Two new species of Cladosporium from leaf spots of Parispolyphylla in north-western Yunnan Province, China.

BACKGROUND: During the survey of pathogenic fungi on medicinal plant leaves in Yunnan Province, China, two Cladosporium-like taxa were isolated from leaf spots of Parispolyphylla. Based on morphological characteristics and phylogenetic analysis of combined ITS, TEF1-α and ACT genes, two new species were discovered. NEW INFORMATION: Two new species Cladosporiumyunnanensis and C.paris are introduced, the detailed descriptions and illustrations are provided. Morphology of the two new species is compared with other related Cladosporium species. This study widens the host diversity of the genus Cladosporium. Yue-Xin Xu, Hong-Wei Shen, Dan-Feng Bao, Zong-Long Luo, Hong-Yan Su, Yu-E Hao.

Introduction

is one of the largest and most heterogeneous genera of hyphomycetous fungi (Dugan et al. 2004). It was initially described by Persoon (1794) from rotten wood as Pers., which was later synonymised by Link (1816) as (Pers.: Fr.). is currently only known as the asexual morph, which is characterised by erect, straight or geniculate conidiophores, abundant branched acropetal chains of smooth to roughened dry conidia produced from mono- or polyblastic conidiogenous cells, the coronate structure of conidiogenous loci and conidial hila, consisting of a central convex dome surrounded by a raised periclinal rim (David 1997, Crous et al. 2007).

To clarify the relationship of species in the complex , subsequent researchers have been constantly revising this genus and the use of molecular analysis is necessary as well as morphological characters (David 1997, Dugan et al. 2004, Heuchert et al. 2005, Schubert 2005, Schubert et al. 2007, Crous et al. 2007, Sandoval-Denis et al. 2016, Bezerra et al. 2017, Bezerra et al. 2017, Abdollahzadeh et al. 2020). Some phylogenetic studies have proposed a multi-locus sequence analysis approach to clarify species diversity within the genus with internal spacers of the rDNA genes (ITS), translation elongation factor 1-α (TEF1-α) and actin (ACT) (Bensch et al. 2012, Bensch et al. 2015, Bensch et al. 2018, Tibpromma et al. 2019, Iturrieta-González et al. 2021, Zimowska et al. 2021). Based on the phylogenetic analyses and morphological features, about 237 species have been accepted within the genus, which are split into three species complexes, (Schubert et al. 2007), (Zalar et al. 2007, Dugan et al. 2008) and (Bensch et al. 2010).

The species of are able to colonise a wide range of substrates and can be isolated in any natural or anthropogenically-affected environment (Flannigan et al. 2002, Bensch et al. 2010, Bensch et al. 2012, Bensch et al. 2018, Sandoval-Denis et al. 2015, Temperini et al. 2018, Chung et al. 2019). They are well known as plant pathogens, which may occur on leaves, stems and fruits on different plants, for example, , , , , and (Schubert 2005, Bensch et al. 2012, Bensch et al. 2015, Marin-Felix et al. 2017, Rosado et al. 2019). Besides, some species have been reported as pathogens of animals and humans, saprobes and endophytes and been also reported as hyperparasites on other fungi (Sandoval-Denis et al. 2015, Sandoval-Denis et al. 2016, Zhou et al. 2016, Velázquez-Jiménez et al. 2019). Furthermore, some species have shown the ability to produce medicinal compounds or their potential as biological agents to control plant diseases (Köhl et al. 2015, Khan et al. 2016, Adorisio et al. 2019).

During the investigation of pathogenic fungi on leaf spots of medicinal plants in Yunnan Province, China, two new species and were identified, based on morphology and multi-gene phylogenetic analysis. Full descriptions, illustrations and update of the phylogenetic backbone tree for are provided as well.

