683 MycoKeys MycoKeys 116: 53-71 (2025) DOI: 10.3897/mycokeys.116.150363 Research Article Morphological and phylogenetic analyses reveal new species and records of Fusarium (Nectriaceae, Hypocreales) from China Congcong Ai’, Qiyun Liu’™®, Yaling Wang', Zhaoxue Zhang™®, Duhua Li’®, Yun Geng?, Xiuguo Zhang™, Jiwen Xia!3® 1 Shandong Provincial Key Laboratory for Biology of Vegetable Diseases and Insect Pests, College of Plant Protection, Shandong Agricultural University, Taian, 271018, China 2 Institute of Crop Germplasm Resources, Shandong Academy of Agricultural Sciences, Jinan, 250100, China 3 College of Agriculture and Forestry, Linyi University, Linyi, Shandong, 276000, China Corresponding authors: Qiyun Liu (liugiyun0416@163.com); Jiwen Xia (xiajiwen1@126.com) OPEN Qaceess Academic editor: Xin-Cun Wang Received: 15 February 2025 Accepted: 28 February 2025 Published: 7 April 2025 Citation: Ai C, Liu Q, Wang Y, Zhang Z, Li D, Geng Y, Zhang X, Xia J (2025) Morphological and phylogenetic analyses reveal new species and records of Fusarium (Nectriaceae, Hypocreales) from China. MycoKeys 116: 53-71. https://doi.org/10.3897/ mycokeys.116.150363 Copyright: © Congcong Ai et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International - CC BY 4.0). Abstract Species of Fusarium are important phytopathogens, saprobes, and endophytes around the world. Some species can affect plant health and cause yield loss of economic plants. Fusarium species are widely distributed in China, and many species were found from dif- ferent plant hosts. The Fusarium incarnatum-equiseti species complex (FIESC) is one of the most significant species complexes within the genus. Based on morphological and three-gene (cal, rob2, and tef7) phylogenetic analyses, two new species are in the Incar- natum clade, and two new host records are identified and described, viz. Fusarium fici sp. nov., Fusarium xylosmatis sp. nov., Fusarium fecundum, and Fusarium weifangense. Key words: Fusarium incarnatum-equiseti species complex, multigene phylogeny, new taxa Introduction Johann Heinrich Friedrich Link first proposed the genus Fusarium (Nectriaceae, Hypocreales) in 1809 and typified it with Fusarium roseum (= F. sambucinum), with falcate or banana-shaped macroconidia and oval, subglobose, or kid- ney-shaped microconidia (Link 1809; Gams et al. 1997; Leslie and Summerell 2006; Liu et al. 2023; Zhang et al. 2023a). Fusarium is one of the most renowned and extensively spread genera in the Kingdom Fungi because of its morpholog- ical and phylogenetic diversity (Leslie and Summerell 2006; Sandoval-Denis et al. 2018; Crous et al. 2021). Fusarium species are known as plant pathogens, endophytes, and saprophytes (Leslie et al. 1990; Bacon and Yates 2006; Mary- ani et al. 2019; He et al. 2024). More than 1800 epithets of Fusarium have been listed in Index Fungorum (https://www.indexfungorum.org), but many species of Fusarium were identified solely based on morphological studies. Excessive overlap of conidial characteristics makes it difficult to morphologically distin- guish Fusarium species. Currently, Fusarium taxonomy is dominated by morpho- logical and molecular phylogenetic studies (Crous et al. 2021; He et al. 2024). At present, Fusarium contains 23 monophyletic species complexes and sev- eral single-species lineages (Xia et al. 2019; O’Donnell et al. 2020; Geiser et al. 93 Congcong Ai et al.: Fusarium incarnatum-equiseti species complex 2021; He et al. 2024). The FIESC includes over 30 recognized phylogenetic spe- cies (O’Donnell et al. 2009; Villani et al. 2016; Maryani et al. 2019; Santos et al. 2019; Wang et al. 2019; Xia et al. 2019). Based on the haplotype nomenclature system, O'Donnell et al. (2009) implemented an informal classification system for FIESC and introduced the Equiseti and Incarnatum clades. The Fusarium camptoceras species complex (FCAMSC) was proposed for three lineages that are sister clades to the FIESC by phylogenetic studies by Xia et al. (2019). However, Han et al. (2023) included the FCAMSC in FIESC as the Camptoceras clade because the FCAMSC and FIESC clearly represent a distinct evolutionary lineage that is strongly supported by the phylogenomic tree. Thus, FIESC com- prises three clades, viz. Camptoceras, Equiseti, and Incarnatum clades. In this study, samples were collected from Hainan, Sichuan, and