The Genus Ravenelia: Insights on Taxonomy, Diversity and Distribution.

Ravenelia is the third largest rust genus of the order Pucciniales with more than 200 described species. It is an important rust genus that has undergone tremendous taxonomic changes. This genus produces teliospores united into a head on a compound pedicel composed of two to several hyphae with autoecious, macro-, demi- to hemi-, and, more rarely, microcyclic modes of their life cycle which provide it a unique identity and have proved helpful in the identification of the genus. The current understanding of the taxonomy, history, diversity and distribution of this genus is discussed in this paper. Both online and offline resources were searched to gather data of the published literature. The data thus obtained were analyzed for numerical and graphical summaries to provide the diversity and distribution of the genus. In addition, a phylogenetic analysis based on the ITS and nLSU DNA sequence data available in GenBank and the published literature was performed to examine the taxonomic placement of different species within the genus. The genus was reported to be distributed over 53 countries of the world. Around 51 plant genera belonging to four plant families, viz., Fabaceae, Phyllanthaceae, Asphodelaceae and Zygophyllaceae were found to be infected with these rust fungi. The phylogenetic analysis based on LSU and ITS sequence data revealed the polyphyletic nature of the genus. A table of 248 species of this genus is also provided with all information of host, distribution and cited reference that can be helpful for mycologists to find all information at one place. Future perspectives for the advancement of this genus are also discussed.

Identification and functional characterization of the npc-2-like domain containing rust effector protein that suppresses cell death in plants.

The MD-2-related lipid-recognition (ML/Md-2) domain is a lipid/sterol-binding domain that are involved in sterol transfer and innate immunity in eukaryotes. Here we report a genome-wide survey of this family, identifying 84 genes in 30 fungi including plant pathogens. All the studied species were found to have varied ML numbers, and expansion of the family was observed in Rhizophagus irregularis (RI) with 33 genes. The molecular docking studies of these proteins with cholesterol derivatives indicate lipid-binding functional conservation across the animal and fungi kingdom. The phylogenetic studies among eukaryotic ML proteins showed that Puccinia ML members are more closely associated with animal (insect) npc2 proteins than other fungal ML members. One of the candidates from leaf rust fungus Puccinia triticina, Pt5643 was PCR amplified and further characterized using various studies such as qRT-PCR, subcellular localization studies, yeast functional complementation, signal peptide validation, and expression studies. The Pt5643 exhibits the highest expression on the 5th day post-infection (dpi). The confocal microscopy of Pt5643 in onion epidermal cells and N. benthamiana shows its location in the cytoplasm and nucleus. The functional complementation studies of Pt5643 in npc2 mutant yeast showed its functional similarity to the eukaryotic/yeast npc2 gene. Furthermore, the overexpression of Pt5643 also suppressed the BAX, NEP1, and H2O2-induced program cell death in Nicotiana species and yeast. Altogether the present study reports the novel function of ML domain proteins in plant fungal pathogens and their possible role as effector molecules in host defense manipulation.

Optimizing an integrated biovigilance toolbox to study the spatial distribution and dynamic changes of airborne mycobiota, with a focus on cereal rust fungi in western Canada.

In the face of evolving agricultural practices and climate change, tools towards an integrated biovigilance platform to combat crop diseases, spore sampling, DNA diagnostics and predictive trajectory modelling were optimized. These tools revealed microbial dynamics and were validated by monitoring cereal rust fungal pathogens affecting wheat, oats, barley and rye across four growing seasons (2015-2018) in British Columbia and during the 2018 season in southern Alberta. ITS2 metabarcoding revealed disparity in aeromycobiota diversity and compositional structure across the Canadian Rocky Mountains, suggesting a barrier effect on air flow and pathogen dispersal. A novel bioinformatics classifier and curated cereal rust fungal ITS2 database, corroborated by real-time PCR, enhanced the precision of cereal rust fungal species identification. Random Forest modelling identified crop and land-use diversification as well as atmospheric pressure and moisture as key factors in rust distribution. As a valuable addition to explain observed differences and patterns in rust fungus distribution, trajectory HYSPLIT modelling tracked rust fungal urediniospores' northeastward dispersal from the Pacific Northwest towards southern British Columbia and Alberta, indicating multiple potential origins. Our Canadian case study exemplifies the power of an advanced biovigilance toolbox towards developing an early-warning system for farmers to detect and mitigate impending disease outbreaks.

Phylogenetics of the rust fungi (Pucciniales) of South Africa, with notes on their life histories and possible origins.