Materials and methods

Isolation and morphological examination

Leaf specimens with disease symptoms of cultivated were collected from Dali, Yunnan Province, China in October and November 2020 and taken back to the laboratory in an envelope. The leaves were kept at 4°C in Zip-lock plastic bags before they were processed in the laboratory. Single spore isolations were made onto potato dextrose agar (PDA). After 8–10 hours, a single germinating conidia was transferred aseptically to a new PDA plate to obtain cultures and grow at 20–25°C in daylight (Chomnunti et al. 2014).

The cultures are deposited in Kunming Institute of Botany, Chinese Academy of Sciences (KUNCC) and China General Microbiological Culture Collection Center (CGMCC). Cultures are deposited at the Herbarium of Cryptogams Kunming Institute of Botany Academia Sinica (Herb. HKAS). Facesoffungi and Index Fungorum numbers were obtained as in Jayasiri et al. (2015) and Index Fungorum.

DNA extraction, PCR amplification and sequencing

Genomic DNA was extracted from fresh mycelium grown on PDA at room temperature (25°C). The TreliefTM Plant Genomic DNA Kit (TSP101) was used to extract DNA according to the manufacturer’s instructions. ITS, TEF1-α and ACT gene regions were amplified using the primer pairs ITS1/ITS4, EF1-728F/EF1-986R and ACT–512F/ACT–783R. The final volume of the PCR reaction was 25 µl and contained 12.5 µl of 2 × Power Taq PCR MasterMix (a premix and ready to use solution, including 0.1 Units/µl Taq DNA Polymerase, 500 µM dNTP Mixture each (dATP, dCTP, dGTP, dTTP), 20 mM Tris–HCl pH 8.3, 100 mM KCl, 3 mM MgCl2, stabiliser and enhancer), 1 μl of each primer (10 μM), 1 µl genomic DNA extract and 9.5 µl deionised water. The PCR thermal cycle programme for ITS, TEF1-α and ACT amplification was as follows: initial denaturation of 94°C for 3 minutes, followed by 35 cycles of denaturation at 94°C for 45 seconds, annealing at 56°C for 50 seconds, elongation at 72°C for 1 minute and the final extension at 72°C for 10 minutes. PCR products were purified using minicolumns, purification resin and buffer according to the manufacturer’s protocols (Amershamproduct code: 27–9602–01). The sequencing works were carried by Tsingke Biological Engineering Technology and Services Co., Ltd (Yunnan, P.R. China).

Phylogenetic analysis

Sequence data for relevant strains were downloaded from GenBank following latest publications (Freitas 2018, Iturrieta-González et al. 2021, Zimowska et al. 2021). The sequences aligned using MAFFTv.7 (http://mafft.cbrc.jp/alignment/server/) (Katoh and Standley 2013) and optimised manually when needed. The aligned dataset was analysed by Maximum Likelihood (ML) and Bayesian Inference (BI).

Maximum Likelihood analysis was performed by using RAxMLGUI v.1.3 (Silvestro and Michalak 2012). The optimal ML tree search was conducted with 1,000 separate runs using the default algorithm of the programme from a random starting tree for each run. The final tree was selected amongst suboptimal trees from each run by comparing the likelihood scores using the GTR+GAMMA substitution model. Maximum Likelihood bootstrap values equal to or greater than 70% were given as the first set of numbers above the nodes in the resulting ML tree (Fig. 1).

Figure 1.

Maximum Likelihood (ML) tree obtained from the combined analysis of ITS, TEF1-α and ACT sequences of 161 strains from . The tree is rooted with (CBS 185.58) and (CBS 126499). Numbers on the branches represent ML bootstrap support values (MLBS) ≥70%, followed by Bayesian posterior probabilities (PP) ≥ 0.95, lower values are indicated as “-”. Names of species newly described are indicated in red and ex-type strains and reference specimens are indicated in bold. Branch lengths are proportional to distance.