Yunnan Provinces of China. Two new species and two new host records were identified and classified by multi-locus analysis of calmodulin (ca/), RNA polymerase II second largest subunit (rpb2), and translations elongation factor 1-alpha (tef7) datasets. They were described and discussed based on their morphological characteristics along with their molecular sequence data. Materials and methods Strain isolation and preservation Plant specimens with necrotic spots were collected from three provinces (Hainan, Sichuan, and Yunnan) of China in 2023. Pure colonies were obtained by tissue iso- lation techniques (Zhang et al. 2024). Fragments (25 mm?) were cut from the edg- es of diseased tissues, immersed in a 75% ethanol solution for 1 min, then rinsed in sterile water for 30 s and 10% sodium hypochlorite solution for 1 min. Frag- ments were rinsed three times with sterile water for 30 s, then using sterilized filter paper to absorb dry, placed on PDA for incubation at 25 °C for 3 days. The strains were preserved in 10% sterilized glycerol and stored them at 4 °C for future de- tailed studies. Specimens were deposited in the Herbarium of the Department of Plant Pathology, Shandong Agricultural University, Taian, China (HSAUP), and the Herbarium Mycologicum Academiae Sinicae, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China (HMAS). The living ex-type cultures were de- posited in the Shandong Agricultural University Culture Collection (SAUCC) and the China General Microbiological Culture Collection Center (CGMCC). DNA extraction, amplification, and sequencing Total genomic DNA was extracted from fresh fungal mycelia grown on potato dextrose agar (PDA) after 7 days using the genomic DNA purification kit (OG- PLF-400, GeneOnBio Corporation, Changchun, China) according to the product manual. The calmodulin (ca/), RNA polymerase second largest subunit (rpb2), and translation elongation factor 1-alpha (tef7) gene loci were amplified using the primer pairs listed in Table 1 (Xia et al. 2019; Han et al. 2023). The reaction was performed in a 25 uL reaction volume, consisting of 12.5 uL of 2 x Hieff Canace® Plus PCR Master Mix (Cat. No. 10154ES03, Yeasen Biotechnology, Shanghai, China), 1 uL each of forward and reverse primer (TsingKe, Qingdao, China), and 1 uL of template genomic DNA, and at last replenished the total MycoKeys 116: 538-71 (2025), DOI: 10.3897/mycokeys.116.150363 54 Congcong Ai et al.: Fusarium incarnatum-equiseti species complex Table 1. Molecular markers and their PCR primers and programs used in this study. Loci| PCR Primers cal |CL1 CL2A rpb2 | 5f2 7cr tef1 | EF-1 EF-2 Sequence (5'—3’') PCR Cycles References GARTWCAAGGAGGCCTTCTC | (94°C: 30s, 55 °C: 30s, 72 °C: 15 s) x 35 cycles | O'Donnell et al. (2020) TTTTTGCATCATGAGTTGGAC GGGGWGAYCAGAAGAAGGC ss: (94°C: 45 s, 57 °C: 45 s, 72 °C: 15 s) x 35 cycles Liu et al. (1999) CCCATRGCTTGYTTRCCCAT ATGGGTAAGGARGACAAGAC | (94°C: 45 s, 55 °C: 45 s, 72 °C: 15 s) x 35 cycles | O'Donnell et al. (1998) GGARGTACCAGTSATCATG volume to 25 uL with double distilled water. PCR products were separated and purified using 1% agarose gel and Safe Red (RM02852 and RM19009 ABclonal, Wuhan, China) and UV light to visualize the fragments. Gel was ex- tracted using a gel extraction kit (Cat. No. AE0101-C, Shandong Sparkjade Biotechnology Co., Ltd., Jinan, China) (Wang et al. 2023). The purified PCR products were sequenced by Youkang Company Limited (Zhejiang, China). All sequences generated in this study were deposited in GenBank under the accession numbers provided in Suppl. material 1. Phylogenetic analyses The reference sequences were downloaded from NCBI's GenBank. All sequenc- es were initially aligned with the MAFFT v. 7 (http://mafft.cbrc.jp/alignment/serv- er/) online service and MEGA 7.0. The concatenated aligned cal, rpb2, and tef17 sequences were used for maximum likelihood (ML) and Bayesian inference (BI), which were run on RaxML-HPC2 with XSEDE v. 8.2.12 and MrBayes v. 3.2.7a with 64 threads on Linux (Zhang et al. 2024). For ML analyses, 100 rapid bootstrap replicates and the GTR+FO+I+G4m model as default parameters were used. For BI analyses, a fast bootstrap algorithm with an automatic stop option was per- formed (Zhang et al. 2023b). The SYM+G model for cal, the SYM+I+G model for rpb2, and the GTR+I+G model for tef7 were selected and incorporated