South Africa has an indigenous rust (Pucciniales) funga of approximately 460 species. This funga was sampled with species from as many genera as possible. The nuclear ribosomal large subunit (28S) region was amplified from samples representing 110 indigenous species, as well as the small subunit (18S) region and the cytochrome c oxidase subunit 3 (CO3) in some cases, and these were used in phylogenetic analyses. One new species is described, 12 new combinations made, six names reinstated, and two life history connections made. The life histories of this funga were summarized; it is dominated by species with contracted life histories. The majority of species are autoecious, with a small proportion being heteroecious. Of the autoecious species, many will likely be homothallic with no spermagonia. A shortened life history with homothallism allows for a single basidiospore infection to initiate a local population buildup under the prevailing unpredictable climatic conditions. Suggestions are made as to the possible origin of this funga based on the development of the modern South African flora. It is postulated that the rusts of South Africa are of relatively recent origin, consisting of three groups. Firstly, there is an African tropical element with members of the Mikronegerineae (Hemileia), the Sphaerophragmiaceae (Puccorchidium, Sphaerophragmium), and certain Uredinineae (Stomatisora). Their immediate ancestors likely occurred in the tropical forests of Africa during the Paleogene. Secondly, there is a pantropical element including the Raveneliaceae (e.g., Diorchidium, Maravalia, Ravenelia sensu lato, Uropyxis). This likely diversified during the Neogene, when the mimosoids became the dominant trees of the developing savannas. Thirdly, the Pucciniaceae invaded Africa as this continent pushed northward closing the Tethys Sea. They diversified with the development of the savannas as these become the dominant habitat in most of Africa, and are by far the largest component of the South African rust funga.

First report of rust caused by Gymnosporangium in Iceland.

Sorbus is a genus of trees and shrubs in the Rosaceae commonly known as rowan and mountain-ash. They are usually found in temperate regions of the Northern Hemisphere and cultivated as ornamental trees for parks and gardens. In September 2023, infection by a rust was observed on a single Sorbus aucuparia tree in Sólbrekkuskógur, Reykjanesbær (64.046645, -22.707276; ~13 m) in Iceland. Infected leaves were collected from this single cultivated tree at an outdoor recreation area in a natural wooded location, with a 2% disease severity. Sori were infrequent, scattered, embedded within circular yellow lesions on leaf margins. On average, one sorus was observed per leaf and only 2% of leaves were infected. Spermogonia epiphyllous, punctate and aggregated, pale yellow to black. Hypophyllous aecia roestelioid with cornute peridium rupturing at apex with peridial cells rhomboidal, aeciospores yellowish brown globoid 17.67-25.17 x 17.20-21.94 µm, walls 1.22-2.28 µm thick (n = 20). The features of this rust and dimensions of spores are consistent with descriptions of Gymnosporangium cornutum (Arthur 1909, Kern 1911). To confirm the identity (specimen MCA9732), a ~620 bp region of the 28S subunit of the ribosomal DNA repeat was sequenced using primers Rust2inv and LR6 following published protocols (Aime 2006). The sequence (GenBank PP413765) shared 100% (649/649 bp) identity with a sequence deposited as Gymnosporangium cornutum (KY764066, BPI910184; J. E. Demers, M. K. Romberg, and L. A. Castlebury, unpublished data) from S. americana and 100% (620/620 bp) identity with G. cornutum (PURN11049) on Sorbus sp. from Canada when blasted against the RustHUBB database (Kaishian et al. 2024). The specimen has been deposited in the Arthur Fungarium at Purdue University as PURN24233. Disease on Sorbus sp. caused by G. cornutum has been reported in various countries in Africa, Asia, North America, and Europe (Kern 1911). To our knowledge, this is the first report of the occurrence of this genus in Iceland from any host. Gymnosporangium cornutum alternates on Juniperus species. In Iceland, J. communis (sect. Oxycedrus) seems to be the only naturally occurring Juniperus species but it is an alternate host for G. cornutum. The presence of the primary and alternative hosts in Iceland and the ability of Gymnosporangium spp. to produce systemic infections in Juniperus spp., represents the potential for reinfection of Sorbus every year, resulting in potential impacts on both host species. With J. communis being the only Juniperus spp. in natural habitats in Iceland, the presence of this rust represents a potential ecological disruption, as repeated infections may reduce host vitality and predispose the host to winter injury and attack from opportunistic pathogens or insects.