Bayesian analysis was conducted with MrBayes v.3.1.2 (Ronquist and Huelsenbeck 2003) to evaluate posterior probabilities (Rannala and Yang 1996) by Markov Chain Monte Carlo sampling (MCMC). The best-fit models of evolution were estimated by MrModeltest v.2.2 (Nylander 2004). ITS selected the SYM+I+G model with inverse gamma-distributed rate in Bayesian analyses. TEF1-α and ACT selected the GTR+I+G model with inverse gamma-distributed rate in Bayesian analyses. The robustness of ML analyses was evaluated by bootstrap support (MLBS). The parameter settings, used in these analyses, were two simultaneous runs of 10,000,000 generations and four Markov chains, sampled every 1,000 generations. The 50% majority rule consensus tree and posterior probability values (PP) were calculated after discarding the first 25% of the samples. A PP value of ≥ 0.95 was considered significant (Hespanhol et al. 2019).

The phylogenetic trees were viewed and optimised in FigTree v.1.2.2 (Rambaut and Drummond 2008) and edited further using Microsoft Office PowerPoint. Newly-generated sequences in this study were deposited in GenBank (Table 1).

Table 1.

Isolates and sequences used in this study (newly-generated sequences are indicated with a “*”, strains isolated from the holotype and reference specimens are indicated in bold).