into the analyses. The Markov chain Monte Carlo (MCMC) analysis of the sequence data was performed over 5,000,000 generations, yielding 34,652 trees. Following the discard of 8,663 trees during the burn-in phase, the remaining trees were used to calculate posterior probabilities in the consensus trees. Morphological characterization Allisolates were inoculated on potato dextrose agar (PDA) medium and oatmeal agar (OA) medium. Colony morphology, pigmentation, and growth rates were recorded. The above and reverse of the PDA and OA flat plates were captured with the Alpha 6400L digital camera (Canon Powershot G7X, Canon, Tokyo, Ja- pan) on the 7* day. Used Carnation leaf agar (CLA; Fisher et al. 1982) medium to describe morphological features, such as shape, size, and septum number of the conidia (Wang et al. 2019). Used a stereomicroscope (Olympus SZ61, Olympus Corporation, Tokyo, Japan) and a microscope (Olympus BX53, Olym- pus Corporation, Tokyo, Japan) with Differential Interference Contrast (DIC) to observe the microscopic morphology. Stereomicroscope and microscope were equipped with BioHD-A20c color digital cameras (FluoCa Scientific, Shanghai, China) to capture the microscopic fungal structures. Microstructures were MycoKeys 116: 53-71 (2025), DOI: 10.3897/mycokeys.116.150363 55 Congcong Ai et al.: Fusarium incarnatum-equiseti species complex randomly measured using Digimizer software v5.6.0 (https://www.digimizer. com, accessed on 18 November 2024) and calculated the mean size (av.). The “n” represents the number of measurements. Results Phylogenetic analyses The combined dataset comprised 133 ingroup strains with Fusarium concolor (NRRL 13459) as the outgroup. The final alignment comprised 1,654 concate- nated characters, spanning from positions 1 to 535 (cal), 536 to 1,192 (rpb2), and 1,193 to 1,654 (tef7). The ML was carried out to be -9,907.383240. MrModelTest recommended using Dirichlet base frequencies for the cal, rpb2, and tef7 data par- titions. The alignment showed a total of 563 unique site patterns (cal: 156, rpb2: 168, tef7: 239). Based on the three-gene (cal, rpb2, and tef7) phylogeny, the 134 strains were classified into 57 species. The topology of the ML tree confirmed the topology obtained from BI, with only the ML tree presented (Fig. 1). Furthermore, single gene trees were evaluated, respectively, for FIESC (Suppl. material 2). Taxonomy Fusarium fecundum S.L. Han, M.M. Wang & L. Cai, Studies in Mycology 104: 87-148. 2023. Fig. 2 Description. On CLA, conidiophores arising from aerial mycelia, 13-71 um long, unbranched or irregularly branched, bearing terminal or lateral phialides, often reduced to single phialides; Periclinal thickening inconspicuous; Aerial conidia hyaline, smooth, rarely ovoid to falcate, on the apical half, the dorsal side is more curved than the ventral side, and the apical cell is either blunt or hooked, basal cell barely to distinctly notched. 1-septate conidia: (16-)22-21(-27) x 4-6 um (av. 20 x 5 um, n = 9); 2-septate conidia: (18—)21-28(—-33) x 5-7 um (av. 26 x 6 um, n = 9); 3-septate conidia: (32—)33-36(—41) x 5-8 um (av. 35 x 7 um, n = 16); 4-septate conidia: (32—)37-43(—43) x 6-9 um (av. 39 x 7 um, n = 18); 5-septate conidia: (41—)43-48(-53) x 7-9 um (av. 46 x 8 um, n= 12). Culture characteristics. Colonies on PDA incubated at 25 °C in the dark, reaching 84-90 mm diameter in 7 d; aerial mycelia dense, white, radiate, colony margin erose; reverse surface greyish yellow in the center, odor absent. On OA in the dark, occupying an entire 90 mm diameter in 7 d; surface white and aerial mycelia scant, crateriform, reverse white, odor absent. Materials examined. CHINA * Yunan Province, Nanuo Mountain, on leaves of Setaria palmifolia, 3 March 2023, Q.Y. Liu (HSAUP41424, HSAUP51424), living cultures CGMCC 3.27792 = SAUCC 2414-4, C6MCC 3.27793 = SAUCC 2414-5. Notes. Phylogenetic analysis showed that isolates (SAUCC 2414-4 and SAUCC 2414-5) were closely related to Fusarium fecundum (LC15875, ex-type strain) (Fig. 1). There are no nucleotide position differences between Fusarium fecundum (SAUCC 2414-4) and Fusarium fecundum (LC15875, ex-type strain). Morphologi- cally, Fusarium fecundum (SAUCC 2414-4) and Fusarium fecundum (LC15875, ex- type strain) are the lack of sporodochia. The aerial conidia of Fusarium fecundum MycoKeys 116: 53-71 (2025), DOI: 10.3897/mycokeys.116.150363 56 Congcong Ai et al.: Fusarium incarnatum-equiseti species complex F. neosemitectum CBS 189.607 96/0.99| F. neosemi ectum CBS 19060 100/1.0. » F. brevicaudatum NRRL 43694 100/1.0/F. brevicaudatum NRRL 43638" IZ brevicaudatum NRRL 45998 90/4.0}1___ IRRL 3: ia. -/0.98 MADER eae ianum CBS 148220 Tr MOSSE oe 148219 F.c croceum CPC 35240 F. croceum CBS 131777" F. croceum CBS 131788 F. croceum NRRL 3214 esielilh croceum NRRL 3020 F. toxicum CBS 406. 