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.


Introduction
Puccinia Pers. is an obligatory plant pathogenic genus of rust fungi that belongs to the family Pucciniaceae of the order Pucciniales (Basidiomycota). It is the largest genus of rust fungi, containing about 4000 species [1][2][3][4][5][6][7], and it has a worldwide distribution. The recently published manuscript "The Outline of Fungi" provides a total of 3300 accepted species of distribution, are also discussed to provide an understanding of the complete distributional range of these fungi. Rusts are obligate parasites that show phenotypic and genetic plasticity because of their complete dependency on the presence of living host plants to complete their life cycles [1,12]. These fungi show unique systematic characteristics by producing interesting morphologically and cytologically distinct spore-producing structures, which have attracted the interest of mycologists for centuries. The species of Puccinia may produce up to five morphologically and cytologically distinct spore-producing structures. The production of these distinctive structures differentiates these rust fungi from other fungal groups. These structures are produced by the species of Puccinia in the infection process of the host-pathogen interaction. These diverse structures are generally the successive stages of reproduction produced by rust fungi, and they may vary from species to species. These fungi commonly appear as yellow-orange or brown pustules on healthy and vigorously growing plant parts, such as leaves, petioles, tender shoots, stems, and fruits. The infection pustules are often associated with chlorotic lesions, which may cause the premature wilt and senesce of infected leaves in cases of severe infection. The spore-producing pustules are present as solitary, scattered, or aggregated in groups, arranged linearly, concentrically, or irregularly, and are often erumpent. The spore-producing structures of the species of Puccinia are the basic spore states, which are generally recognized as spermogonium, aecium, uredinium, telium, and basidium. These states are generally assigned Roman numerals (0, I, II, III, and IV, respectively) during the taxonomic description of rust fungi, including Puccinia spp. Only a few species of Puccinia, such as P. vexans Farl., produce six morphologically and functionally different spore stages. Besides the production of regular pale-colored urediniospores, this species produces thick-walled dark-pigmented urediniospores called amphispores. Two different systems are generally suggested, morphology and ontogeny, and they have been applied in the definition and terminology of the spore states of rust. It is important to discuss this aspect here because the terminology used to describe the biology of Puccinia spp., including the morphology and life cycle, are also the same as that used for all rust fungi. The spore morphology is generally considered the basis for defining the spore states [13,14]. The spore definitions based on this system are as follows:

The Genus
Aeciospores: defined as being produced in chains and with ornamentation that is traditionally known as verrucose; Urediniospores: defined as being always unicellular and borne singly on pedicels, and usually with ornamentation that is traditionally known as echinulate [15]. In the ontogenic system, the position of the spore states in the life cycle rather than the morphology is utilized for defining the spore terminology [16][17][18][19]. The general descriptions of these diverse spores as they are produced by rust fungi (including Puccinia spp.) are as follows: Teliospores: spores that produce basidia (probasidia and hypobasidia); Basidiospores: spores produced on basidia and are haploid and frequently binucleate, but that are not dikaryotic spores; Spermatia: dikaryotizing elements; Aeciospores: dikaryotic nonrepeating spores that are produced in sori typically associated with spermogonia, and that give birth to dikaryotic vegetative mycelia; Urediniospores: repeated dikaryotic mycelia that typically give rise to dikaryotic mycelia on the same host, and that are sometimes referred to as uredospores or urediospores.
The introduction of a taxonomic grouping as Forma specialis (plural formae speciales) is allowed by the International Code of Botanical Nomenclature (ICBN). In the case of fungi, it is applied a taxonomic grouping. It is generally adapted when authors do not feel a subspecies or variety name is appropriate. For example, Puccinia striiformis Westend. consists of several formae speciales based on host specialization, including P. striiformis f.sp. tritici, P. striiformis f.sp. hordei, P. striiformis f.sp. elymi, P. striiformis f.sp. agropyri, and P. striiformis f.sp. secalis. Among the five forms of P. striiformis, the sexual stage was confirmed only for the wheat form of the rust P. striiformis f.sp. tritici, but not known for the other four forms. Based on the host specificity, the numbers of Forma specialis of Puccinia graminis such as P. graminis f.sp. avenae, P. graminis f.sp. secalis, and P. graminis f.sp. tritici,. are available, which were proved to be helpful in the taxonomy of rust fungi including the genus Puccinia [1][2][3].
It is believed that the rust pustules of uredinia that are present on the stem and leaf sheath tissues often survive for a longer duration in comparison with those that are present on the leaves. In the case of spore production, the number of spores produced by leaf pustules is generally higher. Under continuous conditions, stem rust urediniospores show more resistance to atmospheric conditions than leaf rust spores [20,21]. The species of Puccinia are responsible for causing all possible types of rust disease symptoms by producing all five basic spore states, which are generally recognized as spermogonium, aecium, uredinium, telium, and basidium. There is great variation in the production of the spore states by the different species of this rust genus. While some species produce all the spore states, others may produce less than five. The teliosori and teliospores of different Puccinia species are presented in Section 2 and Figure 2, respectively.