First Record of the Rust Fungus Uromyces gageae on Yellow Star-of-Bethlehem (Gagea lutea) in the Netherlands.

Yellow Star-of-Bethlehem (Gagea lutea) is a rare and threatened bulbous plant in the Netherlands, with its largest stronghold in the northern province of Drenthe. In 2022, numerous plants within a population of G. lutea were found to be infected by a rust fungus, which was identified as Uromyces gageae based on morphological characteristics. Further examination of collected teliospores revealed differences from U. acutatus, a closely related rust species known to infect Ornithogalum and Gagea species. Rust symptoms on G. lutea plants were observed within the same population in April 2023, suggesting that teliospores surviving winter conditions serve as a viable source for recurrent infection. DNA of U. gageae and U. acutatus extracted from teliospores was used to obtain partial ribosomal DNA gene fragments by PCR. Amplicon sequencing revealed nucleotide variation between both rust species and verified the identity of the rust fungus on G. lutea as U. gageae. This confirmation substantiates the first documentation of U. gageae in the Netherlands. This study raises new avenues for research on the distribution and host range of U. gageae, as well as additional studies on the population dynamics of this potentially rare, wild plant-rust interaction.

Four new species of Puccinia from herbaceous plants in China.

Members of Puccinia (Pucciniaceae, Pucciniales) are known as plant pathogens worldwide, which are characterized by their morphology, host association, and molecular data of various genes. In the present study, 10 specimens of Puccinia were collected from four herbaceous plants (Anaphalis hancockii, Anthriscus sylvestris, Halenia elliptica, and Pilea pumila) in China and identified based on morphology and phylogeny. As a result, 10 samples represent four undescribed species of Puccinia, viz., P. apdensia, P. decidua, P. dermatis, and P. lianchengensis, spp. nov. P. apdensia is characterized by its smooth teliospores with thickened apex. P. decidua represents the first Puccinia species inhabiting the host Anaphalis hancockii and is distinguished from the other Puccinia species by its telia and uredinia surrounded by the epidermis. P. dermatis from Halenia elliptica differs from the other Puccinia species on the host genus Halenia by the telia that have epidermis and teliospores with sparsely irregular granulated protrusions. P. lianchengensis is characterized by its teliospore surface with fishnet ornamentation and urediniospores without prominent caps. All of the new species are described and illustrated in this study.

Host-Specific and Homologous Pairs of Melampsora larici-populina Effectors Unveil Novel Nicotiana benthamiana Stromule Induction Factors.

The poplar rust fungus Melampsora larici-populina is part of one of the most devastating group of fungi (Pucciniales) and causes important economic losses to the poplar industry. Because M. larici-populina is a heteroecious obligate biotroph, its spread depends on its ability to carry out its reproductive cycle through larch and then poplar parasitism. Genomic approaches have identified more than 1,000 candidate secreted effector proteins (CSEPs) from the predicted secretome of M. larici-populina that are potentially implicated in the infection process. In this study, we selected CSEP pairs (and one triplet) among CSEP gene families that share high sequence homology but display specific gene expression profiles among the two distinct hosts. We determined their subcellular localization by confocal microscopy through expression in the heterologous plant system Nicotiana benthamiana. Five out of nine showed partial or complete chloroplastic localization. We also screened for potential protein interactors from larch and poplar by yeast two-hybrid assays. One pair of CSEPs and the triplet shared common interactors, whereas the members of the two other pairs did not have common targets from either host. Finally, stromule induction quantification revealed that two pairs and the triplet of CSEPs induced stromules when transiently expressed in N. benthamiana. The use of N. benthamiana eds1 and nrg1 knockout lines showed that CSEPs can induce stromules through an eds1-independent mechanism. However, CSEP homologs shared the same impact on stromule induction and contributed to discovering a new stromule induction cascade that can be partially and/or fully independent of eds1. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

First report of Puccinia recondita rust on native jewelweed (Impatiens capensis) in Pennsylvania.