Species	Strain number	GenBank Accession Numbers
		ITS	TEF1-α	ACT
Cladosporiumacalyphae	CBS 125982	NR_119835	HM148235	HM148481
C.aciculare	CBS 140488	KT600411	KT600509	KT600607
C.aerium	DTO 323-G7	MF472899	MF473326	MF473749
C.aggregatocicatricatum	CBS 140493	NR_152300	KT600547	KT600645
C.alboflavescens	UTHSC DI-13-225	LN834420	LN834516	LN834604
C.allicinum	CBS 121624	NR_152266	EF679425	EF679502
C.allicinum	UTHSC DI-13-176	LN834354	LN834450	LN834538
C.allii	CBS 101.81	JN906977	JN906983	JN906996
C.angulosum	COAD 2500	MK253346	MK293786	MK249989
C.angustiherbarum	CBS 140479	NR_152286	KT600475	KT600574
C.angustisporum	CBS 125983	NR_111530	HM148236	HM148482
C.angustiterminale	CBS 140480	NR_152287	KT600476	KT600575
C.antarcticum	CBS 690.92	NR_121332	EF679405	EF679484
C.anthropophilum	CPC 22393	MF472922	MF473349	MF473772
C.aphidis	CBS 132182	JN906978	JN906984	JN906997
C.arenosum	CHFC-EA 566	MN879328	MN890011	MN890008
C.arthropodii	CBS 124043	NR_120011	JN906985	JN906998
C.asperulatum	CBS 126340	NR_119836	HM148239	HM148485
C.australiense	CBS 125984	NR_119837	HM148240	HM148486
C.austroafricanum	CBS 140481	NR_152288	KT600478	KT600577
C.austrohemisphaericum	CBS 140482	KT600382	KT600479	KT600578
C.basiinflatum	CBS 822.84	NR_111531	HM148241	HM148487
C.caprifimosum	FMR 16532	LR813198	LR813210	LR813205
C.chalastosporoides	CBS 125985	NR_119838	HM148242	HM148488
C.chasmanthicola	CPC 21300	NR_152307	KY646227	KY646224
C.chubutense	CBS 124457	NR_119728	FJ936161	FJ936165
C.cladosporioides	CBS 112388	NR_119839	HM148244	HM148490
C.cladosporioides	CBS 113738	HM148004	HM148245	HM148491
C.colocasiae	CBS 386.64	NR_119840	HM148310	HM148555
C.colocasiae	CBS 119542	HM148066	HM148309	HM148554
C.colombiae	CBS 274.80B	NR_119729	FJ936163	FJ936166
C.coprophilum	FMR 16164	LR813201	LR813213	LR813207
C.crousii	CBS 140686	LN834431	LN834527	LN834615
C.cucumerinum	CBS 171.52	NR_119841	HM148316	HM148561
C.cucumerinum	CBS 176.54	HM148078	HM148322	HM148567
C.cycadicola	CPC 17251	KJ869122	KJ869236	KJ869227
C.delicatulum	CBS 126344	MH863920	HM148325	HM148570
C.dominicanum	CBS 119415	DQ780353	JN906986	EF101368
C.echinulatum	CBS 123191	JN906980	JN906987	JN906999
C.europaeum	FP-027-A9	MH102078	MH102121	MH102068
C.exasperatum	CBS 125986	NR_119843	HM148334	HM148579
C.exile	CBS 125987	NR_111532	HM148335	HM148580
C.fildesense	F09-T12-1	JX845290	MN233633	MN233632
C.flabelliforme	CBS 126345	NR_119844	HM148336	HM148581
C.flavovirens	UTHSC DI-13-273	LN834440	LN834536	LN834624
C.floccosum	CBS 140463	LN834416	LN834512	LN834600
C.funiculosum	CBS 122129	NR_119845	HM148338	HM148583
C.funiculosum	CBS 122128	HM148093	HM148337	HM148582
C.fuscoviride	FMR 16385	LR813200	LR813212	LR813206
C.fusiforme	CBS 119414	DQ780388	JN906988	EF101372
C.gamsianum	CBS 125989	NR_111533	HM148339	HM148584
C.globisporum	CBS 812.96	NR_111534	HM148340	HM148585
C.grevilleae	CBS 114271	NR_119960	JF770472	JF770473
C.halotolerans	CBS 119416	DQ780364	JN906989	EF101397
C.herbaroides	CBS 121626	NR_119655	EF679432	EF679509
C.herbarum	CBS 121621	NR_119656	EF679440	EF679516
C.hillianum	CBS 125988	NR_119846	HM148341	HM148586
C.inversicolor	CBS 401.80	NR_111535	HM148345	HM148590
C.ipereniae	CBS 140483	NR_152290	KT600491	KT600589
C.iranicum	CBS 126346	NR_111536	HM148354	HM148599
C.iridis	CBS 138.40	NR_111271	EF679447	EF679523
C.kenpeggii	CPC 19248	KY646222	KY646228	KY646225
C.langeronii	CBS 189.54	DQ780379	JN906990	EF101357
C.lentulum	FMR 16288	LR813203	LR813215	LR813209
C.licheniphilum	CBS 125990	NR_119847	HM148355	HM148600
C.limoniforme	CBS 140484	KT600397	KT600494	KT600592
C.longicatenatum	CBS 140485	NR_152291	KT600500	KT600598
C.longissimum	CBS 300.96	DQ780352	EU570259	EF101385
C.lycoperdinum	CBS 126347	MH863923	HM148356	HM148601
C.lycoperdinum	CBS 574.78C	HM148115	HM148359	HM148604
C.macrocarpum	CBS 121623	NR_119657	EF679453	EF679529
C.macrocarpum	UTHSC DI-13-191	LN834379	LN834475	LN834563
C.magnoliigena	MFLUCC 18-1559	MK347813	MK340864	-
C.montecillanum	CBS 140486	NR_152292	KT600504	KT600602
C.montecillanum	CPC 15605	KT600407	KT600505	KT600603
C.myrtacearum	CBS 126349	MH863925	HM148360	HM148605
C.myrtacearum	CBS 126350	NR_119849	HM148361	HM148606
C.needhamense	Z-1866	MF473142	MF473570	MF473991
C.neopsychrotolerans	CGMCC3.18031	KX938383	KX938400	KX938366
C.ossifragi	CBS 842.91	NR_121333	EF679459	EF679535
C.oxysporum	CBS 125991	NR_152267	HM148362	HM148607