Data Collection and Compilation
This paper was compiled based on the information retrieved from an extensive search of peer-reviewed publications, field guides, monographs, books, conference proceedings, project reports, dissertations, theses, and other offline and online resources. Information based on taxonomic studies, checklists, and new reports, as well as reports on the new taxa, was generally considered in the compilation of this study. The scientific names of the hosts and fungi were then cross-verified for scientific entities. The Plant List (http://www.theplantlist.org, accessed on 20 April 2022) was consulted for the verification of the host plant names, and the fungal databases MycoBank (www.mycobank.org; accessed on 20 April 2022), Species Fungorum (www.speciesfungorum.org; accessed on 20 April 2022), and IndexFungorum (www.indexufngorum.org; accessed on 20 April 2022) were consulted to gather information on the current fungal names, numbers, and distributions. Fungal Databases, US National Fungus Collections, ARS, and USDA, which are important online sources of plant pathogens and their hosts, were also used during the compilation. To understand the general trend of the outline and a higher-rank classification of Puccinia, publications such as [1,4-6,8,22,23] were consulted. An attempt was made to summarize all the collected information in the form of the current statuses of the species numbers, their distributions with respect to hosts, and the trends of the published

Data Collection and Compilation
This paper was compiled based on the information retrieved from an extensive search of peer-reviewed publications, field guides, monographs, books, conference proceedings, project reports, dissertations, theses, and other offline and online resources. Information based on taxonomic studies, checklists, and new reports, as well as reports on the new taxa, was generally considered in the compilation of this study. The scientific names of the hosts and fungi were then cross-verified for scientific entities. The Plant List (http://www.theplantlist.org, accessed on 20 April 2022) was consulted for the verification of the host plant names, and the fungal databases MycoBank (www.mycobank.org; accessed on 20 April 2022), Species Fungorum (www.speciesfungorum.org; accessed on 20 April 2022), and IndexFungorum (www.indexufngorum.org; accessed on 20 April 2022) were consulted to gather information on the current fungal names, numbers, and distri-butions. Fungal Databases, US National Fungus Collections, ARS, and USDA, which are important online sources of plant pathogens and their hosts, were also used during the compilation. To understand the general trend of the outline and a higher-rank classification of Puccinia, publications such as [1,[4][5][6]8,22,23] were consulted. An attempt was made to summarize all the collected information in the form of the current statuses of the species numbers, their distributions with respect to hosts, and the trends of the published literature in the last century and decade. The publication indices of Puccinia spp. in terms of year, decade, and century were also analyzed and are presented in this paper. In addition, the references in other languages were translated into English so that the scientific community could easily understand them. In addition, the role of Puccinia as a threat to biodiversity is also discussed on a global scale in the present paper. A short discussion on the limitations to the current knowledge of Puccina and future recommendations is also presented here.

