Rust Fungi on Medicinal Plants in Guizhou Province with Descriptions of Three New Species

Abstract

During the research on rust fungi in medicinal plants of Guizhou Province, China, a total of 9 rust fungal species were introduced, including 3 new species (Hamaspora rubi-alceifolii, Nyssopsora altissima, and Phragmidium cymosum), as well as 6 known species (Melampsora laricis-populina, Melampsoridium carpini, Neophysopella ampelopsidis, Nyssopsora koelrezidis, P. rosae-roxburghii, P. tormentillae). Notably, N. ampelopsidis and P. tormentillae were discovered for the first time in China, while M. laricis-populina, Me. carpini, and Ny. koelreuteriae were first documented in Guizhou Province. Morphological observation and molecular phylogenetic analyses of these species with similar taxa were compared to confirm their taxonomic identities, and taxonomic descriptions, illustrations and host species of those rust fungi on medicinal plant are provided.

1. Introduction

Rust fungi comprise the largest and most ubiquitous group of obligately biotrophic fungi on vascular plants [1]. The economic impact of rust fungi cannot be ignored. Because of rust fungi, many economic plants suffer diseases and reduce yields [2]. For example, when wheat is harmed by Puccinia striiformis Westend., its yield can be reduced up to 50% [3]. The impact of rust fungi on tropical crops is also immeasurable [4]. The coffee rust fungus has a global distribution and is often found in the coffee growing areas of China, with a greater impact on the main coffee producing countries [2,5,6].

More than 8000 species of rust fungi have been identified, mainly on the basis of their morphological characteristics of teliospores and spermonogia, however, there are still a larger number of genera incertae sedis [7,8,9,10,11]. Rust occurs on ferns to advanced monocots and dicots, and they are obligate biotrophic phytopathogens that produce not only basidiospore but also four other different types of spores: aeciospore, urediniospore, teliospore, and spermatia [8,12,13]. Many species in the order Pucciniales were not described with all types of spores, and have various lifestyles (micro-, hemi-, demi-, or macrocyclic), with alternation on single (autoecious) or two unrelated host plants (heteroecious) [7,14]. To date, approximately 8400 rust species are currently recognized worldwide, and 71 genera and 1175 species have been discovered in China so far [11]. However, species diversity, host alternation and geographic distribution of rust fungi in China remain poorly understood.

Medicinal plants are also infected by rust fungi on a large scale [15]. By the end of 2020, a total of 79 rust species have been reported on 76 medicinal plant species from 33 families, and these rust fungi restricts the development and utilization of medicinal plants and affects the quality of botanicals [16,17]. In recent years, as many as 3924 species of medicinal plants are cultivated in Guizhou province, and the number of medicinal plants is increasing number year by year [18]. As important pathogenic fungi, rust can infect the leaf and stem of a variety of medicinal plants and affect their quality and yield which eventually hampered the development and utilization of medicinal plant resources [19]. For example, rust diseases are frequently found on pepper leaves in Guizhou province, and those diseases seriously affect the normal development of pepper, with their incidence reaching 90% in serious infections [20]. However, there are few researches on rust species infecting medicinal plant diseases in Guizhou province [21]. Therefore, it is of great significance to investigate the species diversity of rust fungi on important medicinal plants for local medicinal production.

In 2021, an investigation of rust fungi on the medicinal plants was carried out in Guizhou province, China. Nine species including three new species were found on medicinal plants. Detailed descriptions and illustrations of all those novel species and other species on the medicinal plants are provided.

2. Materials and Methods

2.1. Sample Collections

Rust infected specimens were collected from Guiyang, Qingzhen and Anshun cities in Guizhou province, China. All hosts and habitats information of specimens was recorded. For each specimen, part of specimens was kept in a refrigerator at 4 °C, and the other part was made as a dry specimen [22]. Specimens were deposited in both Mycological Herbarium of the Chifeng University, Inner Mongolia, China (CFSZ) and Herbarium of Guizhou Medical University (GMB).

2.2. Morphology

The specimens were observed under a stereomicroscope (Nikon SMZ745T, Nikon Corporation, Tokyo, Japan) and shot with a Canon digital camera (Canon EOS 1500D, Canon Inc., Tokyo, Japan) fitted on. Microscope images of the samples were taken by a Canon EOS 700D digital camera fitted on the Nikon ECLIPSE Ni compound microscope (Nikon, Japan). Measurements were taken with the Tarosoft (R) Image Frame Work (v.0.9.7). More than 30 morphological characteristics such as teliospores, urediniospores, and paraphyses were measured for each specimen. Photo plates were arranged by using Adobe Photoshop CS6 v. 13 (Adobe Systems Software Ireland Ltd, San Jose, USA). The different spore stages of rust fungi are designated by the following Roman numerals: spermogonia/spermatia (0), aecia/aeciospores (I), uredinia/urediniospores (II), telia/teliospores (III), and basidia/basidiospore (IV). We applied the definitions of spore stage based on Cummins and Hiratsuka [7], and followed morphological types of spermogonia designated by Hiratsuka and Hiratsuka [23].

2.3. DNA Extraction, Polymerase Chain Reaction (PCR), and Sequencing

The rust sori were picked out into a sterilized centrifuge tube with a sterilized fine needle for DNA extraction. Genomic DNA was extracted following the manufacturer’s protocol of the OMEGA E.Z.N.A.^® Fungal Genomic DNA Extraction Kit (D3390, Guangzhou Feiyang Bioengineering Co., Ltd., Guangzhou, China). DNA extracts were stored at –20 °C. PCR was carried out in a volume of 25 μL containing 9.5 μL of ddH₂O, 12.5 μL of 2 × Taq PCR Master Mix (2 × Taq Master Mix with dye, TIANGEN, China), 1 μL of DNA extraction and 1 μL of forward and reverse primers (10 µm each) in each reaction. Primers pairs, ITS4/ITS5 and LR0R/LR5 (Sangon Biotech, Shanghai, China) were used to amplify the regions of internal transcribed spacer (ITS) and large subunit ribosomal (LSU), respectively [8,24,25,26]. PCR profiles for the ITS and LSU were: initially at 95 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 52 °C for 1 min, polymerization at 72 °C for 1.5 min and a final extension at 72 °C for 10 min. PCR products were sequenced by Sangon Biotech (Shanghai) Co., Ltd., China.

2.4. Phylogenetic Studies

All sequences used for phylogenetic tree construction were listed in

Table 1. GenBank accession number and information of taxa used for phylogenetic analyses.