Jewelweed (Impatiens spp., Balsaminaceae) is a common native annual plant within Pennsylvania wetland ecosystems, many of which are under threat from invasive non-native plants, and is an important wetland indicator plant (code FACW; facultative wetland). In May 2014, rust disease symptoms on native jewelweed (Impatiens capensis Meerb.) were observed within a small (0.1 ha) wet area in York County, southeastern Pennsylvania (39.9080648oN, -77.2472024oW). Rust symptoms were noted on most jewelweed plants within the wet area. Foliar symptoms included chlorosis and premature defoliation; infected stems were distorted. Infected leaves and stems contained orange, erumpent aecia with white fragmented peridia (Fig. 1). Symptomatic leaves and stem sections were collected from five infected plants within one small (5 X 5 m) plot in the center of the wet area and taken to the laboratory for microscopic observations and morphological measurements. Mean aecia diameter was 299.2 ± 55.0 µm (n = 60). Aeciospores were single-celled, orange, and generally globose (Fig. 2) with a mean diameter of 24.4 ± 1.4 µm, (n = 60). Disease symptomology and aecia morphology were consistent with Puccinia recondita Dietel & Holw. DNA extraction (from infected stem material), polymerase chain reactions, and DNA sequencing of the 28S region of the nuclear ribosomal DNA repeat was conducted following protocols in Aime (2006) and Aime et al. (2018). The sequence shares 99.34% identity (903 / 909 bp) with P. recondita (BPI 910319) collected in California (KY798399). A voucher specimen has been deposited in the Arthur Fungarium at Purdue University (PUR N24229) with corresponding 28S sequence (GenBank accession OR648406). P. recondita has been reported on native I. capensis in Indiana (Koslow and Clay 2010) and North Carolina (Grand 1985), but not in Pennsylvania to the best of our knowledge (Farr and Rossman 2022). If this rust disease becomes severe on native jewelweeds in Pennsylvania, it may adversely affect our ability to accurately classify native wetlands in the state. In addition, P. recondita is a heteroecious rust that is a major pathogen of grain crops of economic importance (i.e., wheat, barley, oats), which are grown in southeastern Pennsylvania near the infested area. Further research is warranted to understand if native, annual jewelweed can serve as a secondary or alternate host to cause rust disease in major cereal crops.

Uromyces savulescui on statice (Limonium sinuatum) in the Americas and reevaluation of its reported geographic range.

Limonium sinuatum (Plumbaginaceae) is the most commonly cultivated recognizable cut flower crop in the genus Limonium. It is known by several common names including statice and sea lavender, due to its lilac-colored flowers and the fact that it naturally inhabits mainly coastal areas (Mellesse et al, 2013). Limonium sinuatum is native to the Mediterranean, although as a popular garden plant, has been naturalized in other parts of the world including coastal areas of California (USDA NRCS 2020). Cultivated L. sinuatum is used in fresh and dry flower arrangements in the Americas, comprising approximately 20% of the floriculture cultivated area in Ecuador (Vega and Morales 2011; Abascal Cañas 2017). In December 2014, L. sinuatum plants in the public park "Baños del Inca" in Cajamarca, Peru (S 7 9'46"; W 78 27'53"), were found infected with a rust disease. The plants were scattered in the park but infection incidence was 100% as individual plants were all found to be infected (Fig 1). Based on the percentage of symptomatic areas, including the yellow halos around pustules, calculated with ImageJ (Collins, 2007) from field photographs, the disease severity was estimated to be 58.9% in average, ranging from 19.8% up to 90.0%. Uredinia were present on both sides of the leaves as well as on stems and were roundish, oblong, pulverulent, and cinnamon brown in color; urediniospores 25.5 to 35.0 × 22.5 to 31.0 µm, were globoid to ellipsoid; urediniospore walls were cinnamon-brown, 2.5 to 3.0 µm thick, densely verrucose, with 2 to 3 equatorial germ pores. Few telia were present on leaves; these were scattered roundish or oblong, and greyish in color; teliospores 26.5 to 41.0 × 16.0 to 25.0 µm, were ellipsoid to obovoid, mostly attenuated at the apex; teliospore walls were colorless, 2-3 µm thick at sides, and up to 10 µm thick at apex. Teliospores readily germinated in sori producing basidia and basidiospores (Fig. 2). The rust features and dimensions of rust spores are consistent with available descriptions of Uromyces savulescui Rayss (Guyot 1951; Vakalounakis and Malathrakis 1987). To confirm identity, a 576 bp region of the 28S subunit of the ribosomal DNA repeat was sequenced following previously published protocols and primers (Aime 2006, Aime et al. 2018). The resulting sequence (GenBank Accession No. OR291160) shared 99.83% (573/574 bp) identity with a sequence deposited as Uromyces limonii (DC.) Lev. (accession KY764194, BPI910295, Demers et al. unpublished) from L. sinuatum in Ethiopia. However, U. limonii produces orange uredinia, thin-walled yellow-orange urediniospores, teliospores with mostly light chestnut brown wall and infects different hosts (Savile and Conners 1951). It is likely that KY764194 represents a misannotated record of U. savulescui. While Koch's postulates can be a useful tool for establishing causality in certain infectious diseases, their use may be limited when it comes to rust diseases based on old herbarium specimens. In our case, due to the age of the specimen, which is almost nine years old, various other methods were employed to identify the pathogen. These methods included microscopic examination for morphological criteria of the urediniospores and teliospores, as well as molecular techniques like 28S rDNA sequencing. Rust disease on L. sinuatum has been previously reported in Ecuador but the causal agent was identified as a Puccinia sp. and reported that the rust was able to destroy entire plots in humid conditions (Vega and Morales 2011). Whether this report also represent U. savulescui is not certain, but given that the urediniospores of Puccinia species are generally 2-celled, it is unlikely. García-Hernández et al. (2008) reported U. limonii on Limonium spp. from Chile, and Coca (2020) also reported U. limonii on Limonium sp., from Bolivia. However, judging from the photomicrographs (Coca 2020), the rust in the latter report is definitely U. savulescui and not U. limonii. Uromyces savulescui has been previously reported from the Mediterranean region and the Canary Islands (Vakalounakis and Malathrakis 1987). To our knowledge there is no report of this rust in the Americas, excepting the probable misidentifications already listed herein. The specimen has been deposited in the Arthur Fungarium at Purdue University as PURN15037.