Phylogenetic Analyses
We worked on the Puccinia species phylogeny, and the NCBI search showed 292,000 Puccinia hits, of which most were repeated and whole-genome sequences. An NCBI search with "Puccinia and type" showed 52,446 sequence results, of which 13 sequences were found to be type sequences. We chose Index Fungorum to search for species deposited more than two decades between 2000 and 2022 (till July), for which a total of 228 species were found.
From the above two sources, we collected the ITS (69), LSU (65), SSU (15), cytochrome oxidase COX (9), TUB (8), RPB2 (1), and TEF1 sequences. Because most regions have a small number of sequences, we selected the ITS and LSU sequences to construct the multigene phylogeny. The DNA sequence data of the Puccinia species from the LSU and ITS rDNA were downloaded from GenBank and earlier published literature. Individual nucleotide sequences of the LSU and ITS were distinctly aligned using the MAFFT v7.450 online server (https://mafft.cbrc.jp/alignment/server/; accessed on 20 April 2022) and exported to aligned sequence data [24], and they were then manually checked and edited where necessary in BioEdit v.7.0.9 [25]. The sequences of taxa containing weakly aligned portions, incomplete data, missing sequence data, and gaps were removed. The separate aligned gene regions of the LSU and ITS were combined in BioEdit. The combined multigene sequence alignment was converted to the PHYLIP format (.phy) using the alignment transformation environment (http://sing.ei.uvigo.es/ALTER/; accessed on 20 April 2022) for randomized accelerated maximum likelihood (RAxML) analysis. The aligned LSU and ITS single-gene datasets and a concatenated dataset of LSU and ITS genes were analyzed with maximum likelihood using the RAxML-HPC2 on XSEDE (8.2.8) [26,27] on the CIPRES Science Gateway platform [28] using the GTR + I + G model of evolution. Maximum likelihood bootstrap values equal to or greater than 70% were given above each node. Phylogenetic trees were visualized with the FigTree v.1.4.0 program [29] and reorganized in Microsoft PowerPoint. A checklist of molecular studies on Puccinia spp., along with the names of the isolates, was also prepared and is presented in Table 1. Most of the Puccinia species were identified based on the morphology and microscopic characteristics of the uredia and telia, or based on other successive stages observed on the collected samples. The identification of this rust genus based on molecular parameters is not up to the mark and still requires extensive studies. In the phylogenetic results, the Puccinia species were separated into two complexes in both the ITS and LSU sequence data. Both complexes of the ITS and LSU share many similar sequences. The incomplete sequences were mostly found in the Puccinia sequence dataset (e.g., ITS1 and 5.8S or ITS1, 5.8S complete, and ITS partial or 28S partial). Approximately, 50% of the sequences had up to 300 nucleotides, while the remaining sequences had up to 800 nucleotides. Incomplete sequences can result in two complexes in a single genus. Therefore, complete gene sequences from the ITS and LSU are needed to analyze these complex clades. The phylogenetic analyses exposed the polyphyletic nature of this genus, which requires further DNA-based analyses of the rust disease caused by Puccinia to develop a better understanding of its taxonomic placement. The genus Uromyces also showed a polyphyletic nature during a study carried out by the authors of [30]. A study carried out by the authors of [6,31,32] also confirmed the polyphyletic nature of the rust genus Puccinia (Figure 3). plete sequences can result in two complexes in a single genus. Therefore, complete gene sequences from the ITS and LSU are needed to analyze these complex clades. The phylogenetic analyses exposed the polyphyletic nature of this genus, which requires further DNA-based analyses of the rust disease caused by Puccinia to develop a better understanding of its taxonomic placement. The genus Uromyces also showed a polyphyletic nature during a study carried out by the authors of [30]. A study carried out by the authors of [6,31,32] also confirmed the polyphyletic nature of the rust genus Puccinia (Figure 3).