86" F. toxicum NRRL 43636 97/.0/F. toxicum CBS 219.63 F. toxicum CBS 130385 100/1.0 100/1.0 extenuatum LLC1501° F. extenuatum LLC 1492 he, 100/1.0 Bait. of] Oe Speen CBS 186.3157 oot. 'F. COR ERIE eS Thee 31 \ | F B3/1.0 é ua Ri F. lacertarum NRRL 36123 F. lacertarum NRRL 204237 92/0.99 100/1.0 F. clavum NRRL 32871 F. clavum NRRL 34032 F. clavum CBS 126202" ee F. clavum CBS 119881 70/1.0 S F. clavum CBS 394.93 se F. cated NRRL Panes __.F. mucidum CBS 102394 400/1.0| 'F. mucidum CBS 1023957 F. mucidum \|ndo 175 100/1.0 Figure 1. Phylogeny inferred based on the combined cal-rpb2-tef1 sequence dataset of the Fusarium incarnatum-equiseti species complex (FIESC), with Fusarium concolor (NRRL 13459) as the outgroup. The RAxML Bootstrap support values (MLBS = 70%) and Bayesian inference posterior probabilities (BIPP = 0.90) were shown at the nodes. Ex-type, ex-epitype, and ex-neotype strains were indicated by T, ET, and NT, respectively. Strains isolated in this study were indicated in red. MycoKeys 116: 538-71 (2025), DOI: 10.3897/mycokeys.116.150363 57 Congcong Ai et al.: Fusarium incarnatum-equiseti species complex F. xylosmatis CGMCC 3.27794" F. xylosmatis CGMCC 3.27795 F. humuli LC12159 100/1.0;F. humuli LC4490 F. humuli LC2158 F. luffae NRRL 32522 100/1.0/F. luffae CBS 131097 F. luffae NRRL 31167 |, F. sulawesiense InaCC F941 F. sulawesiense |InaCC F940° peessiin || i‘ 4a 100/1.0+— |_|F. pernambucanum CBS 132194 F. pernambucanum CBS 133024 100/1.0__ | F. caatingaense CBS 976.97 F. caatingaense NRRL 34003 96/1.0. 7 ie] s E F. bubalinum CBS 161.257 100/1.0 l 100/1.0| | | F. persicinum CBS 143596 83/0.99 F. persicinum CBS 143600 F. persicinum CBS 143598 99/0.96ILF. persicinum CBS 143606 F persicinum CBS 479.83' ic Pe r|| oon GN IF: thinolophicola KUMCC 21-0450 F. rhinolophicola KUMCC 21-0449° F. aberrans CBS 131387 F. aberrans CBS 1313857 F. aberrans CBS 119866 .F. aberrans CBS 131388 7710.94 100/1.0, 0.05 as 4x asi ¥ ae F. concolor NRRL 13459 Figure 1. Continued. MycoKeys 116: 538-71 (2025), DOI: 10.3897/mycokeys.116.150363 58 Congcong Ai et al.: Fusarium incarnatum-equiseti species complex Figure 2. Fusarium fecundum (SAUCC 2414-4) a colony on PDA after 7 days at 25 °C (left: above, right: reverse) b colony on OA after 7 days at 25 °C (left: above, right: reverse) c, d conidiophore on aerial mycelium with monophialides e aerial conidia. Scale bars: 10 um (c-e). (SAUCC 2414-4) are smaller than those of Fusarium fecundum (LC15875, ex-type strain). Fusarium fecundum was previously isolated from wheat and rice, and it has now been reported for the first time on Setaria palmifolia (Han et al. 2023). Fusarium fici Q.Y. Liu, X.G. Zhang & J.W. Xia, sp. nov. MycoBank No: 856644 Fig. Etymology. Referring to the genus name of the host plant Ficus fistulosa. Typus. CHINA « Hainan Province, Baoting Li and Miao Autonomous County, on leaves of Ficus fistulosa, 10 April 2023, Q.Y. Liu (HMAS 353395, holotype), ex-holotype culture CGMCC 3.27796 = SAUCC 3249C-3. Description. Conidiophores arising from aerial mycelium, 17-21 ym long, un- branched, reduced to single phialidic pegs, subulate to subcylindrical; aerial conid- ia hyaline, smooth, and thin-walled, rarely ellipsoidal to falcate, straight to curved dorsiventrally, a blunt apical cell and barely notched basal cell, 1-3(—5)-septate; 1-septate conidia: (12—)12-16(—28) x 3-5 um (av. 17 x 3 um, n = 18); 2-sep- tate conidia: (16—-)17-21 (—26) x 3-5 um (av. 19 x 4 um, n = 17); 3-septate co- nidia: (20—)22—28 (—36) x 3-6 um (av. 26 x 4 um, n = 31); 4-septate conidia: MycoKeys 116: 53-71 (2025), DOI: 10.3897/mycokeys.116.150363 59 Congcong Ai et al.: Fusarium incarnatum-equiseti species complex Figure 3. Fusarium fici (CGMCC 3.27796) a colony on PDA after 7 days at 25 °C (left: above, right: reverse) b colony on OA after 7 days at 25 °C (left: above, right: reverse) ¢ sporodochia on