Diploid Nuclei Occur throughout the Life Cycles of Pucciniales Fungi.

Within Eukaryotes, fungi are the typical representatives of haplontic life cycles. Basidiomycota fungi are dikaryotic in extensive parts of their life cycle, but diploid nuclei are known to form only in basidia. Among Basidiomycota, the Pucciniales are notorious for presenting the most complex life cycles, with high host specialization, and for their expanded genomes. Using cytogenomic (flow cytometry and cell sorting on propidium iodide-stained nuclei) and cytogenetic (FISH with rDNA probe) approaches, we report the widespread occurrence of replicating haploid and diploid nuclei (i.e., 1C, 2C and a small proportion of 4C nuclei) in diverse life cycle stages (pycnial, aecial, uredinial, and telial) of all 35 Pucciniales species analyzed, but not in sister taxa. These results suggest that the Pucciniales life cycle is distinct from any cycle known, i.e., neither haplontic, diplontic nor haplodiplontic, corroborating patchy and disregarded previous evidence. However, the biological basis and significance of this phenomenon remain undisclosed. IMPORTANCE Within Eukaryotes, fungi are the typical representatives of haplontic life cycles, contrasting with plants and animals. As such, fungi thus contain haploid nuclei throughout their life cycles, with sexual reproduction generating a single diploid cell upon karyogamy that immediately undergoes meiosis, thus resuming the haploid cycle. In this work, using cytogenetic and cytogenomic tools, we demonstrate that a vast group of fungi presents diploid nuclei throughout their life cycles, along with haploid nuclei, and that both types of nuclei replicate. Moreover, haploid nuclei are absent from urediniospores. The phenomenon appears to be transversal to the organisms in the order Pucciniales (rust fungi) and it does not occur in neighboring taxa, but a biological explanation or function for it remains elusive.

Insights into Diversity, Distribution, and Systematics of Rust Genus Puccinia.

Puccinia, which comprises 4000 species, is the largest genus of rust fungi and one of the destructive plant pathogenic rust genera that are reported to infect both agricultural and nonagricultural plants with severe illnesses. The presence of bi-celled teliospores is one of the major features of these rust fungi that differentiated them from Uromyces, which is another largest genus of rust fungi. In the present study, an overview of the current knowledge on the general taxonomy and ecology of the rust genus Puccinia is presented. The status of the molecular identification of this genus along with updated species numbers and their current statuses in the 21st century are also presented, in addition to their threats to both agricultural and nonagricultural plants. Furthermore, a phylogenetic analysis based on ITS and LSU DNA sequence data available in GenBank and the published literature was performed to examine the intergeneric relationships of Puccinia. The obtained results revealed the worldwide distribution of Puccinia. Compared with other nations, a reasonable increase in research publications over the current century was demonstrated in Asian countries. The plant families Asteraceae and Poaceae were observed as the most infected in the 21st century. The phylogenetic studies of the LSU and ITS sequence data revealed the polyphyletic nature of Puccinia. In addition, the presences of too short, too lengthy, and incomplete sequences in the NCBI database demonstrate the need for extensive DNA-based analyses for a better understanding of the taxonomic placement of Puccinia.