Current Status of Numbers of Species
The occurrence of the rust genus Puccinia is considered cosmopolitan. All the continents, except Antarctica, show the presence of many species of the rust genus Puccinia. The genus is one of the broadly studied rust genera, and the fungi of this genus also possess broad host ranges and distributions. Nearly all categories of plants belonging to

Current Status of Numbers of Species
The occurrence of the rust genus Puccinia is considered cosmopolitan. All the continents, except Antarctica, show the presence of many species of the rust genus Puccinia. The genus is one of the broadly studied rust genera, and the fungi of this genus also possess broad host ranges and distributions. Nearly all categories of plants belonging to approximately all the families have been found to be infected with these fungi. Similar to the trends of the occurrences of different rust fungi on their hosts, the occurrence of Puccinia rust has also been reported to be the most profuse on plant hosts that belong to the families Asteraceae, Poaceae, and Ranunculaceae. When we came across the number of research papers previously published by several researchers, a similar trend of the occurrence of Puccinia rust was observed. Similarly, plant families such as Apiaceae, Polygonaceae, Rubiaceae, Cyperaceae, Acanthaceae, Berberidaceae, Lamiaceae, and Saxifragaceae are among the most infected plant families with Puccinia rust; however, the infection and host range of Puccinia rust is not only limited to these plant families. A tentative distribution of Puccinia rust in the major plant families is summarized and presented in Figure 4. When we talk about the species boundaries of Puccinia rust, a total of 5450 epithets are available on Index Fungorum (www.indexfungorum.com; accessed on 20 April 2022). However, a total of 3300 species of Puccinia have been reported all over the world on a variety of hosts [1][2][3][4][5][6][7][8]22,33].

Trends in Published Literature
In this section, the research on Puccinia rust reported and published in various journals is presented. A total of 988 papers were published from the year 1794 to 2020, and they were included in the present study to understand the general publication trend. The data from different online platforms, as well as offline resources, were retrieved to compile the information on these rust fungi. To understand the decadal trend, the numbers of publications were counted per ten years and are presented according to century and some more criteria. Publications on the new records, reports, and taxa (genus/species) were generally included in this analysis and are presented in Table 2.

Trends in Published Literature
In this section, the research on Puccinia rust reported and published in various journals is presented. A total of 988 papers were published from the year 1794 to 2020, and they were included in the present study to understand the general publication trend. The data from different online platforms, as well as offline resources, were retrieved to compile the information on these rust fungi. To understand the decadal trend, the numbers of publications were counted per ten years and are presented according to century and some more criteria. Publications on the new records, reports, and taxa (genus/species) were generally included in this analysis and are presented in Table 2.
The results revealed that the research on Puccinia rust was quite encouraging during the 19th and 20th centuries. A total of 277 papers were published during the 19th century, while 627 were published during the 20th century. The trend of research publications for the current century (up to 2020) has also been high in terms of both qualitative and quantitative aspects. Similarly, in the decadal analysis of the published research on Puccinia rust, the highest numbers of papers were published at the end of the 19th century and the beginning of the 20th century (121 and 144, respectively)  Morphotaxonomy alone is not enough to describe a new taxon. The molecular aspects play an important role in resolving the correct taxonomic placement of all fungi, including rust fungi. This might explain the variations in the number of publications during the current century. Not all researchers can afford a good laboratory and access to resources. Nowadays, insufficient funding is also a major constraint in the performance of basic taxonomic research.