carnation leaves d lateral phialidic peg on aerial mycelium e monophialide f, g sporodochial conidiophores h, i monophialides on aerial mycelium j sporodochial conidia k aerial conidia. Scale bars: 10 um (d-k). MycoKeys 116: 53-71 (2025), DOI: 10.3897/mycokeys.116.150363 60 Congcong Ai et al.: Fusarium incarnatum-equiseti species complex (28-)31-34 (-39) x 4-5 um (av. 33 x 5 um, n = 14); 5-septate conidia: (23-)32- 33 (-36) x 4-5 um (av. 31 x 4 um, n= 5). Sporodochia salmon to saffron, formed abundantly on surface of carnation leaves. Sporodochial conidiophores densely and bearing apical whorls of 1 phialide; sporodochial phialides subulate to sub- cylindrical, 9-11 x 3-4 um, smooth, thin-walled, with inconspicuous periclinal thickening; sporodochial conidia falcate, straight to curved dorsiventrally, taper- ing towards both ends, with slightly papillate, a conical to slightly papillate apical cell, a notched to foot-like basal cell, (0-)1—3(—5)-septate, hyaline, smooth, and thin-walled; 0-septate conidia: (10-)15-20(-21) x 2-4 um (av. 16 x 3 um, n = 9); 1-septate conidia: (13—)15-22(-—25) x 2-5 um (av. 18 x 4 um, n = 23); 2-septate conidia: (13-)16-18(—23) x 2-5 um (av. 18 x 3 um, n = 23); 3-septate conidia: (19-)20-25(-29) x 3-5 um (av. 24 x 4 um, n = 37); 4-septate conidia: (28—)31- 34(-36) x 4-5 um (av. 33 x 4 um, n = 12); 5-septate conidia: (34—)34-36(-38) x 3-5 um (av. 35 x 4 um, n = 5). Chlamydospores not observed. Culture characteristics. Colonies on PDA incubated at 25 °C in the dark, reaching 76-80 mm diameter in 7 d, flat, convex, with abundant aerial myce- lium, colony margin lightly erose; surface white, odor absent; reverse yellowish white, odor absent. On OA in the dark, reaching 85-90 mm diameter in 7 d; aerial mycelium scant in the center forming a vacant circle, reverse white, odor absent. Additional material studied. CHINA * Hainan Province, Baoting Li and Miao Autonomous County, on leaves of Ficus fistulosa, 10 April 2023, Q.Y. Liu (HSAUP44932), living culture CGMCC 3.27797 = SAUCC 3249C-4. Notes. Phylogenetic analyses of three combined sequences (cal, rpb2, and tef1) showed that F. fici constitutes a distinct clade, closely related to F. aber- rans. Between F. fici (CGMCC 3.27796) and F. aberrans (CBS 131385), there were 11/535 differences in cal, 13/657 in rpb2, and 34/462 in tef1. The my- celium on OA of F. fici (CGMCC 3.27796) is sparser than that of F. aberrans (CBS 131385). Morphologically, F. fici (CGMCC 3.27796) and F. aberrans (CBS 131385) have different sporodochial conidial septa (0—5-septate in F. fici vs. 1—-3-septate in F aberrans) and sporodochial phialides (1 phialide in F. fici vs. 2-3 phialides in F aberrans). The aerial conidiophores of F. aberrans (16-110 Um) are longer than F. fici (17-21 ym) (Xia et al. 2019). Fusarium weifangense S.L. Han, M.M. Wang &L. Cai, Studies in Mycology 104: 87-148. 2023. Fig. 4 Synonym. Fusarium caulendophyticum H. Zhang & Y.L. Jiang, Mycosphere 14(1): 2092-2207. 2023. Description. Conidiophores arising from aerial mycelium, 14-18 um long, un- branched or irregularly branched, often reduced to single phialides; aerial phialides monophialidic, subulate to subcylindrical, smooth- and thin-walled, with inconspic- uous or absent periclinal thickening, 9.2-12.2 x 4.0—4.4 um; aerial conidia hyaline, rarely ellipsoidal to falcate, slightly curved with almost parallel sides, tapering to- wards both ends, with a blunt to conical and slightly curved apical cell, blunt to bare- ly notched basal cell, smooth and thin-walled, (1—)3-5-septate; 1-septate conidia: (14-)15-19(—20) x 3-4 um (av. 17 x 3 um, n = 8); 2-septate conidia: (19-)19-21(- 24) x 3-5 um (av. 21 x 4 um, n = 14); 3-septate conidia: (22—)26-31(-34) x 3-6 um MycoKeys 116: 53-71 (2025), DOI: 10.3897/mycokeys.116.150363 61 Congcong Ai et al.: Fusarium incarnatum-equiseti species complex Figure 4. Fusarium weifangense (SAUCC 5208C-2) a colony on PDA after 7 days at 25 °C (left: above, right: reverse) b col- ony on OA after 7 days at 25 °C (left: above, right: reverse) ¢ sporodochia on carnation leaves d polyphialide e monophi- alide f sporodochial conidiophores g sporodochial conidia h aerial conidia. Scale bars: 10 um (d-h). MycoKeys 116: 53-71 (2025), DOI: 10.3897/mycokeys.116.150363 Congcong Ai et al.: Fusarium incarnatum-equiseti species complex (av. 28 x 4 um, n = 22); 