C.oxysporum	CBS 126351	MH863927	HM148363	HM148608
C.paracladosporioides	CBS 171.54	NR_119850	HM148364	HM148609
C.paralimoniforme	CGMCC3.18103	KX938392	KX938409	KX938375
C.paralimoniforme	CGMCC3.18104	KX938393	KX938410	KX938376
C.parapenidielloides	CBS 140487	NR_152293	KT600508	KT600606
C.parasubtilissimum	CPC 22396	MF473171	MF473594	MF474019
C.paris sp. nov.*	KUN HKAS 121701*	OK338503*	OL825681*	OL466938*
C.penidielloide	CBS 140489	KT600412	KT600510	KT600608
C.perangustum	CBS 125996	NR_119851	HM148365	HM148610
C.phaenocomae	CBS 128769	NR_119950	JF499875	JF499881
C.phlei	CBS 358.69	NR_120013	JN906991	JN907000
C.phyllactiniicola	CBS 126355	NR_111537	HM148397	HM148642
C.phyllophilum	CBS 125992	NR_111538	HM148398	HM148643
C.pini-ponderosae	CBS 124456	NR_119730	FJ936164	FJ936167
C.prolongatum	CGMCC3.18036	KX938394	KX938411	KX938377
C.pseudiridis	CBS 116463	NR_111272	EF679461	EF679537
C.pseudochalastosporoides	CBS 140490	NR_152296	KT600513	KT600611
C.pseudocladosporioides	CBS 125993	NR_119852	HM148402	HM148647
C.pseudotenellum	FMR 16231	LR813145	LR813196	LR813146
C.psychrotolerans	CBS 119412	DQ780386	JN906992	EF101365
C.puris	COAD 2494	MK253338	MK293778	MK249981
C.puyae	CBS 274.80A	NR_152298	KT600516	KT600614
C.ramotenellum	CBS 121628	NR_119658	EF679462	EF679538
C.rectoides	CBS 125994	NR_111539	HM148438	HM148683
C.rectoides	CBS 126357	MH863933	HM148439	HM148684
C.rhusicola	CBS 140492	NR_152299	KT600539	KT600637
C.rubrum	CMG 28	MN053018	MN066644	MN066639
C.ruguloflabelliform	CBS 140494	KT600458	KT600557	KT600655
C.rugulovarians	CBS 140495	KT600459	KT600558	KT600656
C.salinae	CBS 119413	DQ780374	JN906993	EF101390
C.scabrellum	CBS 126358	NR_119853	HM148440	HM148685
C.silenes	CBS 109082	NR_111270	EF679429	EF679506
C.sinense	CBS 143363	MF473252	MF473675	MF474102
C.sinuatum	CGMCC3.18096	KX938385	KX938402	KX938368
C.sinuosum	CBS 121629	NR_119659	EF679464	EF679540
C.soldanellae	CPC 13153	NR_120014	JN906994	JN907001
C. sp.	UTHSC DI-13-227	LN834422	LN834518	LN834606
C. sp.	UTHSC DI-13-245	LN834429	LN834525	LN834613
C. sp.	UTHSC DI-13-265	LN834435	LN834531	LN834619
C. sp.	UTHSC DI-13-218	LN834418	LN834514	LN834602
C. sp.	UTHSC DI-13-210	LN834414	LN834510	LN834598
C.sphaerospermum	CBS 193.54	NR_111222	EU570261	EU570269
C.spinulosum	CBS 119907	NR_119660	EF679466	EF679542
C.subcinereum	UTHSC DI-13-257	NR_148193	LN834529	LN834617
C.subinflatum	UTHSC DI-13-189	LN834391	LN834487	LN834575
C.subinflatum	CBS 121630	NR_119661	EF679467	EF679543
C.submersum	FMR 17264	LR813144	LR813197	LR813195
C.subtilissimum	CBS 113754	NR_111273	EF679475	EF679551
C.subtilissimum	CBS 113753	EF679396	EF679474	EF679550
C.subuliforme	CBS 126500	NR_119854	HM148441	HM148686
C.subuliforme	CPC 15833	KT600453	KT600552	KT600650
C.succulentum	CBS 140466	LN834434	LN834530	LN834618
C.tenellum	CBS 121634	NR_119662	EF679479	EF679555
C.tenellum	CPC 22410	MF473280	MF473703	MF474130
C.tenellum	CPC 12051	EF679400	EF679478	EF679554
C.tenellum	CPC 22291	MF473279	MF473702	MF474129
C.tenellum	CPC 22290	MF473278	MF473701	MF474128
C.tenuissimum	CBS 125995	NR_119855	HM148442	HM148687
C.tianshanense	CGMCC3.18033	KX938381	KX938398	KX938364
C.tuberosum	UTHSC DI-13-219	LN834419	LN834515	LN834603
C.uredinicola	CPC 5390	AY251071	HM148467	HM148712
C.uwebrauniana	DTO 072-D8	MF473306	MF473729	MF474156
C.uwebraunianum	DTO 305-H9	MF473307	MF473730	MF474157
C.variabile	CBS 121635	NR_119663	EF679481	EF679557
C.varians	CBS 126362	NR_119856	HM148470	HM148715
C.velox	CBS 119417	DQ780361	JN906995	EF101388
C.verrucocladosporioides	CBS 126363	NR_111540	HM148472	HM148717
C.verruculosum	CGMCC3.18099	KX938388	KX938405	KX938371
C.verruculosum	CGMCC3.18100	KX938389	KX938406	KX938372
C.versiforme	CBS 140491	NR_152297	KT600515	KT600613
C.vicinum	CPC 22316	MF473311	MF473734	MF474161
C.vignae	CBS 121.25	HM148227	HM148473	HM148718
C.welwitschiicola	CPC 18648	NR_152308	KY646229	KY646226
C.westerdijkiae	CBS 113746	HM148061	HM148303	HM148548
C.wyomingense	CPC 22310	MF473315	MF473738	MF474165
C.xanthochromaticum	CBS 126364	HM148122	HM148366	HM148611
C.xantochromaticum	CBS 140691	LN834415	LN834511	LN834599
C.xylophilum	CBS 125997	NR_111541	HM148476	HM148721
C.xylophilum	CBS 113749	HM148228	HM148474	HM148719
C.yunnanensis sp. nov.*	KUN HKAS 121704*	OK338502*	OL825680*	OL466937*
Toxicocladosporiumirritans	CBS 185.58	NR_152316	-	LT821375
Toxicocladosporiumprotearum	CBS 126499	NR_152321	-	LT821379