Threat to Biodiversity
Rust fungi are considered one of the most serious threats to both agricultural (e.g., wheat, soybean, or coffee), and non-agricultural crops and tree species as well. Puccinia is one of the harmful biotrophic fungal genera that seriously harm major cereal crops (except rice) and nonagricultural plants all over the world. The type species of Puccinia (Puccinia graminis) is one of the destructive rust fungi reported to cause the mass destruction of wheat and barley stem rust (black rust). Similarly, Puccinia striiformis f. sp. tritici, wheat stripe rust (yellow rust), and P. triticina, wheat leaf rust (brown rust), are also destructive rusts that are distributed all around the globe. To understand why the species of Puccinia are a threat to biodiversity, some examples of rust diseases caused by them are presented in this section.
Puccinia graminis Pers., Neues Mag. Bot. 1: 119 (1794) This is a macrocyclic heteroecious rust that has devastated wheat for many decades, and it is one of the most studied rust fungi. It causes the black stem rust of wheat and poses a serious threat to food security. It may cause crop losses of up to 70%. The Berberis, Berberis, Mahoberberis, and Mahonia serve as alternate hosts for this fungus. This fungus occurs in all major wheat-growing areas around the world. Puccinia graminis has also been studied in detail and has long been used as a model for studying the cytology, physiology, biochemistry, and molecular aspects of rust fungus biology [34][35][36]. Another extremely contagious race of Puccinia graminis, TTKSK (Ug99), was discovered in Uganda. Because it does not recognize any national borders and can infect fields anywhere, it poses a serious threat to wheat growers all over the world. This strain is aggressive against numerous resistance genes that have previously shielded wheat against stem rust. It can cause losses of the victim crop of up to 100%. Although there are Ug99-resistant wheat variants available, their cultivation range is not broad [37][38][39][40].
Puccinia striiformis Westend., Bull. Acad. R. Sci. Belg., Cl. Sci. 21 (no. 2): 235 (1854) This is a biotrophic and heteroecious rust pathogen that has been reported to cause yellow (stripe) rust. At least two lineages are exclusive to grasses, while one lineage primarily infects cereals. The pathogen has the widest host range within the tribe Triticeae (in the plant genera Aegilops, Elymus, Hordeum, and Triticum) and Berberis spp. as the alternate host or sexual host. It is reported to be one of the most damaging cereal rusts compared with other rusts. This fungus reduces the photosynthetic area and the production of sugars for the plant. As the fungus mainly infects the leaf, it also causes substantial water loss while erupting the epidermis. Based on the disease severity and susceptibility of the variety, this rust can cause mild to very high losses; however, a 30% loss is common in susceptible varieties [41].
Puccinia coronata Corda, Icon. Fung. (Prague) 1: 6 (1837) This rust is reported to cause crown rust disease in cultivated and wild oat (Avena spp.). It infects two hosts to complete its life cycle: oat (asexual phase) and Rhamnus spp. (sexual phase) as the primary and alternate hosts, respectively. Oat crop cultivation areas with warm temperatures (20-25 • C) and high humidity are more prone to this rust epidemic. Infection by the pathogen leads to plant lodging and shriveled grains of poor quality. This rust pathogen can infect 290 species of grass hosts [42][43][44].
Puccinia psidii G. Winter, Hedwigia 23: 171 (1884) The rust Puccinia psidii is a pathogen with a broad host range in the myrtle family (Myrtaceae). However, the common guava (Psidium guajava) and Eucalyptus spp. are at more risk, as it causes severe infection in these plants [45][46][47]. A severe infection of P. psidii was reported in Brazil, which caused damage to various members of the family Myrtaceae [46,48,49]. Similarly, this fungus causes eucalyptus rust in Australia and poses a threat to the biodiversity in this country, as well as to the eucalyptus forest industry worldwide [45]. In 2017, based on a DNA-based molecular analysis of rust samples, the names were synonymized by Beenken in a new genus as Austropuccinia psidii [50]. Apart from the abovementioned diseases caused by the rust genus Puccinia, these fungi are reported to cause several diseases on other plants. Several research and review papers are available on different online and offline platforms that describe the diversities, distributions, and host ranges of rust fungi, including Puccinia [31,32,[51][52][53][54][55][56][57][58]. A list of the species of Puccinia that cause destructive diseases in agricultural and nonagricultural crops is presented in Table 3.