4-septate conidia: (30-)35-36(—45) x 3-6 um (av. 36 x 5 um, n = 17); 5-septate conidia: (31—-)34-37(—46) x 4-6 um (av. 38 x 5 um, n= 15). Sporodochia salmon to orange, formed abundantly on surface of carnation leaves. Sporodochial conidiophores densely, bearing apical whorls of one phialide; spo- rodochial phialides monophialidic, subulate to subcylindrical, 16-24 x 2-3 um, smooth. Sporodochial conidia falcate, slightly curved, tapering towards both ends, with a slightly elongated conical or whip-like curved apical cell, a foot-like to notched basal cell, (0Q-)4—5-septate, hyaline, thin, and smooth-walled; 0-septate conidia: 26-28 x 3-4 um; 1-septate conidia: (17—)26-36(-37) x 3-6 um (av. 28 x 4 um,n = 10); 2-septate conidia: (20—)21-37 x 3-5 um (av. 25 x 4 um, n = 7); 3-sep- tate conidia: 21-33(-38) x 3-5 um (av. 32 x 5 um, n = 12); 4-septate conidia: (31- )32-35(-44) x 3-6 um (av. 36 x 4 um, n = 22); 5-septate conidia: (34—)40—45(—48) x 3-6 um (av. 42 x 4 um, n = 16). Chlamydospores not observed. Culture characteristics. Colonies on PDA incubated at 25 °C in the dark, reaching 86-90 mm diameter in 7 d; surface white, flat, felty to velvety, aerial mycelia dense, colony margin entire; reverse white, odor absent. Colonies on OA incubated at 25 °C in the dark, reaching 85-89 mm diameter in 7 d; surface white and aerial mycelia scant, radiate, reverse white, radiate, odor absent. Materials examined. CHINA « Sichuan Province, Baoting Li and Miao Autono- mous County, on leaves of Prunus salicina, 2 July 2023, Q.Y. Liu (HSAUP20852, HSAUP30852), living cultures SAUCC 5208C-2 = CGMCC3.27939, SAUCC 5208C-3. Notes. Fusarium weifangense (LC18333, ex-type strain) was proposed by Han et al. (2023). Fusarium caulendophyticum (CGMCC 3.25474, ex-type strain) was proposed by Zhang et al. (2023a). Fusarium weifangense (LC18333, ex- type strain) was the first to be discovered. Fusarium weifangense (LC18333 and LC18243) are clustered with Fusarium caulendophyticum (CGMCC 3.25474 and GUCC 191050.2) clade in the combined phylogenetic tree (Fig. 1, Suppl. material 4). Fusarium weifangense (LC18333, ex-type strain) and Fusarium caulendophyt- icum (CGMCC 3.25474, ex-type strain) were similar in cal (0/535), rpb2 (1/657), and tef1 (2/462) sequences. We therefore considered the Fusarium caulendophyti- cum synonym of Fusarium weifangense. In this study, our strains (SAUCC 5208C-2 and SAUCC 5208C-3) are clustered with the Fusarium weifangense (LC18333 and LC18243) clade in the combined phylogenetic tree (Fig. 1). SAUCC 5208C-2 and SAUCC 5208C-3 were similar to the latter in cal (with 100% sequence identity), rpb2 (99.85%), and tef7 (98.70%) sequences. Fusarium weifangense was previous- ly isolated from wheat, Capsicum sp., Triticum sp., Medicago sativa, Lactuca sativa, Chenopodium quinoa, and Rosaceae roxburghii, and it has now been reported for the first time on Prunus salicina (Wang et al. 2019; Xia et al. 2019; Yin et al. 2021; Han et al. 2023; Zhang et al. 2023a) (Suppl. material 3). Fusarium xylosmatis Q.Y. Liu, X.G. Zhang & J.W. Xia, sp. nov. MycoBank No: 856642 Pig: 5 Etymology. Referring to the genus name of the host plant Xylosma congesta. Typus. CHINA * Yunan Province, Nanuo Mountain, on leaves of Xylosma con- gesta, 3 March 2023, Q.Y. Liu (HMAS 353394, holotype), ex-holotype culture CGMCC 3.27794 = SAUCC 2416-1. MycoKeys 116: 53-71 (2025), DOI: 10.3897/mycokeys.116.150363 63 Congcong Ai et al.: Fusarium incarnatum-equiseti species complex Figure 5. Fusarium xylosmatis (CGMCC 3.27794) a colony on PDA after 7 days at 25 °C (left: above, right: reverse) b colo- ny on OA after 7 days at 25 °C (left: above, right: reverse) c sporodochia on carnation leaves d chlamydospores e polyph- ialide f monophialide g, h sporodochial conidiophores i sporodochial conidia j aerial conidia. Scale bars: 10 um (d-j). MycoKeys 116: 53-71 (2025), DOI: 10.3897/mycokeys.116.150363 64 Congcong Ai et al.: Fusarium incarnatum-equiseti species complex Description. Conidiophores arising from aerial mycelium, 25-35 um long, un- branched or irregularly branched, often reduced to single phialides, subulate to subcylindrical, smooth, 12-15 x 4-5 um, periclinal thickening inconspicuous; aerial conidia ellipsoidal to falcate, slightly curved, tapering towards both ends, with a blunt to conical and slightly curved apical cell and papillate