Taxon treatments

H.W. Shen, Y.X. Xu, H.Y. Su & Z.L. Luo sp. nov.

C6DA32AB-EEE6-595C-98BA-4DA0C7F227ED

558843

Facesoffungi number: FoF 10538

Type status: Holotype. Occurrence: recordedBy: Yue-Xin Xu; Taxon: scientificName: ; kingdom: ; phylum: ; class: ; order: ; family: ; genus: ; Location: locationRemarks: China, Yunnan Province, Dali, on diseased leaves of ; Event: day: 2020; habitat: leaf spots of ; Record Level: collectionID: 1CL JD 5-1-4; collectionCode: Y-23

Description

Asexual morph: hyphomycetous (Fig. 2). Mycelium superficial and immersed, composed of septate, branched, subhyaline, smooth-walled, 1–3 μm wide. Conidiophores macronematous, 127–190 × 4–6 μm (x̄ = 158.2 × 5.1 μm, n = 15), solitary or in small loose groups, erect to slightly flexuous, non-nodulose, sometimes subnodulose at the uppermost apex, unbranched, 0–6 septate, sometimes slightly constricted at septa, pale brown, smooth, sometimes somewhat irregularly rough-walled or verruculose. Conidiogenous cells terminal and intercalary, loci crowded at the apex forming clusters of pronounced scars, 1–2 conidiogenous loci formed at about the same level, loci often situated at lateral shoulders due to sympodial proliferation, loci 1–2 μm diam. Conidia solitary or in short unbranched chains, straight to slightly curved, cylindrical-oblong, 7–19 × 5–7 μm (x̄ = 13.2 × 5.7 μm, n = 30), 0–3 septate, sometimes slightly constricted at the septa, pale to pale medium olivaceous-brown. Sexual morph: Undetermined.