Phylogeny and taxonomy of three species of Blastospora (Pucciniales) from East Asia.

Three species of the rust fungus genus Blastospora, Bl. betulae, Bl. itoana, and Bl. smilacis, have been reported in East Asia. Although their morphological characteristics and life cycles have been investigated, their phylogenetic positions have not been clarified sufficiently. Phylogenetic analysis showed that these three species were included into Zaghouaniaceae of Pucciniales. However, Bl. betulae was phylogenetically distinct from Bl. itoana and Bl. smilacis and different from other genera. Based on this result, and applying recent International Code of Nomenclature decisions/recommendations/requirements, Botryosorus, gen. nov., and Bo. deformans,, comb. nov., were applied for Bl. betulae. Two new combinations, Bl. radiata for Bl. itoana and Bl. makinoi for Bl. smilacis, were also applied. Their host plants and distribution were described based on literature records. Zaghouania yunnanensis, comb. nov., was proposed for Cystopsora yunnanensis as a result of this analysis.

Raveneliopsis, a new genus of ravenelioid rust fungi on Cenostigma (Caesalpinioideae) from the Brazilian Cerrado and Caatinga.

The multicellular discoid convex teliospore heads represent a prominent generic feature of the genus Ravenelia. However, recent molecular phylogenetic work has shown that this is a convergent trait, and that this genus does not represent a natural group. In 2000, a rust fungus infecting the Caesalpinioid species Cenostigma macrophyllum (= C. gardnerianum) was described as Ravenelia cenostigmatis. This species shows some rare features, such as an extra layer of sterile cells between the cysts and the fertile teliospores, spirally ornamented urediniospores, as well as strongly incurved paraphyses giving the telia and uredinia a basket-like appearance. Using freshly collected specimens of Rav. cenostigmatis and Rav. spiralis on C. macrophyllum, our phylogenetic analyses based on the nuc 28S, nuc 18S, and mt CO3 (cytochrome c oxidase subunit 3) gene sequences demonstrated that these two rust fungi belong in a lineage within the Raveneliineae that is distinct from Ravenelia s. str. Besides proposing their recombination into the new genus Raveneliopsis (type species R. cenostigmatis) and briefly discussing their potentially close phylogenetic affiliations, we suggest that five other Ravenelia species that are morphologically and ecologically close to the type species of Raveneliopsis, i.e., Rav. corbula, Rav. corbuloides, Rav. parahybana, Rav. pileolarioides, and Rav. Striatiformis, may be recombined pending new collections and confirmation through molecular phylogenetic analyses.

Applying early divergent characters in higher rank taxonomy of Melampsorineae (Basidiomycota, Pucciniales).

Rust fungi in the order Pucciniales represent one of the largest groups of phytopathogens, which occur on mosses, ferns to advanced monocots and dicots. Seven suborders and 18 families have been reported so far, however recent phylogenetic studies have revealed para- or polyphyly of several morphologically defined suborders and families, particularly in Melampsorineae. In this study, a comprehensive phylogenetic framework was constructed based on a molecular phylogeny inferred from rDNA sequences of 160 species belonging to 16 genera in Melampsorineae (i.e. Chrysomyxa, Cerospora, Coleopuccinia, Coleosporium, Cronartium, Hylospora, Melampsora, Melampsorella, Melampsoridium, Milesina, Naohidemyces, Pucciniastrum, Quasipucciniastrum, Rossmanomyces, Thekopsora, Uredinopsis). Our phylogenetic inference indicated that 13 genera are monophyletic with strong supports, while Pucciniastrum is apparently polyphyletic. A new genus, Nothopucciniastrum was therefore established and segregated from Pucciniastrum, with ten new combinations proposed. At the family level, this study further demonstrates the importance of applying morphologies of spore-producing structures (basidia, spermogonia, aecia, uredinia and telia) in higher rank taxonomy, while those traditionally applied spore morphologies (basidiospores, spermatia, aeciospores, urediniospores and teliospores) represent later diverged characters that are more suitable for the taxonomy at generic and species levels. Three new families, Hyalopsoraceae, Nothopucciniastraceae and Thekopsoraceae were proposed based on phylogenetic and morphological distinctions, towards a further revision of Pucciniales in line with the phylogenetic relationships.