basal cell, (0O-)3-5-septate; 0-septate conidia: 16-20 x 3-4 um (av. 21 x 4 um, n = 5); 1-septate conidia: (12—)15-19(-29) x 3-4 um (av. 18 x 4 um, n = 33); 2-septate conidia: (16—)16-23(—29) x 3-5 um (av. 21 x 4 um, n = 18); 3-septate conidia: (20-)30-36(-41) x 4-5 um (av. 31 x 5 um, n = 45); 4-septate conidia: (31—)30- 36(-34) x 4-6 um (av. 34 x 5 um, n = 26); 5-septate conidia: (30—)37-41(-43) x 4-6 um (av. 38 x 5 um, n = 26). Sporodochia pale orange, formed abundantly on surface of carnation leaves. Sporodochial conidiophores densely and irregularly branched, 15-19 x 2-3 um, bearing apical whorls of 1-2 phialides; sporodochi- al phialides monophialidic, subulate to subcylindrical, 10-12 x 2-3 um, smooth, and thin-walled; sporodochial conidia falcate, curved dorsiventrally, straight to slightly curved, tapering towards both ends, with slightly papillate, curved apical cell and a notched to foot-like basal cell, (0Q-)3—4(—5)-septate, hyaline, smooth, and thin-walled; 0-septate conidia: 28-30 x 3—4 um (av. 29 x 4 um, n = 5); 1-sep- tate conidia: (16—)21-—32(-36) x 3-5 um (av. 27 x 4 um, n= 11); 2-septate conid- ia: 22-23 x 3-4 um (av. 23 x 4 um, n = 4); 3-septate conidia: (22—)25-33(-41) x 3-6 um (av. 32 x 4 um, n = 38); 4-septate conidia: (33-)35-38(-43) x 4-6 um (av. 37 x 5 um, n = 26); 5-septate conidia: (36-)38-40(-44) x 4-6 um (av. 40 x 5 um, n = 16). Chlamydospores abundant, globose, subglobose to ellipsoid, ter- minal or intercalary, solitary, in pairs, or forming long chains, 8-12 um diameter. Culture characteristics. Colonies on PDA incubated at 25 °C in the dark, reach- ing 71-79 mm diameter in 7 d; aerial mycelia dense, flat, white, colony margin entire; reverse yellowish white, radiate, aerial mycelia dense, odor absent. Colo- nies on OA grown in the dark, reaching 69-77 mm diameter after 7 d at 25 °C, flat, aerial mycelia scant, colony margin entire, white; reverse white, odor absent. Additional material studied. CHINA * Yunan Province, Nanuo Mountain, on leaves of Xylosma congesta, 3 March 2023, Q.Y. Liu (HSAUP21624), living cul- ture CGMCC 3.27795 = SAUCC 2416-2. Notes. Phylogenetically, F xylosmatis (CGMCC 3.27794) is closely relat- ed to the species F. weifangense (LC18333); there were 7/535 differences in cal, 9/657 in rpb2, and 8/462 in tef1. Morphologically, F xylosmatis (CGMCC 3.27794) is distinguished from F. weifangense (LC18333) by the number of spo- rodochial conidial septa (0—5-septate in F. xylosmatis (CGMCC 3.27794) vs. 3-7-septate in F. weifangense (LC18333)) (Han et al. 2023; Zhang et al. 2023a). Discussion The genus and species concepts in Fusarium have endured significant chang- es (Leslie and Summerell 2006; Crous et al. 2021; He et al. 2024). Traditionally, the identification of Fusarium is mainly based on morphological characteristics (Wollenweber and Reinking 1935; Snyder and Hansen 1940; Toussoun and Nel- son 1968; Gerlach and Nirenberg 1982; Leslie and Summerell 2006). However, identification is difficult due to the high morphological variation that compli- cates morphological identification among the closely related species (Leslie and Summerell 2006; Crous et al. 2021). Therefore, it is important to identify Fusarium MycoKeys 116: 53-71 (2025), DOI: 10.3897/mycokeys.116.150363 65 Congcong Ai et al.: Fusarium incarnatum-equiseti species complex species through molecular analysis (Wang et al. 2019; Xia et al. 2019; Crous et al. 2021; Wang et al. 2022; He et al. 2024). The internal transcribed spacer (ITS), the large subunit (LSU), ATP citrate lyase (ac/7), calmodulin (ca/), RNA polymerase II largest subunit (rpbb7), RNA polymerase second largest subunit (rpb2), translation elongation factor 1-alpha (tef7), and beta-tubulin (tub2) are used in current stud- ies (Lombard et al. 2015; Sandoval-Denis et al. 2018; Xia et al. 2019; Crous et al. 2021; Suwannarach et al. 2023; He et al. 2024). However, the identification of Fu- sarium at the species level could not be resolved using the ribosomal DNA gene (ITS and LSU) alone (Balajee et al. 2009; O’Donnell et al. 2015; Suwannarach et al. 2023). Thus, the protein-coding genes (acl!7, cal, rob1, rpb2, tef1, and tub2) are added (Xia et al. 2019; Crous et al. 2021; Suwannarach et al. 2023; He et al. 2024). Different complexes of Fusarium require different gene combinations to identify. In this study, we collected