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

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.


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.

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).

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].

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 ddH 2 O, 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.
Hamaspora rubi-alceifolii Q. Z. Wu, T. Z. 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           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.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].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] 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.
Materials examined: CHINA, Guizhou Province, Anshun City, Longdong Scenic Area (26 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 (Figures 5 and 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.
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.      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.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.

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

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 DNAbased 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.

Figure 1 .
Figure 1.RAxML tree of the family Melampsoraceae based on rDNA ITS and LSU sequence.ML bootstrap supports (≥70%) and Bayesian posterior probability (≥0.90) are indicated as ML/BYPP.The tree is rooted to Rossmanomyces monesis and Chrysomyxa empetri [11].The type specimens are shown as boldface.New sequences are in red.

Figure 1 .
Figure 1.RAxML tree of the family Melampsoraceae based on rDNA ITS and LSU sequence.ML bootstrap supports (≥70%) and Bayesian posterior probability (≥0.90) are indicated as ML/BYPP.The tree is rooted to Rossmanomyces monesis and Chrysomyxa empetri [11].The type specimens are shown as boldface.New sequences are in red.

Figure 2 .
Figure 2. The RAxML tree of the family Neophysopellaceae and Phakopsoraceae based on rDNA ITS and LSU sequences.ML bootstrap supports (≥70%) and Bayesian posterior probability (≥0.90) are indicated as ML/BYPP.The tree is rooted to G. asiaticum and G. clavariiforme [11].The type specimens are shown as boldface.New sequences are in red.

Figure 2 .
Figure 2. The RAxML tree of the family Neophysopellaceae and Phakopsoraceae based on rDNA ITS and LSU sequences.ML bootstrap supports (≥70%) and Bayesian posterior probability (≥0.90) are indicated as ML/BYPP.The tree is rooted to G. asiaticum and G. clavariiforme [11].The type specimens are shown as boldface.New sequences are in red.

Figure 3 .
Figure 3. RAxML tree of the family Phragmidiaceae based on rDNA ITS and LSU sequences.ML bootstrap supports (≥70%) and Bayesian posterior probability (≥0.90) are indicated as ML/BYPP.The tree is rooted to G. asiaticum and G. clavariiforme [11].The type specimens are shown as boldface.New sequences are in red.

Figure 3 .
Figure 3. RAxML tree of the family Phragmidiaceae based on rDNA ITS and LSU sequences.ML bootstrap supports (≥70%) and Bayesian posterior probability (≥0.90) are indicated as ML/BYPP.The tree is rooted to G. asiaticum and G. clavariiforme[11].The type specimens are shown as boldface.New sequences are in red.

Figure 4 .
Figure 4. RAxML tree of the family Pucciniastraceae and Hyalopsoraceae based on rDNA ITS and LSU sequence.ML bootstrap supports (≥70%) and Bayesian posterior probability (≥0.90) are indicated as ML/BYPP.The tree is rooted to G. asiaticum and G. clavariiforme [11].New sequences are in red.

Figure 4 .
Figure 4. RAxML tree of the family Pucciniastraceae and Hyalopsoraceae based on rDNA ITS and LSU sequence.ML bootstrap supports (≥70%) and Bayesian posterior probability (≥0.90) are indicated as ML/BYPP.The tree is rooted to G. asiaticum and G. clavariiforme [11].New sequences are in red.

Figure 3 .
Figure 3. RAxML tree of the family Phragmidiaceae based on rDNA ITS and LSU sequences.ML bootstrap supports (≥70%) and Bayesian posterior probability (≥0.90) are indicated as ML/BYPP.The tree is rooted to G. asiaticum and G. clavariiforme[11].The type specimens are shown as boldface.New sequences are in red.

Figure 4 .
Figure 4. RAxML tree of the family Pucciniastraceae and Hyalopsoraceae based on rDNA ITS and LSU sequence.ML bootstrap supports (≥70%) and Bayesian posterior probability (≥0.90) are indicated as ML/BYPP.The tree is rooted to G. asiaticum and G. clavariiforme [11].New sequences are in red.

Figure 5 .
Figure 5.The RAxML tree of the families Gymnosporangiaceae, Sphaerophragmiaceae and Uredinineae incertae sedis based on rDNA ITS and LSU sequences.ML bootstrap supports (≥70%) and Bayesian posterior probability (≥0.90) are indicated as ML/BYPP.The tree is rooted to Melampsoridium botulinum [40].The type specimens are shown as boldface.New sequences are in red.

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