Family Name	Species	Specimen No.	Host	Province, Country	GenBank Accession No.		Reference
					ITS	LSU
Chrysomyxaceae	Chrysomyxa empetri	QFB 25015	Empetrum nigrum	Ste-Anne-des-Monts, Quebec, Canada	GU049434	GU049526	[36]
	Rossmanomyces monesis	DAOM 221982	Moneses (=Pyrola) uniflora	Graham Island, British Columbia, Canada	GU049476	GU049547	[36]
Gymnosporangiaceae	Gymnosporangium asiaticum	CUP-0016	Juniperus chinensis	Gifu prefecture, Japan	MN642593	MN642617	[37]
	Gymnosporangium clavariiforme	BRIP 59471	Crataegus sp.	—	—	MW049261	[10]
	Gymnosporangium confusum	LD 1021	Crataegus monogyna	Turkey	HM114219	HM114219	—
	Gymnosporangium cupressi	RSP9998	—	—	KJ720169	KJ720169	—
	Gymnosporangium juniperi-virginianae	MCA3585	Cupressaceae juniperus	Virginia, America	—	MG907217	[10]
	Gymnosporangium sabinae	TNM F0030477	Pyrus communis	Bulgaria: Sofia	KY964764	KY964764	[38]
	Gymnosporangium trachysorum	RN91	—	—	KJ720184	KJ720184	—
Hyalopsoraceae	Coleopuccinia sinensis	BJFC:R02506	Cotoneaster microphyllus	China	—	MF802285	[39]
	Hyalopsora aspidiotus	PUR N4641	Gymnocarpium dryopteris	China	—	MW049264	[40]
	Melampsoridium alni	H 7019539	Alnus mandshurica	Finland	KF031557	KF031534	[41]
	Melampsoridium betulinum	BPI 871107 (MCA2884)	Alnus sp.	Costa Rica	—	DQ354561	[8]
	Melampsoridium hiratsukanum	PDR #1480181	Alnus rhombifolia	USA: Santa Cruz County, California	KC313888	KC313888	[42]
	Melampsoridium carpini	KR-M-0048587	Carpinus betulus	—	MH908486	MH908486	[43]
	Melampsoridium carpini	GMB0112	Carpinus turczaninowii	Guizhou, China	OQ067091	—	This study
Melampsoraceae	Melampsora abietis-canadensis	1399MEA-POG-USA	Populus grandidentata	Wisconsin, America	JN881733	JN934918	[44]
	Melampsora abietis-populi	TSH-R20042 *	Populus yunnanensis	Yunnan, China	JN881739	JN934933	[44]
	Melampsora apocyni	LYR3	Apocynum venetum	Xinjiang, China	KR296802	KR296803	—
	Melampsora caprearum	NYS-F-003819	Salix caprea	Germany	KU550034	KU550033	[45]
	Melampsora coleosporioides	HNMAP3114	Salix sp.	Japan	KF780755	KF780638	[46]
	Melampsora epiphylla	TSH-R3884	Salix bakko	Japan	KF780787	KF780670	[45]
	Melampsora epitea	TNS-F-121034	Salix viminalis	Germany	KX386070	KX386097	[35]
	Melampsora euphorbiae	HMAS350000	Euphorbia kansui	Guangxi, China	MK518875	MK518545	[11]
	Melampsora euphorbiae	BRIP 39560	Euphorbia peplus	Queensland, Australia	—	EF192199	[40]
	Melampsora euphorbiae	BPI 871135 (U681)	Euphorbia heterophylla	Oman	—	DQ351722	[47]
	Melampsora humilis	TSH-R7550	Salix koriyanagi	Miyagi, Japan	KF780812	—	[46]
	Melampsora hyperici-sampsonii	HMAS350001 *	Hypericum sampsonii	Guangxi, China	MK518877	MK518547	[11]
	Melampsora iranica	HMAAC4055	Salix sp.	China	MK372158	MK372191	[48]
	Melampsora kamikotica	HNMAP3186	Chosenia arbutifolia	China	KF780760	KF780643	[46]
	Melampsora laricis-pentandrae	HNMAP3201	Salix pentandra	Inner Mongolia, China	KF780801	KF780684	[46]
	Melampsora laricis-populina	HMAS247977	Populus simonii	Haixi Qinghai, China	MK028583	MK064524	[49]
	Melampsora laricis-populina	GMB0097	Populus lasiocarpa	Guizhou, China	OQ067085	—	This study
	Melampsora larici-tremulae	PFH04-5	Populus tremula	France	JN881744	JN934956	[44]
	Melampsora medusae	BPI 0021209	—	America	JX416848	JX416843	[44]
	Melampsora medusae f.sp. deltoidis	98D10	Populus × euramericana	South Africa	GQ479307	JN934962	[44]
	Melampsora medusae f.sp. tremuloidis	1028ME-LAL-LJ	Larix laricina	Quebec, Lac Saint Jean, Peribonka, Canada	GQ479883	JN934965	[44]
	Melampsora microsora	TSH-R7335	Salix subfragilis	Hiroshima, Japan	KF780833	KF780730	[46]
	Melampsora microspora	PUR:F17540	populus nigra	Iraq	JN881737	JN934931	[44]
	Melampsora occidentalis	MO96H	Populus trichocarpa	Idaho, America	JN881740	JN934934	[44]
	Melampsora pakistanica	BA13c	Euphorbia helioscopia	Pakistan	KX237555	KX237556	[50]
	Melampsora populnea	664ME-POA-BC45.1	Populus alba	British Columbia, Canada	EU808021	FJ666510	[51]
	Melampsora populnea	97MP10	—	France	EU808035	FJ666523	[51]
	Melampsora populnea	AAH00-1	Populus alba	England	AY444772	AY444786	[52]
	Melampsora pruinosae	BPI 1109446	Populus diversifolia	Xinjiang Uygur Zizhiqu, Shule, China	GQ479899	JN934938	[44]
	Melampsora pulcherrima	O8ZK4	Mercurialis annua	Italy	GQ479320	JN934941	[44]
	Melampsora ribesii-purpureae	PURP897-1	Salix purpurea	—	AY444770	AY444791	[52]
	Melampsora euphorbiae	PDD 98363	Ricinus communis	—	—	KJ716352	[36]
	Melampsora rostrupii	01G1.1	Mercurialis perennis	France	EU808038	JN934942	[44]
	Melampsora salicis-albae	NWC-06210	Salix alba	Rothamsted, England	KF780757	KF780640	[46]
	Melampsora salicis-argyraceae	HMAS52894 *	Salix argyracea	Xinjiang, China	KF780733	KF780616	[46]
	Melampsora salicis-bakko	TSH-R3879 *	Salix bakko	—	KC631854	KC685611	[45]
	Melampsora salicis-cavaleriei	HMAAC4043	Salix serrulatifolia	China	MK277296	MK277301	[48]
	Melampsora salicis-delavayanae	HMAS248120 *	Salix delavayana	Yunnan, China	MK518954	MK518651	[11]
	Melampsora salicis-purpureae	HMAS62584 *	Salix purpurea	Shandong, China	KF780766	KF780649	[46]
	Melampsora salicis-viminalis	HMAS38658 *	Salix viminalis	Tibet, China	KF780732	KF780615	[46]
	Melampsora sp.	HMAS350003		Xinjiang, China	MK518844	MK518499	[11]
Neophysopellaceae	Neophysopella ampelopsidis	IBA-8618	Ampelopsis brevipedunculata	Kagoshima, Japan	AB354774	AB354741	[11]
	Neophysopella ampelopsidis	GMB0110	Ampelopsis sinica	Guizhou, China	OQ067090	—	This study
	Neophysopella meliosmae	IBAR-10037	Meliosma myriantha	Ibaraki, Japan	KC815591	KC815650	[53]
	Neophysopella meliosmae-myrianthae	AGLR56	—	—	MK290819	MK290819	—
	Neophysopella meliosmae-myrianthae	IBA-8584	Vitis coignetiae	Tochigi, Japan	AB354785	AB354748	[53]
	Neophysopella orientalis	IBAR-9942	Meliosma tenuis	Tochigi, Japan	KC815597	KC815656	[54]
	Neophysopella vitis-davidii	HMAS350005	Viola faurieana	Chongqing, China	MK518870	MK518536	[11]
	Neophysopella vitis-davidii	HMAS248115 *	Vitis davidii	Yunnan, China	—	MK518593	[11]
Phakopsoraceae	Masseeëlla capparis	RIP 56844	Flueggea virosa	Northern Territory, Australia	JX136798	JX136798	[13]
	Nothoravenelia japonica	HMJAU8598	—	China	—	MK296509	—
	Phakopsora argentinensis	ZT:RB 8248	Croton cf. anisodontus	—	KF528009	KF528009	[55]
	Phakopsora crcis-filii	ZT Myc 48990	Annona paludosa	Sinnamary, French Guiana	KF528016	KF528016	[55]
	Phakopsora myrtacearum	PREM 61155 *	Eucalyptus grandis	Maragua, Kenya	NR_132913	KP729473	[56]
	Phakopsora neocherimoliae	RB 3096 in ZT	Annona cherimola	San José Costa Rica	KF528011	KF528011	[55]
	Phakopsora pachyrhizi	BRIP 56941	Neonotonia wightii	Warrumbungle, New South Wales, Australia	—	KP729475	[56]
	Phakopsora phyllanthi	RB 8581	Phyllanthus acidus	Ceará, Brazil	KF528025	KF528025	[55]
	Phakopsora pistila	ZT Myc 48992	Annona sericea	French GuianaIracubo, French Guiana	KF528026	KF528026	[55]
	Phakopsora sophorae	HMAS248098 *	Leptopus chinensis	Beijing, China	—	MK518628	[11]
Phragmidiaceae	Arthuriomyces peckianus	BPI 879271	Rubus occidentalis	New York, America	GU058010	GU058010	[57]
	Gerwasia pittieriana	BPI 843556	Rubus sp.	—	KY764065	KY764065	—
	Gerwasia rubi	HMAS249978	Rubus parkeri	Yunnan, China	MK519039	MK518737	[11]
	Gerwasia rubi	HMAS249983	Rubus setchuenensis	Fujian, China	—	MK518734	[11]
	Gerwasia rubi	BRIP:58369	Rubus sp.	—	—	KT199397	[13]
	Gerwasia rubi-playfairiani	HMAS249840 *	Rubus playfairianus	Guangxi, China	MK518976	—	[11]
	Hamaspora acutissima	BRIP:55606	Rubus rolfei	Philippines	—	KT199398	[13]
	Hamaspora rubi-alceifolii	GMB0116	Rubus alceaefolius	Guizhou, China	—	OQ067533	This study
	Hamaspora rubi-alceifolii	GMB0109 *	Rubus alceaefolius	Guizhou, China	—	OQ067532	This study