parasitic or saprotrophic fungi from terrestrial habitats in Hainan, Sichuan, and Yunnan Provinces of China on four plant spec- imens: Setaria palmifolia, Ficus fistulosa, Prunus salicina, and Xylosma conges- ta. Morphologically, these species exhibit a range of variations in spore size, shape, and ornamentation, as well as colony characteristics such as growth rate, pigmentation, and texture. We also conducted phylogenetic analyses us- ing cal, rob2, and tef1 sequences and can be recognized as two new phyloge- netic species (Fusarium. fici sp. nov. and Fusarium xylosmatis sp. nov.), along with two known species (Fusarium fecundum and Fusarium weifangense). The discovery of two new species underscores the rich fungal diversity in Hainan, Sichuan, and Yunnan Provinces and emphasizes the need for further explora- tion of understudied habitats. Fusarium fecundum was first reported from Se- taria palmifolia; Fusarium weifangense was first reported from Prunus salicina. It can contribute to our knowledge of host specificity and ecological adaptation in fungal pathogens. These findings have significant implications for fungal tax- onomy, ecology, and potential applications in plant pathology and biocontrol. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement No ethical statement was reported. Funding This research was funded by the National Natural Science Foundation of China (nos. 32370001, 32270024, 31900014, U2002203), the Key Technological Innovation Program of Shandong Province, China (no. 2022CXGC020710), the Jinan City’s ‘New University 20 Policies’ Initiative for Innovative Research Teams Project (no. 202228028), and the Innovative Agricultural Application Technology Project of Jinan City (no. CX202210). Author contributions Sampling, molecular biology analysis: Qiyun Liu and Congcong Ai; fungal isolation: Yal- ing Wang; description and phylogenetic analysis: Zhaoxue Zhang; microscopy: Duhua Li and Yun Geng; writing—original draft preparation: Qiyun Liu; writing—review and editing: Jiwen Xia and Xiuguo Zhang. All authors read and approved the final manuscript. MycoKeys 116: 538-71 (2025), DOI: 10.3897/mycokeys.116.150363 66 Congcong Ai et al.: Fusarium incarnatum-equiseti species complex Author ORCIDs Qiyun Liu © https://orcid.org/0009-0009-9545-7962 Zhaoxue Zhang ® hitps://orcid.org/0000-0002-4824-9716 Duhua Li © https://orcid.org/0009-0006-5200-2034 Xiuguo Zhang ® https://orcid.org/0000-0001-9733-8494 Jiwen Xia © https://orcid.org/0000-0002-7436-7249 Data availability The sequences were deposited in the GenBank database. References Bacon CW, Yates IE (2006) Endophytic root colonization by Fusarium species: histolo- gy, plant interactions, and toxicity. 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MycoKeys 106: 303-325. https://doi.org/10.3897/mycok- eys.106.127355 Supplementary material 1 GenBank accession numbers of the taxa used in phylogenetic reconstruction Authors: Congcong Ai, Qiyun Liu, Yaling Wang, Zhaoxue Zhang, Duhua Li, Yun Geng, Xiuguo Zhang, Jiwen Xia Data type: docx Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/mycokeys.116.150363.suppl1 MycoKeys 116: 538-71 (2025), DOI: 10.3897/mycokeys.116.150363 70 Congcong Ai et al.: Fusarium incarnatum-equiseti species complex Supplementary material 2 Phylogeny of the Fusarium incarnatum-equiseti species complex (FIESC) inferred based on the cal (a), rpb2 (b), and tef17 (c) loci, respectively Authors: Congcong Ai, Qiyun Liu, Yaling Wang, Zhaoxue Zhang, Duhua Li, Yun Geng, Xiuguo Zhang, Jiwen Xia Data type: docx Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/mycokeys.116.150363.suppl2 Supplementary material 3 GenBank accession numbers of the taxa used in phylogenetic reconstruction (Suppl. material 4) Authors: Congcong Ai, Qiyun Liu, Yaling Wang, Zhaoxue Zhang, Duhua Li, Yun Geng, Xiuguo Zhang, Jiwen Xia Data type: docx Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/mycokeys.116.150363.suppl3 Supplementary material 4 Phylogeny inferred based on the combined cal-rpb2-tef1 sequence dataset with Fusarium concolor (NRRL 13459) as the outgroup Authors: Congcong Ai, Qiyun Liu, Yaling Wang, Zhaoxue Zhang, Duhua Li, Yun Geng, Xiuguo Zhang, Jiwen Xia Data type: docx Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/mycokeys.116.150363.suppl4 MycoKeys 116: 53-71 (2025), DOI: 10.3897/mycokeys.116.150363 71