Gapless Genome Assembly of Puccinia triticina Provides Insights into Chromosome Evolution in Pucciniales.

Chromosome evolution drives species evolution, speciation, and adaptive radiation. Accurate genome assembly is crucial to understanding chromosome evolution of species, such as dikaryotic fungi. Rust fungi (Pucciniales) in dikaryons represent the largest group of plant pathogens, but the evolutionary process of adaptive radiation in Pucciniales remains poorly understood. Here, we report a gapless genome for the wheat leaf rust fungus Puccinia triticina determined using PacBio high-fidelity (HiFi) sequencing. This gapless assembly contains two sets of chromosomes, showing that one contig represents one chromosome. Comparisons of homologous chromosomes between the phased haplotypes revealed that highly frequent small-scale sequence divergence shapes haplotypic variation. Genome analyses of Puccinia triticina along with other rusts revealed that recent transposable element bursts and extensive segmental gene duplications synergistically highlight the evolution of chromosome structures. Comparative analysis of chromosomes indicated that frequent chromosomal rearrangements may act as a major contributor to rapid radiation of Pucciniales. This study presents the first gapless, phased assembly for a dikaryotic rust fungus and provides insights into adaptive evolution and species radiation in Pucciniales. IMPORTANCE Rust fungi (Pucciniales) are the largest group of plant pathogens. Adaptive radiation is a predominant feature in Pucciniales evolution. Chromosome evolution plays an important role in adaptive evolution. Accurate chromosome-scale assembly is required to understand the role of chromosome evolution in Pucciniales. We took advantage of HiFi sequencing to construct a gapless, phased genome for Puccinia triticina. Further analyses revealed that the evolution of chromosome structures in rust lineage is shaped by the combination of transposable element bursts and segmental gene duplications. Chromosome comparisons of Puccinia triticina and other rusts suggested that frequent chromosomal arrangements may make remarkable contributions to high species diversity of rust fungi. Our results present the first gapless genome for Pucciniales and shed light on the feature of chromosome evolution in Pucciniales.

Genetic time traveling: sequencing old herbarium specimens, including the oldest herbarium specimen sequenced from kingdom Fungi, reveals the population structure of an agriculturally significant rust.

Sequencing herbarium specimens can be instrumental in answering ecological, evolutionary, and taxonomic inquiries. We developed a protocol for sequencing herbarium specimens of rust fungi (Pucciniales) and proceeded to sequence specimens ranging from 4 to 211 yr old from five different genera. We then obtained sequences from an economically important biological control agent, Puccinia suaveolens, to highlight the potential of sequencing herbarium specimens in an ecological sense and to evaluate the following hypotheses: (1) The population structure of a plant pathogen changes over time, and (2) introduced pathogens are more diverse in their native range. Our efforts resulted in sequences from 87 herbarium specimens that revealed a high level of diversity with a population structure that exhibited spatial-temporal patterns. The specimens sequenced from Europe showed more diversity than the ones from North America, uncovering an invasion pattern likely related to its European native host in North America. Additionally, to the best of our knowledge, the specimen from France collected in c. 1811 is the oldest herbarium specimen sequenced from kingdom Fungi. In conclusion, sequencing old herbarium specimens is an important tool that can be extrapolated to better understand plant-microbe evolution and to evaluate old type specimens to solidify the taxonomy of plant pathogenic fungi.

Haplotype-phased and chromosome-level genome assembly of Puccinia polysora, a giga-scale fungal pathogen causing southern corn rust.

Rust fungi are characterized by large genomes with high repeat content and have two haploid nuclei in most life stages, which makes achieving high-quality genome assemblies challenging. Here, we described a pipeline using HiFi reads and Hi-C data to assemble a gigabase-sized fungal pathogen, Puccinia polysora f.sp. zeae, to haplotype-phased and chromosome-scale. The final assembled genome is 1.71 Gbp, with ~850 Mbp and 18 chromosomes in each haplotype, being currently one of the two giga-scale fungi assembled to chromosome level. Transcript-based annotation identified 47,512 genes for the dikaryotic genome with a similar number for each haplotype. A high level of interhaplotype variation was found with 10% haplotype-specific BUSCO genes, 5.8 SNPs/kbp, and structural variation accounting for 3% of the genome size. The P. polysora genome displayed over 85% repeat contents, with genome-size expansion and copy number increasing of species-specific orthogroups. Interestingly, these features did not affect overall synteny with other Puccinia species having smaller genomes. Fine-time-point transcriptomics revealed seven clusters of coexpressed secreted proteins that are conserved between two haplotypes. The fact that candidate effectors interspersed with all genes indicated the absence of a "two-speed genome" evolution in P. polysora. Genome resequencing of 79 additional isolates revealed a clonal population structure of P. polysora in China with low geographic differentiation. Nevertheless, a minor population differentiated from the major population by having mutations on secreted proteins including AvrRppC, indicating the ongoing virulence to evade recognition by RppC, a major resistance gene in Chinese corn cultivars. The high-quality assembly provides valuable genomic resources for future studies on disease management and the evolution of P. polysora.