	Hamaspora longissima	BPI 871506	Rubus rigidus	Eastern Cape, South Africa	—	MW049262	[40]
	Hamaspora sinica	HMAS249989	Rubus setchuenensis	Guangdong, China	MK519049	MK518636	[11]
	Kuehneola japonica	BPI 910185	Rosa sp.	—	KY764067	—	—
	Kuehneola uredinis	LD1029	Rubus sp.	New York, America	GU058013	GU058013	[57]
	Kuehneola uredinis	R216	Rubus fruticosus	Belgium	EU014068	EU014068	[58]
	Phragmidium andersonii	HMAS53231	Potentilla fruticosa	China	—	MG669120	[59]
	Phragmidium barclayi	HMAS67281	Rubus austrotibetanus	China	—	MG669117	[59]
	Phragmidium barnardii	BRIP:56945	Rubus multibracteatus	Queensland, Australia:	—	KT199402	[13]
	Phragmidium biloculare	U564	Potentilla flabellifolia	Washington, America	—	JF907670	[60]
	Phragmidium brevipedicellatum	HMUT100463	Potentilla multifida	Xinjiang, China	—	KU059170	—
	Phragmidium butleri	HMAS67841	Rosa macrophylla	China	—	MG669118	[59]
	Phragmidium chayuensis	BJFC:R02532 *	Rosa duplicata	China	MH128374	NG_064492	[59]
	Phragmidium cibanum	BJFC:R02528 *	Rubus niveus	China	MH128370	NG_064491	[59]
	Phragmidium cymosum	GMB0115	Rosa cymosa	Guizhou, China	OQ067097	OQ067531	This study
	Phragmidium cymosum	GMB0108 *	Rosa cymosa	Guizhou, China	OQ067096	OQ067530	This study
	Phragmidium duchesnea-indica	HMAS249846 *	Duchesnea indica	Yunnan, China	—	MK518681	[11]
	Phragmidium fragariae	WM 1317	Potentilla sterilis	—	—	AF426217	[61]
	Phragmidium fructigenum	HMUT100472	Rosa glomerata	Chongqing, China	—	KU059168	—
	Phragmidium fusiforme	BJFC:R00942	Rosa hugonis	China	—	KP407632	[62]
	Phragmidium griseum	HMAS56906	Rubus crataegifolius	Beijing, China	MH128377	MG669115	[59]
	Phragmidium griseum	HMAS350016	Rosa sp.	Beijing, China	—	MK518530	[11]
	Phragmidium handelii	BJFC:R01030	Rosa webbiana	China	—	KP407631	[62]
	Phragmidium ivesiae	U35	Potentilla gracilis	Utah, America	—	JF907672	[60]
	Phragmidium japonicum	HMAS41585	Rosa laevigata	Fujian, China	MN264716	MN264734	[63]
	Phragmidium jiangxiense	BJFCR 03453 *	Rosa laevigata	Jiangxi, China	MN264715	MN264733	[63]
	Phragmidium kanasense	HMAS248114 *	Rosa fedtschenkoana	Xinjiang, China	—	MK518464	[11]
	Phragmidium leucoaecium	BJFCR 02118 *	Rosa sp.	Yunnan, China	MN264719	MN264737	[63]
	Phragmidium mexicanum	HMAS350019	Phoenix acaulis	Yunnan, China	MK518980	MK518678	[11]
	Phragmidium mexicanum	HMAS350020	Duchesnea indica	Yunnan, China	MK518982	MK518680	[11]
	Phragmidium mexicanum	MCA2496	Potentilla indica	Maryland, America	—	JF907660	[60]
	Phragmidium montivagum	FO 47828	Rosa cf. woodsii	—	—	AF426213	[61]
	Phragmidium mucronatum	U-671	Rosa sp.	Oman	—	HQ412646	[64]
	Phragmidium mucronatum	BRIP:60097	Rosa rubiginosa	—	—	MW049275	[40]
	Phragmidium nambuanum	HMUT100465	Rubus innominatus	Chongqing, China	—	KU059165	—
	Phragmidium octoloculare	HMAS140416	Rubus biflorus	China	MH128376	MG669119	[59]
	Phragmidium pauciloculare	HMUT100466	Rubus innominatus	Chongqing, China	—	KU059162	—
	Phragmidium pauciloculare	HMAS350023	Rubus corchorifolius	Guangxi, China	MK518874	MK518542	[11]
	Phragmidium potentillae	BRIP:60089	Acaena novae-zelandiae	Tasmania, Australia	—	KT199403	[13]
	Phragmidium potentillae-canadensis	MCA2858	Potentilla sp.	New York, America	—	JF907666	[60]
	Phragmidium punjabense	BA65A *	Rosa sp.	Murree, Ghora Gali, Pakistan	KX358856	KX358854	[50]
	Phragmidium rosae-californicae	MVAP0000154	Rosa californica	Putah Creek Reserve, Davis, California, America	MK045315	MK045315	—
	Phragmidium rosae-moschatae	BPI 893257	Rosa macrophylla	—	—	KY798368	—
	Phragmidium rosae-multiflorae	HMAS71053	Rosa multiflora	Shanxi, China	—	KU059174	[65]
	Phragmidium rosae-multiflorae	HMAS94924	Rosa multiflora	Zhejiang, China	—	KU059175	[65]
	Phragmidium rosae-multiflorae	BJFCR 03454 *	Rosa multiflora	Jiangxi, China	MN264721	MN264739	[63]
	Phragmidium rosae-roxburghii	HGUP21025 *	Rosa roxburghii	Guizhou, China	OL684818	OL684831	[65]
	Phragmidium rosae-roxburghii	GMB0104	Rosa xanthina	Guizhou, China	OQ067092	—	This study
	Phragmidium rubi-idaei	HMUT100470	Rubus saxatilis	Chongqing, China	—	KU059163	—
	Phragmidium rubi-oldhami	HMAS64306	—	China	—	MG669116	[59]
	Phragmidium tormentillae	GMB00114	Potentilla simulatrix	Guizhou, China	OQ067093	—	This study
	Phragmidium tormentillae	BPI 843392	Duchesnea sp.	Maryland, America	—	DQ354553	[8]
	Phragmidium tuberculatum	BPI 877977	Rosa floribunda	Massachusetts, America	—	KJ841923	[66]
	Phragmidium tuberculatum	BPI 877980	Rosa floribunda	Oregon, America	—	KJ841922	[66]
	Phragmidium violaceum	PDD 99246	Rubus sp.	New Zealand	—	KJ716351	[36]
	Phragmidium yangii	BJFC:R00338 *	Rosa sp.	China	—	NG_060138	[62]
	Phragmidium zangdongii	BJFC:R02447 *	Rosa tibetica	Tibet, China	MH128372	NG_064490	[59]
	Phragmidium zhouquense	BJFC:R01516 *	Rosa omeiensis	China	—	NG_060139	[62]
	Trachyspora alchemillae	WM 1019	Alchemilla vulgaris	—	—	AF426220	[61]
	Trachyspora alchemillae	BPI 843828	Alchemilla vulgaris	Switzerland	—	DQ354550	[8]
	Xenodochus carbonarius	PUR N15566	Sanguisorba officinalis	—	—	MW049289	[40]
Pucciniastraceae	Melampsorella elatina	WM 1092	Abies alba	—	—	AF426232	[61]
	Melampsorella elatina	PUR 82	Cerastium fontanum	Minnesota, America	—	MG907233	[10]
	Pucciniastrum circaeae	RB 2098	Circaea lutetiana	—	—	AF426227	[61]
	Pucciniastrum corni	TSH-R13510	Cornus kuosa	Tottori, Japan	AB221437	AB221409	[67]
	Pucciniastrum epilobii	PUR N11088(MCA5308)	Epilobium angustifolium	—	—	MW147083	[40]
	Pucciniastrum fagi	TSH-R10724	Fagus crenata	Tochigi, Japan	AB221425	AB221378	[67]
	Pucciniastrum hikosanense	TSH-R4287 (IBA2565)	Acer rufinerve	Yamanashi Japan	AB221441	AB221388	[67]
	Pucciniastrum hydrangeae	MCA2837	Hydrangea arborescens	North Carolina, America	—	MG907240	[10]
	Pucciniastrum kusanoi	HiroU21509	Clethra barbinervis	Miyazaki, Japan	AB221426	AB221402	[67]
	Pucciniastrum yoshinagai	TSH-R4272 IBA8430	Stewartia monadelpha	Nara, Yoshino-gun, Japan	AB221434	AB221411	[67]
	Thekopsora minima	MCA2988	Vaccinium angustifolium	Maryland, America	—	MG907243	[10]
Sphaerophragmiaceae	Austropuccinia psidii	BRIP:58164	Rhodamnia angustifolia	Brisbane, Queensland. Australia	—	KF318449	[67]
	Dasyspora amazonica	BPI US0116382 *	Xylopia amazonica	Brazil	JF263460	JF263460	[68]
	Dasyspora gregaria	ZT Myc 3397	Xylopia cayennensis	French Guiana	JF263477	JF263477	[68]
	Dasyspora guianensis	ZT Myc 3413 *	Xylopia benthamii	French Guiana	JF263479	JF263479	[68]
	Dasyspora mesoamericana	PUR 42390 *	Xylopia frutescens	Panama	JF263480	JF263480	[68]
	Dasyspora segregaria	PMA MP4941	Xylopia aromatica	Panama	JF263488	JF263488	[68]
	Dasyspora winteri	S F30078	Xylopia sericea	Brazil	JF263492	JF263492	[68]
	Puccorchidium polyalthiae	ZT HeRB 251	Polyalthia longifolia	India	JF263493	JF263493	[68]
	Sphaerophragmium acaciae	BRIP:56910	Albizzia sp.	Kununurra, Western Australia, Australia	—	KJ862350	[69]
	Sphaerophragmium longicorne	PUR N16513	Dalbergia hostilis	—	—	MW147053	[40]
	Sphenorchidium xylopiae	PC 0096711	Xylopia aethiopica	western Central Africa.	KM217355	KM217355	[70]
Uredinineae incertae sedis	Allodus podophylli	BPI 842277	Podophyllum peltatum	Maryland, America	DQ354543	DQ354543	[67]
	Neopuccinia bursa	RB 75707	Protium heptaphyllum	Brazil	—	MH047186	—
	Nyssopsora altissima	GMB0103 *	Ailanthus altissima	Guizhou, China	OQ067089	OQ067529	This study
	Nyssopsora echinata	KR0012164	Meum athamanticum	—	—	MW049272	[40]
	Nyssopsora koelreuteriae	BBSW-1	Koelreuteria bipinnata	—	KT750965	KT750965	[71]
	Nyssopsora koelreuteriae	GMB0105	Koelreuteria bipinnata	Guizhou, China	OQ067088	—	This study
	Nyssopsora thwaitesii	AMH:9528	Schefflera wallichiana	India	KF550283	—	[72]