Figure 2.

(KUN-HKAS 121704, holotype). a Colonies; b-c Conidiophores; d-g Conidiogenous cells with conidia; h-m Conidia; n Germinating conidium; o Culture on PDA from above and reverse. Scale bars: b-d = 20 μm; e-k = 15 μm; l = 30 μm.

Culture characteristics: Colonies on PDA attaining 25 mm diam. after 7 d, 45 mm diam. after 14 d and covering the whole Petridish after 30 d, dark green to olive green, velvety, furrowed; reverse dark green to black.

Material examined: China, Yunnan Province, Dali, on diseased leaves of , 2 October 2020, Y.X. Xu, Y-23. (KUN-HKAS 121704, holotype), ex-type living culture CGMCC 3.20622 = KUNCC 21-10712

Etymology

“” refers to Yunnan Province, China, where the species was collected.

Distribution

China, Yunnan Province, Dali, on diseased leaves of

Notes

Based on the multi-locus phylogenetic analysis (Fig. 1), grouped in a well-supported clade, together with and . However, the genetic distance allows it to be considered a distinct species within the clade (Fig. 1). Morphologically, has much shorter conidiophores than (up to 190 μm vs. up to 350 μm), but longer than (up to 190 μm vs. up to 150 μm). Moreover, the new species differs from by the smaller conidiogenous cells (7–19 × 5–7 μm vs. 4–18 × 2–5 μm), but larger than (7–19 × 5–7 μm vs. 4–18 × 2–5 μm) (Bensch et al. 2012, Jayasiri et al. 2019). The BLAST analysis of TEF1-α and ACT shows that (KUN-HKAS 121704) is different from (CBS 112388) by 16 and 10 nucleotide differences, respectively and the comparison of TEF1-α between (KUN-HKAS 121704) and (CBS 140463) reveals 33 nucleotide differences.

H.W. Shen, Y.X. Xu, H.Y. Su & Z.L. Luo sp. nov.

3C7F2601-1E71-567A-9D18-73083DAB684B

558844

Facesoffungi number: FoF 10539

Type status: Holotype. Occurrence: recordedBy: Yue-Xin Xu; Taxon: scientificName: ; kingdom: ; phylum: ; class: ; order: ; family: ; genus: ; Location: locationRemarks: China, Yunnan Province, Dali, on diseased leaves of ; Event: year: 2020; habitat: leaf spots of ; Record Level: collectionID: 2CL JD 18-2-1; collectionCode: Y-27

Asexual morph: hyphomycetous (Fig. 3). Mycelium immersed and superficial, composed of septate, constricted at septa, unbranched, subhyaline, smooth hyphae, 2–6 μm wide. Conidiophores macronematous, 209–285 × 5–8 μm (x̄ = 246.9 × 6.5 μm, n = 15), solitary or in small fascicles, erect to slightly flexuous, sometimes slightly geniculate, non-nodulose, sometimes subnodulose at the uppermost apex, unbranched, 0–6 septate, sometimes slightly constricted at septa, pale to olivaceous-brown, smooth or almost so. Conidiogenous cells cylindrical, sometimes geniculate-sinuous, proliferation of sympodia with up to 5 conidiogenous loci, often crowded at the apex. Conidia 13–21 × 7–12 μm (x̄ = 17 × 9.7 μm, n = 30), solitary or catenate, usually in simple chains, broadly ellipsoid-ovoid, rather pale, pale olivaceous or olivaceous-brown, verruculose, ends usually broadly rounded. Sexual morph: Undetermined.

Figure 3.

(KUN-HKAS 121701, holotype). a Colonies on leaves; b,c Conidiophores; d Conidiophore with conidium; e,f Conidiogenous cells with conidia; g-l Conidia; m Germinating conidium; n,o Culture on PDA from above and reverse. Scale bars: b-d = 50 μm; e,f = 30 μm; g-m = 20 μm.