Exogenous double-stranded RNA inhibits the infection physiology of rust fungi to reduce symptoms in planta.

Rust fungi (Pucciniales) are a diverse group of plant pathogens in natural and agricultural systems. They pose ongoing threats to the diversity of native flora and cause annual crop yield losses. Agricultural rusts are predominantly managed with fungicides and breeding for resistance, but new control strategies are needed on non-agricultural plants and in fragile ecosystems. RNA interference (RNAi) induced by exogenous double-stranded RNA (dsRNA) has promise as a sustainable approach for managing plant-pathogenic fungi, including rust fungi. We investigated the mechanisms and impact of exogenous dsRNA on rust fungi through in vitro and whole-plant assays using two species as models, Austropuccinia psidii (the cause of myrtle rust) and Coleosporium plumeriae (the cause of frangipani rust). In vitro, dsRNA either associates externally or is internalized by urediniospores during the early stages of germination. The impact of dsRNA on rust infection architecture was examined on artificial leaf surfaces. dsRNA targeting predicted essential genes significantly reduced germination and inhibited development of infection structures, namely appressoria and penetration pegs. Exogenous dsRNA sprayed onto 1-year-old trees significantly reduced myrtle rust symptoms. Furthermore, we used comparative genomics to assess the wide-scale amenability of dsRNA to control rust fungi. We sequenced genomes of six species of rust fungi, including three new families (Araucariomyceaceae, Phragmidiaceae, and Skierkaceae) and identified key genes of the RNAi pathway across 15 species in eight families of Pucciniales. Together, these findings indicate that dsRNA targeting essential genes has potential for broad-use management of rust fungi across natural and agricultural systems.

[Comparative transcriptomic analysis of the haustoria of Gymnosporangium yamadae and G. asiaticum].

To provide a theoretical basis for controlling the spread of rust disease, cultivating disease-resistant varieties and reducing yield losses, we investigated the transcriptome differences between Gymnosporangium yamadae and Gymnosporangium asiaticum at the haustorial stage and revealed a specialized selection mechanism for Gymnosporangium species to infect host plants. We sequenced the transcriptomes of the haustoria in rust-infected leaves when basidiospores of G. yamadae and G. asiaticum infected their hosts, and obtained 21 213 and 13 015 unigenes, respectively. Real-time fluorescence quantitative PCR validation of five genes selected from G. yamadae and G. asiaticum, respectively, showed that their expression profiles were generally consistent with the results of transcriptome analysis, demonstrating the reliability of the transcriptome data. We used seven databases such as Nr, GO, KEGG, and KOG to perform gene function annotation and enrichment analysis, and found that the genes from both rusts were mainly enriched in cellular processes, translation, and metabolism-related pathways. Moreover, we used SignalP, TMHMM online website and other software such as dbCAN, BLSAT, HMMER to show that there were 343 (2.51%) and 175 (2.79%) candidate effector proteins containing 14 and 5 proteases and 10 and 3 lipases in the haustoria of G. yamadae and G. asiaticum, respectively. Furthermore, we used OrthoFinder, BLAST and KaKs Calculator software to analyze the evolutionary relationship of the two fungi. Among one-to-one homologous genes, gene pairs with > 82% alignment were considered to be under conservative selection, and 12.37% under positive selection. Five effectors of G. asiaticum were under positive selection, and one of which was a lipase. No significant differences were found in the enrichment of expressed genes between G. yamadae and G. asiaticum, indicating the biological processes involved in haustoria were relatively conserved, despite the typical host selectivity between species. The low protein similarity between the two species suggested that they were under greater host selective pressure and there was significant evolutionary divergence, which might be related to the host-specific selection mechanism. In the haustorial, the main purpose of the effectors might be to regulate physiological processes in the plants rather than attacking the host directly, and G. yamadae and G. asiaticum might use plant lipids as energy sources.