Notes: —: no data available; *: type specimens.

. Sequences were aligned by MAFFT v. 7.394 (https://www.ebi.ac.uk/Tools/msa/mafft/) (accessed on 12 September 2023) [27] and adjusted to ensure maximum similarity using TrimAl v1.4.1 [28]. Alignments were converted from FASTA to PHYLIP format by using Alignment Transformation Environment online (https://sing.ei.uvigo.es/ALTER/) (accessed on 12 September 2023) [29]. Maximum Likelihood (ML) analyses and Bayesian posterior probabilities (BYPP) based on a combination of ITS and LSU sequence data were performed using RAxML-HPC 7.4.2 BlackBox [30] and MrBayes v. 3.2.7 tools in the CIPRES Science Gateway platform [31,32]. GTR+I+G was estimated as the best-fit substitution model by jModelTest2 on XSEDE v.2.1.6 [33,34]. The Bootstrap values of ML analyses were obtained by running 1000 replicates by using a Markov chain Monte Carlo (MCMC) method to approximate the posterior probabilities of trees. Six simultaneous Markov Chains were run for 3,000,000 generations and trees were sampled every 1000th generation. Finally, the trees were visualized in FigTree v.1.4.4 [35] and edited by using Adobe Photoshop CS6 v. 13 software. The final alignment and phylogenetic trees were deposited in TreeBASE v. 2 under the submission ID30041 (http://www.treebase.org/) (accessed on 12 September 2023).