Culture characteristics: Colonies on PDA attaining 21 mm diam. after 7 d, 40 mm diam. after 14 d and covering the whole Petridish after 30 d, radially folded, furrowed, margin irregularly undulate; reverse olivaceous grey.

Material examined: China, Yunnan Province, Dali, on diseased leaves of , 16 October 2020, Y.X. Xu, Y-27. (KUN-HKAS 121701, holotype), ex-type living culture CGMCC 3.20623 = KUNCC 21-10713.

“” refers to the host genus, .

China, Yunnan Province, Dali, on diseased leaves of

Phylogenetic analysis showed that is closely related to (Fig. 1). Morphologically, our new isolate is distinguished from by its longer conidiophores (up to 285 μm vs. up to 100 μm) and larger conidiogenous cells (13–21 × 7–12 μm vs. 8–15 × 6–8.5 μm). In addition, conidia of are 0–3 septate, while are 0–1 septate (Sandoval-Denis et al. 2016). A comparison of the TEF1-α and ACT between (KUN-HKAS 121701) and (CBS 140463) reveals 3 and 16 nucleotide differences, respectively, which indicates that they are distinct taxa.

Analysis

Phylogenetic analysis

The combined ITS, TEF1-α and ACT dataset consisted of 161 sequences representing all genera of the with (CBS 185.58) and (CBS 126499) as outgroup taxa. The best scoring RaxML tree with the final ML optimisation likelihood value of -24601.202740 is shown here (Fig. 1). The alignment comprised 1297 characters including gaps. The matrix had 775 distinct alignment patterns, with 15.38% undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.228337, C = 0.293636, G = 0.250484, T = 0.227544; substitution rates AC = 1.726214, AG = 3.618770, AT = 1.752951, CG = 1.098108, CT = 5.802327, GT = 1.000000; Tree-Length = 7.357731.

Phylogenetic analyses of combined ITS, TEF1-α and ACT sequence data showed that the two new isolates of (KUN-HKAS 121704) and (KUN-HKAS 121701) grouped with members of . (KUN-HKAS 121704) clustered with (CBS 112388 and CBS 113738) and (MFLUCC 18-1559), but in an independent lineage with significant bootstrap (86 ML/1.00 PP). (KUN-HKAS 121701) formed a distinct lineage and sister to (CBS 140463) and basal to the genus with highly-supported value (94 ML/0.98 PP).

Discussion

In our study, based on the typical morphological features (Schubert et al. 2007, Zalar et al. 2007, Dugan et al. 2008, Bensch et al. 2010), and belong to the and species complex, respectively. The ITS sequences of the two new species are identical under the common barcode for fungi as previously reported studies for many other species (Bensch et al. 2010, Bensch et al. 2012, Marin-Felix et al. 2017). Therefore, multi-gene phylogenetic analysis (ITS, TEF1-α and ACT) can further prove the taxonomy of the two species in , which is consistent with the result by morphological features.

species are found as the dominant fungal genera in indoor and outdoor environments and are also important as saprobes and endophytes which have been screened from grains, fruits and chilled meat (Fradkin et al. 1987, Bullerman 2003, Hassan et al. 2021). However, and have been isolated from leaves of in Yunnan Province, China for the first time. Studies indicate that investigation on new hosts for fungi diversity would lead to the discovery of new fungal species and expand species resources (Hyde et al. 2018, Hyde et al. 2020). Certain species have been reported as producers of mycotoxin and to cause fungal allergies, particularly rhinitis and asthma. (Horner et al. 1995, Kurup 2003, Matheson et al. 2005, Simon-Nobbe et al. 2008, Mercier et al. 2013, Alwatban et al. 2014 , Segers et al. 2015). Both new species are isolated from diseased spots on plant leaves and many species of this genus are reported as plant pathogens, so they also have the potential to cause plant diseases. To determine whether these fungi are plant pathogens or have long-term adverse reactions on human health, pathogenicity determination and secondary metabolites of can be the focus of our future research.