shivas 2023a). Both lineages have identical ITS sequences, while rpb2 sequences are not available for Bisifusarium sp. 1. Both taxa differ by 51 bp in the combined alignment (tef1 39, and tub2 12 bp). Morphologically, Bisifusarium sp. 1 differs from its closest phylogenetic relatives (B. delphinoides, B. lovelliae and B. nectrioides) by the absence of microconidia, and its often well-developed macroconidial foot cells. Bisifusarium sp. 2. Fig. 14. Sporodochia orange, formed abundantly on carnation leaves, giving rise to a creamy conidial mass. Sporodochial conidiophores verticillately branched and densely packed; sporodochial phialides subulate to subcylindrical, 10–20 × 3–4 µm, smoothand thin-walled. Sporodochial macroconidia slender, falcate, slightly curved with almost parallel sides tapering slightly towards both ends, with a papillate to hooked, curved apical cell and a blunt to rarely foot-like basal cell, aseptate, rarely 1-septate, hyaline, smoothand thinwalled, (16.8–)18.0–21.0(–23.1) × (2.4–)2.6–3.0(–3.3) µm. Chlamydospores and microconidia not observed. Culture characteristics: Colonies in the dark for 7 d at 25 °C: on OA reaching 6–6.5 cm diam., raised, aerial mycelium dense, colony margin entire, surface and reverse brownish orange. Colonies on PDA reaching 7.5–8 cm diam., raised, aerial mycelium dense, colony margin erose, surface and reverse white, pale brown in centre. Colonies on SNA reaching 6.5–7 cm diam., flat, aerial mycelium scant, colony margin erose, snowflake-like, surface and reverse white; odour absent. Material examined: India, Gijarat, Ahmedabad, from human cornea, collection date and collector unknown, culture CBS 135686. Notes: Bisifusarium sp. 2 is phylogenetically closely related to but different from B. dimerum by 51 bp in the combined alignment (ITS 9, and rpb2 42 bp, while tef1 and tub2 sequences are not available for the novel lineage). Morphologically, Bisifusarium sp. 2 is characterized by its wider macroconidia (width up to 3.3 µm in Bisifusarium sp. 2 in comparison to less than 3 µm wide in B. dimerum). Furthermore, conidia of Bisifusarium sp. 2 are mostly aseptate, while those of B. dimerum are 1-septate, and Bisifusarium sp. 2 lacks chlamydospores and microconidia in culture (Schoers et al. 2009). DISCUSSION The family Nectriaceae (Hypocreales) includes numerous saprotrophic, plant and human pathogenic fungi, many of which are also of interest due to their various industrial applications (Lombard et al. 2015). Bisifusarium (based on B. dimerum, formerly the F. dimerum species complex) was introduced by Lombard et al. in 2015, and at the time included seven species. Being phylogenetically and morphologically distinct from Fusarium s. str., species of Bisifusarium are primarily recognized by their 1–2-septate macroconidia. Subsequent to the description of the genus, several additional species have been described from soil, plants, food products, or opportunistic human infections (Schoers et al. 2009, do Carmo et al. 2016, Park 2019, Brent et al. 2023, Frederick et al. 2023). This study has added eight new species to the 16 previously known in Bisifusarium. The new species include B. namibense from Namib desert soil, a species from the dry Namaqualand region of South Africa (B. colchici), and other species from plants that normally grow in areas having warm climates (B. hedylamarriae on Lederbouria floribunda and leaves of Aloe dyeri in South Africa), B. africanum on an Encephalartos leaf (South Africa), B. australianum on poultry feed (Australia), B. salinasense from air (Salinas, California, USA), B. sechiicola on Sechium edule (Mexico), and B. cacticola on Opuntia ficus-indica (Italy and South Africa). In addition to the eight novel species, we have recognised two additional but poorly defined phylogenetic lineages that likely represent novel taxa, which we have referred to as Bisifusarium sp. 1 from soil (Australia), and Bisifusarium sp. 2 from a human corneal sample (India). Given the limited available data and lack of phylogenetic support for these putative novel species, future research should focus on acquiring and analysing a larger number of isolates corresponding to these lineages from diverse geographic locations and substrates. The results of this study suggest that Bisifusarium spp. favour extreme environments such as desert soils, plants that grow in arid regions and generally harsh substrates. Future studies considering the biology of these fungi, should consider this fact. An interesting characteristic of Bisifusarium is that some species are used in cheese making. One species in particular, B. domesticum, is utilised to reduce surface stickiness for many washed-rind cheeses (Bachmann et al. 2003, 2005). A recent study by Savary et al. (2021) introduced two additional species from cheese, namely B. allantoides (soft cheeses from France and Spain), and B. penicilloides (Mont d’Or soft cheese from France). Savary et al. (2021) also showed that these species produced no known mycotoxins. Later, Savary et al. (2023) reported that B. penicilloides could be used as a ripening culture in the dairy industry, while B. domesticum had potential bioprotective antifungal activities, and could be used to improve overall cheese quality and safety. This association with cheese production also suggests adaptation to extreme environments and would justify further studies of these fungi. Bisifusarium species, particularly B. domesticum, have very few biosynthetic gene clusters and a low number of carbohydrate-active enzymes (CAZymes), which aligns with its association with saprotrophism (Ulaszewski et al. 2025). However, the genus also has a significant number