3. Results

In this study, 11 samples were collected from 9 species of medicinal plants in nine genera in seven families in Guizhu Province. Ten species including three new species were identified based on morphological and molecular phylogenetic studies. Morphological and phylogenetically allied taxa were selected for the final phylogenetic analyses, mainly following Zhao et al. [11]. The alignment for Melampsoraceae includes 1200 character (ITS: 532 bp, LSU: 1200 bp) (Figure 1). The dataset of phylogenetic tree from Neophysopellaceae and Phakopsoraceae has 1162 characters (ITS: 474 bp, LSU: 1162 bp) including gaps (Figure 2). The alignment for Neophysopellaceae and Phakopsoraceae phylogenetic tree contained 20 taxa. The taxa from genera Gerwasia, Hamaspora, Kuehneola, Phragmidium, Trachyspora, and Xenodochus were included in the phylogenetic tree of the family Phragmidiaceae (Figure 3). The alignment is made up of 71 species and has 1244 characters including gaps (ITS: 392 bp, LSU: 1244 bp). The phylogenetic tree of the families Pucciniastraceae and Hyalopsoraceae includes the taxa from genera Coleopuccinia, Hyalopsora, Melampsorella, Melampsoridium, and Pucciniastrum (Figure 4). In the phylogenetic tree of Pucciniastraceae, there are 20 taxa with 1244 characters, including gaps (ITS: 351 bp, LSU: 1244 bp). Twenty-seven representative species from the Gymnosporangiaceae, Sphaerophragmiaceae, and Uredinineae incertae sedis were chosen for Figure 5, which included 1272 characters including gaps (ITS: 398, LSU: 1272). Based on morphological and multi-locus phylogenetic characterisation, 13 specimens were identified to 9 species in 6 genera 5 families (Hamaspora rubi-alceifolii sp. nov., Nyssopsora altissima sp. nov., Phragmidium cymosum sp. nov., Melampsora laricis-populina, Melampsoridium carpini, Neophysopella ampelopsidis, Ny. koelreuteriae, P. rosae-roxburghii and P. tormentillae). Among them, three novel species were recognized, moreover, 2 species were reported first in China, and 4 species were first reported on medicinal plant.

Taxonomy

Based on morphology and molecular phylogeny, all collected rust specimens on medicinal plants were identified as three new species (Hamaspora rubi-alceifolii sp. nov., Nyssopsora altissima sp. nov., Phragmidium cymosum sp. nov.) and six known species (Melampsora laricis-populina, Melampsoridium carpini, Neophysopella ampelopsidis, Ny. koelreuteriae, P. rosae-roxburghii and P. tormentillae) All species parasitic on medicinal plants are described and illustrated below.

Hamaspora rubi-alceifolii Q. Z. Wu, T. Z. Liu, P. Zhao & Q. R. Li, sp. nov. Figure 6

MycoBank number: MB847104

Etymology: Epithet follows the epithet of host species, Rubus alceifolius Poir.

Holotype: GMB0109

Parasitic on the leave and stem of R. alceifolius. Telia up to 5 mm long, mostly hypophyllous, occasionally amphigenous and stem, caespitose, filiform, fluffy, golden yellow when fresh, white when dry; Teliospores 116–230 × 20–10 µm (av. = 184 × 17 µm, n = 30), fusiform, hyaline 3–6 septate, mostly 5–6 septate, smooth, the contents yellow when fresh, solid apex 9–30 µm (av. = 23 µm, n = 30). Spermogonia, aecia and uredinia were not observed.

Materials examined: CHINA, Guizhou Province, Guiyang City, Campus of Guizhou Medical University (26°22′48.37″ N, 106°37′30.33″ E), III on leaf of R. alceifolius Poir., 6 October 2021, Q. Z. Wu and L. L. Liu, GMB0109, holotype, CFSZ 50531, isotype; CHINA, Guizhou Province, Guiyang City, Huaxi District, III on leaf of R. alceifolius Poir., 20 October 2021, Q. Z. Wu and L. L. Liu, GMB0116.

Notes: Hamaspora rubi-alceifolii is characterized by 5–6 septate teliospore with long solid apex up to 30 µm, and hypophyllous telia (Figure 6). Phylogenetically, it formed a distinct clade sister to H. acutissima with high support values (100% ML, 1 BYPP; Figure 3). Morphologically, the differences between H. rubi-alceifolii and H. acutissima are in the number of septa in the teliospores (5–6 vs. 2–3), smaller teliospores (116–230 × 10–20 µm vs. 158–205 × 18–25 µm), and smaller solid apex (9–30 µm vs. 20–40 µm). Both H. rubi-sieboldii and H. rubi-alceifolii exhibit similar teliospore morphology with consistent solid apex size. However, the difference between H. rubi-alceifolii and H. rubi-sieboldii is that the former has more cells (5–6 vs. 4) and smaller solid apex (116–230 × 10–20 µm vs. 118–240 × 15–23 µm) [73,74,75,76].

Roots and leaves of Rubus alceifolius is a traditional Chinese medicine, which can be used for treatment of acute and chronic hepatitis, hepatosplenomegaly and other liver damage diseases [77,78,79,80,81]. Previously, thirteen Hamaspora species have already been reported on Rubus species [76]. For the convenience of recognition, a worldwide identification key for the Hamaspora has been provided.

Melampsora laricis-populina Kleb., Z. PflKrankh. 12: 43 (1902) Figure 7

Mycobank number: MB18930

Uredinia, mostly hypophyllous, seldom epiphyllous, in little groups, 0.25 mm, Bright yellow. Urediniospores oblong to broadly ellipsoid or obovoid, hyaline to light yellow with yellowish granules, 33–45 × 21–26 µm (av. = 37 × 24 µm, n = 30), wall 1.8–3 µm thick, echinulate except at the smooth apex; Paraphyses clavate to capitate, hyaline to pale yellow, 50–71 × 12–20 µm (av. = 62 × 17 µm, n = 30), wall thick, up to 9 µm at the apex.

Materials examined: CHINA, Guizhou Province, Kaili City, Xiasi Town (26°26′29.48″ N, 107°47′30.33″ E) II on Populus lasiocarpa Oliv., 1 October 2021, Q. Z. Wu, GMB0097.

Notes: Melampsora laricis-populina is characterized by the echinulate urediniospores that are obovate or oval, with golden yellow cytoplasm; roughly spherical paraphyses with swollen tips. The uredinial morphologies of our specimen (GMB0097) are consistent with those of M. laricis-populina [82,83]. Phylogenetic tree (Figure 1) showed that our specimen (GMB0097) was clustered with M. laricis-populina with the high bootstrap supports (83/-). Thus, here we confirmed the rust fungus on Populus lasiocarpa as M. laricis-populina. Populus lasiocarpa has hemostatic function and mainly used for treatment for bleeding from trauma [84,85]. Previously, M. laricis-populina has already been reported on Populus lasiocarpa in Japan [86] and Norway [87], and it has been reported in northwest region of China [88].

Melampsoridium carpini (Nees) Dietel, in Engler & Prantl, Nat. Pflanzenfam., Teil. I (Leipzig) 1(1**): 551 (1900) Figure 8

Basionym: Caeoma carpini Nees, Syst. Pilze (Würzburg): 16 (1816) [1816-17]

Mycobank number: 205589

Uredinia, hypophyllous, scattered or grouped on yellow spots, 0.2 mm diam., yellow. Urediniospores long obovoid, clavate or pear shaped, 19–31 × 13–18 µm (av. = 26 × 15 µm, n = 30), yellow; spore wall hyaline, echinulate, on apex smooth, 0.8–1.5 µm thick.

Materials examined: CHINA, Guizhou Province, Qingzhen City, Xiasi Town (26°27′18.48″ N, 107°20′7.33″ E) II on Carpinus turczaninowii Hance, 8 October 2021, Q. Z. Wu and L. L. Liu, GMB0112, CFSZ 50543.

Notes: Carpinus turczaninowii is commonly used to treat bruises, canker sores and swellings, as recorded in the Pharmacopoeia of the People’s Republic of China. Carpinus turczaninowii can alleviate arterial damage and inflammation caused by hyperglycemia [89]. It contains Pheophorbide A, which has anti-cancer and anti-inflammatory activity [90,91]. We collected rust infected Carpinus turczaninowii in Guizhou province in China, and the urediospores of our specimen (GMB0112) are consistent with those of Me. carpini, which is characterized by sparse thorns on the surface and a smooth top of urediospores [92,93,94]. According to the phylogenetic tree (Figure 4.), the new collection (GMB0112) was clustered with Me. carpini with the high bootstrap values (99/-). This species has been found in Anhui, Chongqing, Sichuan, Taiwan and other provinces in China [94], which is the first record of Me. carpini from Guizhou province, China.

Neophysopella ampelopsidis (Dietel & P. Syd.) Jing X. Ji & Kakish., in Ji, Li, Li & Kakishima, Mycol. Progr. 18(6): 863 (2019) Figure 9

Basionym: Phakopsora ampelopsidis Dietel & P. Syd. [as ’ampelosidis’], in Dietel, Hedwigia 37: 217 (1898)

Mycobank number: MB830298

Uredinia hypophyllous, grouped on brown or yellow spots. the paraphyses, incurved, 30–43 × 6–14 µm (av. = 37 × 11 µm, n = 30), wall 2–3 µm thick (av. = 2.5 µm, n = 30). Urediniospores, obovoid or obovoid-ellipsoid, dark yellow or brown, 20–32 × 15–21 µm (av. = 25 × 18 µm, n = 30), walls hyaline, echinulate. The wall was colorless or pale yellowish, equally ca 0.8–1.5 µm thick, and evenly echinulate.

Materials examined: CHINA, Guizhou, Qingzhen City, Xiasi Town (26°27′18.18″ N, 107°20′7.13″ E) II on Ampelopsis sinica (Mig.) W.T. Wang., 22 July 2021. Q. Z. Wu and L. L. Liu, GMB0110, CFSZ 50532.

Notes: Ampelopsis sinica root (ASR) is a traditional Chinese medicine known to have a hepatoprotective function. Moreover, it has been proven having anti-hepatocellular carcinogenic activity and to inhibit Hepatitis B virus activity [95,96,97]. Our rust collection (GMB00110) on Ampelopsis sinica is compatible with Neophysopella ampelopsidis [98]. Phylogenetically, our collection clustered with N. ampelopsidis (IBA-8618) with the high bootstrap supports (100/1, in Figure 2). N. ampelopsidis has been previously introduced from Japan, Philippines and Taiwan provinces of China [98]. This is the first record of N. ampelopsidis from the Chinese mainland.

Nyssopsora altissima Q. Z. Wu, T. Z. Liu, P, Zhao & Q. R. Li, sp. nov. Figure 10

MycoBank number: MB847103

Etymology: Epithet follows the epithet of host species, Ailanthus altissima (Mill.) Swingle.

Holotype: GMB0103

Parasitic on the leave of A. altissima. Uredinia, usually amphigenous, sparse or aggregated, pulverulent, golden; Urediniospores subglobose or ellipsoid, 18–23 × 16–24 µm (av. = 21 × 20 µm, n =30), cell wall 1.2–2.4 µm (av. = 1.5 µm, n = 30) thick, echinulate. Telia soft hypophyllous, aggregated, rounded, pulverulent, dark; Teliospores subglobose or globose-trigonal, septa constricted slightly, 2–3 cells are mostly 3, reddish-brown to opaque, 28–38 × 20–39.5 µm (av. = 33.5 × 33 µm, n = 30), projections up to 14 (av. = 12, n = 30), 1–6 (av. = 2, n = 30) apical furcations, 2.8–7.8 µm (av. = 4.5 µm n = 30) long and 1.3–3.3 µm (av. = 2 µm, n = 30) thick, each cell has 1–2 germination pores; pedicel hyaline, persistent, about 33–57 µm (av. = 44 µm, n = 30) long, 5.5–9 µm (av. = 7.8 µm, n = 30) thick.

Materials examined: CHINA, Guizhou Province, Guiyang City, Campus of Guizhou Medical University (26°22′48.37″ N, 106°37′30.33″ E), II, III on leave of A. altissima, 8 July 2021, Q.Z. Wu and L.L. Liu, GMB0103, holotype, CFSZ 50535, isotype.

Notes: Phylogenetically, Nyssopsora altissima was phylogenetically allied to Ny. echinata with a high bootstrap support (86/0.99, Figure 5). Morphologically, Ny. altissima differs from Ny. echinata by the number of germination pores in teliospores (3 vs. 2), the bigger teliospores (28–38 × 20–39.5 µm vs. 25–30 × 23–27 µm), and the shorter processes (2.8–7.8 µm vs. 6.6–12 µm). Morphologically, Ny. altissima can be distinguished from Ny. cedrelae by several morphological differences that bigger urediniospores (18–23 × 16–24 µm compared to 15–19 × 13–17 µm), smaller teliospores (28–38 × 20–39.5 µm vs. 28–45 × 18–43 µm), and fewer projections (1–14 vs. 15–22). Additionally, Ny. altissima has shorter hyaline (33–57 µm vs. 40–65 µm) and finer hyaline (5.5–9 µm vs. 10–12 µm) than those of Ny. cedrelae [76,99].

Ailanthus altissima has been used as a medicinal herb to hemostasis and Anti-diarrhea documented in Chinese pharmacopoeia [100]. Ailanthus altissima has the anti-malarial, anti-viral and anti-tumor active ingredient and shows potential as a novel drug for the treatment of prostate cancer [101,102,103]. To date, another rust species, Ny. cedrelae, has already been reported on Ailanthus altissima [76], here we reported another Nyssopsora species on Ailanthus altissima. Compared to Ny. cedrelae, Ny. altissima exhibits larger-sized urediniospores (18–23 × 16–24 µm vs. 15–19 × 13–17 µm), shorter apical furcations (2.8–7.8 µm vs. 2–10 µm), and a shorter pedicel (33–57 µm vs. 120 µm).

Nyssopsora koelreuteriae (Syd. & P. Syd.) Tranzschel, J. Soc. bot. Russie 8: 132 (1925) [1923] Figure 11

Basionym: Triphragmium koelreuteriae Syd. & P. Syd. 1913

Mycobank number: MB335240

Parasitic on leaves of Koelreuteria bipinnata, surrounded by yellowish margins spermagonia, aecia and uredinia unknown. Telia amphigenous, scattered or slightly clustered, densely aggregated in groups, confluent, naked, erumpent, blackish, chestnut-brown. Teliospores 27–31 × 24–32 µm (av. = 28 × 27 µm, n = 30), 3-celled with a single proximal cell and two collateral distal cells, triquetrous pyriform, strongly constricted at septa, blackish–brown; walls uniformly 1.5–3 µm thick, pale yellow when young and becoming blackish–brown when older. Projections up to 18 and branched at the tips, 2–3 branched at apex. Pedicel 20–26 × 4.5–6.5 µm (av. = 23 × 6 µm, n = 30), persistent, hyaline.

Materials examined: CHINA, Guizhou Province, Anshun City, Longdong Scenic Area (26°6′32.28″ N, 105°52′30.23″ E) III on Koelreuteria bipinnata Franch., July 10, 2021 Q.Z. Wu, GMB0105.

Notes: The leaves of Koelreuteria bipinnata have strong antimicrobial activity, and their extracts contain a variety of components that can inhibit bacteria and fungi [104,105]. We collected rust samples on the leaves of Koelreuteria bipinnata, and it can be identified as Ny. koelreuferiae based on both morphological and molecular evidences (Figure 5 and Figure 11). This rust fungus has already been reported in Zhejiang province, China in 1928 [106], and here we reported it on the same host species in Guizhou province.

Phragmidium cymosum Q. Z. Wu, T. Z. Liu, P, Zhao & Q. R. Li, sp. nov. Figure 12

MycoBank number: MB847102

Etymology: Epithet follows to the epithet of host species, Rosa cymosa Tratt.

Holotype: GMB0108

Parasitic on the leaves of Rosa cymosa. Uredinia Uredo-type (Uraecium type II), hypophyllous, yellowish, aggregated, 0.1–0.4 mm (av. = 0.2 mm, n = 30), with thick-walled, gradual thickening by the roots of the paraphyses, incurved, intermixed paraphyses, 25–70 × 9–15 µm (av. = 44 × 11 µm, n = 30), wall 3–11 µm thick (av. = 7 µm, n = 30), top width 5–13 µm thick (av. = 8.5 µm, n = 30), Urediniospores borne singly, mostly echinulate, globose, broadly or obovoid, echinulate, 19–27 × 21–29 µm (av. = 24 × 26 µm, n = 30), cytoplasm orange, wall 1–2.5 µm thick (av. = 1.5 µm, n = 30).

Materials examined: CHINA, Guizhou Province, Guiyang City, Campus of Guizhou Medical University (26°22′46.37″ N, 106°37′29.33″ E), II on R. cymosa, 6 October 2021, Q. Z. Wu, GY-XGQW GMB0108, holotype, CFSZ 50542, isotype; CHINA, Guizhou Province, Guiyang City, Campus of Guizhou Medical University (26°22′48.22″ N, 106°37′30.12″ E), II on R. cymosa, 6 October 2021, Q. Z. Wu, GMB0115.

Notes: Phragmidium cymosum formed a distinct lineage in the phylogenetic tree (Figure 3) with a high support rate (98/1). Morphologically, it differs from those species by its paraphyses with relatively thick wall, by the width of the urediniospores (21–29 µm in P. cymosum; 18–21 µm in P. japonicum; 11–18 µm in P. jiangxiense; 15–20 in P. rosae-multiflorae) [63,76]. In addition, the paraphyses of P. cymosum (25–70 × 9–15 µm) are thicker compared to those of P. jiangxiense (22–41 × 6–10 µm), and the cell walls of the paraphyses in P. cymosum (3–11 µm) are thicker than those of P. rosae-multiflorae (1 µm). Phragmidium rosae-multiflorae was once reported on the same host species in China [107].

The leaves, flowers and roots of Rosa cymosa can be used as the Chinese herbal medicine. Moreover, it has anti-inflammatory components, can be used to treat burns, analgesic [108,109,110].

Phragmidium rosae-roxburghii J.E. Sun & Yong Wang bis Figure 13

MycoBank number: MB845041

Parasitic on the leave of Rosa roxburghii Tratt. Uredinia Uredo-type (Uraecium type II), hypophyllous, yellowish, aggregated, with thick-walled, gradual thickening by the roots of the paraphyses, incurved, intermixed paraphyses, 23–50 × 8–13.5 µm (av. = 36.5 × 10 µm, n = 30), wall 2–8.5 µm thick (av. = 5.5 µm, n = 30), Urediniospores borne singly, mostly echinulate, globose, broadly or obovoid, echinulate, 18–29 × 17–24 µm (av. = 24 × 20 µm, n = 30), cytoplasm orange, wall 0.5–1.5 µm thick (av. = 1 µm, n = 30).

Materials examined: CHINA, Guizhou province, Anshun City, Longdong Scenic Area (26°6′46.32″ N, 105°52′32.23″ E) II on Rosa roxburghii Tratt, 10 July 2021 Q.Z. Wu, GMB0104, CFSZ 50540.

Notes: Rosa roxburghii as a Chinese herbal medicine is used as a remedy for respiratory diseases. It has been recently reported to be an antioxidant and anticoagulant, and also be used to treat dyspepsia, dysentery, hypo immunity, and neurasthenia [65,111]. Our collection (GMB0104) and P. rosae-roxburghii form a clade in phylogenetic tree with a high support value (100/1). The morphological characteristics of GMB0104 are consistent with those of P. rosae-roxburghii. Phragmidium rosae-roxburghii was introduced based on the specimen collected from Guizhou province [65]. Here we reported it on medicinal plant in the same province.

Phragmidium tormentillae Fuckel, Jb. nassau. Ver. Naturk. 23-24: 46 (1870) [1869-70] Figure 14

Mycobank number: MB177066

Spermogonia were not observed. Uredinia hypophyllous, scattered, subepidermal and erumpent becoming pulverulent, 0.5 mm diam. Urediniospores circular or obovoid, pale yellow, 18–24 × 14–20 µm (av. = 211 × 17 µm, n = 30), walls hyaline, echinulate, 1–1.5 mm thick.

Materials examined: CHINA, Guizhou Province, Qingzhen City, Xiasi Town (26°27′16.48″ N, 107°20′8.23″ E) II on Potentilla simulatrix Th. Wolf, 22 July 2021 Q.Z. Wu, GMB0114.

Notes: Potentilla is documented in most areas of China, which was used as a traditional Chinese medicine for hemostasis and treatment of malaria [112]. The pharmacological activities of Potentilla are mainly related to antioxidant, hypoglycemic, anti-inflammatory, antibacterial, antitumor, and cardiovascular system protective effects [113]. It also has positive effects on hemorrhagic cystitis [114]. Our collection on Potentilla simulatrix (GMB0110) is located in the same clade with P. tormentillae (Figure 3). Based on the size, shape and wall thickness of the urediniospores, as well as the presence of sparse spines on the surface of the urediniospores, new collection is identified as P. tormentillae [60,115,116]. However, no spermatozoa and paraphyses were observed in new collections. Phragmidium tormentillae was firstly collected in Norway in 1895 [116]. This is the first record for Chinese mainland.

4. Discussion

The research on rust diseases in medicinal plants has been relatively less compared to those in economic crops, mainly because in the past, medicinal plants were mostly sourced from the wild, with fewer incidents of rust diseases and thus had not received much attention. However, as the cultivation area of medicinal plants continues to increase, rust diseases have gradually become one of the important diseases affecting the quality and yield of medicinal materials. Attention on rust diseases in medicinal plants have been steadily increasing over time [17].

A total of 79 rust species were found to cause diseases on 76 species of medicinal plants from 33 families in China [16,17]. Rust diseases have become the primary diseases on some important medicinal plants in their primary growth regions, with strong prevalence and large damage areas, such as safflower rust disease (Puccinia carthami), Japanese yam rust (Puccinia dioscoreae), Radix glehniae rust (Puccinia phellopteri), and Rust on bulbus fritillariae ussuriensis (Uromyces aecidiiformis) [117,118,119,120]. Most researches on rusts on medicinal plants have been focused on the descriptions of symptoms, the incidence scopes and geographic distribution and the rough morphological descriptions of some spores [17]. However, high morphological variations, wide host range and complicated life cycles, identification of rust fungi is very difficult solely based on morphologies or host specificity. Herein, with the aid of morphological and molecular data, ten rust species have been found on medicinal plants collected from Guizhou province, including three new species and six known species. Among them, Hamaspora rubi-alceifolii, Nyssopsora altissima and Phragmidium cymosum were introduced as new to science. Neophysopella ampelopsidis, Phragmidium tormentillae was firstly introduced in Chinese mainland. Melampsora laricis-populina, Melampsoridium carpini, and Nyssopsora koelreuteriae were documented for the first time in Guizhou province. The accurate identification of rust fungi on medicinal plants will lay the foundation for disease control of medicinal plants.

The use of DNA sequences is becoming more and more important in the identification of rust fungi. Despite early research on rust fungi, the taxonomic system remains perplexing [121]. Distinguishing between individual rust fungi based solely on morphology is challenging [13,122,123]. Because the vast majority of rust fungi cannot be cultured on the artificial medium, pure culture strain cannot be obtained. Therefore, there is no enough DNA sequence available for a large number of rust species for a long time. As DNA extraction techniques continue to improve, valid DNA sequences will become increasingly available [61], and phylogenetic and morphological-based approaches will resolve the taxonomic confusion in rust fungi [11,124]. There were 337 species of rust fungi in 76 genera of 14 families using both morphological and molecular data from 86 natural reserves and national parks in the past five years [11]. Because molecular phylogenetic approaches can be used to connect the telial and aecial stages of rust fungi, they used more additional characters for species recognition [125]. Thus, their studies using DNA-based phylogenetic approach have facilitated precise identification of rust fungi at familial, generic, and species level. These studies can present a significant contribution to the knowledge of rust flora in China, especially those on medicinal plants.