Puccinia in the Present Century
To understand the status of the rust genus Puccinia in the current century, the published literature on new genera and species and new geographical records were taken into consideration, and the data obtained were compiled concerning the host, host family, yearly publication details, and distribution throughout the regions, countries, and continents around the globe. Data based on other aspects of Puccinia rust, such as physiology and biochemistry, were not considered in this study. The publication details of the last two decades of the current century reveal that a total of 82 papers on Puccinia rust were published, 42 of which were published during 2001-2010, and 40 of which were published from 2010 to the present date ( Figure 5).
After analyzing the yearly data, it was observed that the publication record numbers reached up to seven in many years, while the lowest number (one) was also observed for many years. Further, a total of 126 records in the form of new geographical records from 62 regions belonging to 37 countries were recorded during the last two decades. All seven continents showed distributions of the species of Puccinia. If we compare the continental distribution of these rust fungi during the current century, then the highest number of records is found in Asia (42 records from 10 countries), followed by South America (27 records in four countries), Africa (23 records in three countries), Europe (17 records in seven countries), North America (13 records in 11 countries), Oceania (six records in two countries), and Australia (with three records) ( Figure 6). 62 regions belonging to 37 countries were recorded during the last two decades. All seven continents showed distributions of the species of Puccinia. If we compare the continental distribution of these rust fungi during the current century, then the highest number of records is found in Asia (42 records from 10 countries), followed by South America (27 records in four countries), Africa (23 records in three countries), Europe (17 records in seven countries), North America (13 records in 11 countries), Oceania (six records in two countries), and Australia (with three records) ( Figure 6).  The rich biodiversity and variable climatic conditions of Asia might be responsible for the occurrence of rust fungi (Puccinia spp.) in high numbers. The same explanation is applicable to America and Africa, while the lowest reports from Oceania and Australia directly correlate with the agroclimatic conditions of these two continents. When we analyze all 126 records, only 34 species of Puccinia have been identified at the molecular level using multigene analysis. From the data obtained on the host distribution, a total of 124 plant species belonging to 90 plant genera of 34 plant families have been found to be infected with different species of Puccinia. As observed in the previous section, Asteraceae and Poaceae have been found to be highly infected with different species of Puccinia. The data on Puccinia rust during the present century is summarized and graphically presented in Figures 7 and 8. The rich biodiversity and variable climatic conditions of Asia might be responsible for the occurrence of rust fungi (Puccinia spp.) in high numbers. The same explanation is applicable to America and Africa, while the lowest reports from Oceania and Australia directly correlate with the agroclimatic conditions of these two continents. When we analyze all 126 records, only 34 species of Puccinia have been identified at the molecular level using multigene analysis. From the data obtained on the host distribution, a total of 124 plant species belonging to 90 plant genera of 34 plant families have been found to be infected with different species of Puccinia. As observed in the previous section, Asteraceae and Poaceae have been found to be highly infected with different species of Puccinia. The lyze all 126 records, only 34 species of Puccinia have been identified at the molecular level using multigene analysis. From the data obtained on the host distribution, a total of 124 plant species belonging to 90 plant genera of 34 plant families have been found to be infected with different species of Puccinia. As observed in the previous section, Asteraceae and Poaceae have been found to be highly infected with different species of Puccinia. The data on Puccinia rust during the present century is summarized and graphically presented in Figures 7 and 8.

Limitations of Current Knowledge
The current research on rust fungi is mainly based on morpho-taxonomy (the morphologies of the shapes and sizes of certain spore stages), while, the inclusion of recent technologies, and specifically DNA-based techniques, brings a new turn to the taxonomy of rust fungi. When it is not possible to differentiate two similar fungal species based on their morphological characteristics, molecular techniques can be used to successfully differentiate them, even at the difference of one base pair of nucleotides. However, few studies are based on the use of modern instruments and molecular-based techniques for fungal taxonomy, and specifically DNA-based techniques. The fundamental reasons for the slow adoption of molecular techniques in taxonomic studies on the rust fungi of Puccinia are the same as those that apply to all other rust fungi. When studying the species of Puccinia, mycologists encounter several problems/limitations, including the lack of necessary databases, resources, and funding, the reduced interest of budding researchers, and the pricey services offered by many agencies. To understand these limitations, a detailed explanation is given below:

Limitations of Current Knowledge
The current research on rust fungi is mainly based on morpho-taxonomy (the morphologies of the shapes and sizes of certain spore stages), while, the inclusion of recent technologies, and specifically DNA-based techniques, brings a new turn to the taxonomy of rust fungi. When it is not possible to differentiate two similar fungal species based on their morphological characteristics, molecular techniques can be used to successfully differentiate them, even at the difference of one base pair of nucleotides. However, few studies are based on the use of modern instruments and molecular-based techniques for fungal taxonomy, and specifically DNA-based techniques. The fundamental reasons for the slow adoption of molecular techniques in taxonomic studies on the rust fungi of Puccinia are the same as those that apply to all other rust fungi. When studying the species of Puccinia, mycologists encounter several problems/limitations, including the lack of necessary databases, resources, and funding, the reduced interest of budding researchers, and the pricey services offered by many agencies. To understand these limitations, a detailed explanation is given below:

•
Apart from the availability of 5000 species of Puccinia, only a few species are known to have DNA sequence data. The difficulty in culturing rust fungi is one of the possible reasons behind the reduced availability of molecular data. Further, DNA isolation directly from rust fungi present on a natural host and further processing are not easy tasks. In addition, the available sequences are not up to the mark. While others are too long, some sequences are too short, and some are incomplete. While some sequences are up to 300 nucleotides long, others are up to 800 nucleotides long, reflecting the intricacy of their taxonomic evaluation. Incomplete sequences in the Puccinia sequence dataset can result in two complexes in a single genus. Therefore, to investigate these complicated clades, entire gene sequences are required. The phylogenetic studies revealed the polyphyletic nature of the species of Puccinia, which require more DNA-based analyses for a better understanding of their taxonomic placement. The nonavailability of molecular data for all collections of Puccinia all over the globe is another limitation that highlights the requirement for fresh collections of Puccinia species and their molecular characterizations to generate molecular data so that their phylogenetic relationships can be explained more precisely. Although country-wide databases of rust fungi are available on various online platforms, the lack of a universal platform exclusively for global rust fungi is also a major limitation in the research on rust fungi, including Puccinia.

•
When we talk about the day-by-day decreased interest of budding scientists/researchers in the field of the basic taxonomy of fungi, the reasons behind this are complex, such as insufficient funds, expensive outsourced mycological services, and, overall, the difficulty in publishing taxonomies in high-impact journals without modern techniques. These issues are leading to the decreased interest of mycologists in fungal taxonomy, which is ultimately decreasing the number of fungal taxonomists [32]. • Publication in high-impact journals has now become a criterion to assess the quality of research and the performance of researchers/scientists/academicians, or to appraise whether they should be promoted. However, taxonomy based on DNA-based molecular techniques has now become a minimum criterion to process any submitted manuscript, even for initial review. Luckily, few journals are still focusing on the novelty of the research, and most are now considering fungal manuscripts that are purely based on morpho-taxonomy.

•
Despite being less expensive to support than applied research, basic fungus research is no longer prioritized for funding. This scenario is common in developing countries. The fundamental inventorying and identification of fungi is not everyone's cup of tea, similar to obtaining funding for applied research (ideally in biotechnology). Due to a lack of sufficient funding, laboratories working on taxonomic studies of fungi continue to lack current equipment (e.g., that which is used in DNA isolation, amplification (PCR), and sequencing), and they are gradually turning their attention to the practical elements of the field. Additionally, not every mycologist can afford the service fees for the molecular techniques offered by many agencies/institutions of national and worldwide reputation, and particularly researchers working on a self-finance basis.

Conclusions
In conclusion, it was observed that Puccinia is the largest genus of rust fungi that infect a wide variety of host plants of both agricultural and nonagricultural importance. The genus shows variation in its diversity and distribution worldwide. Based on compiled data, a total of 5450 epithets (3300 species) are available [70]. In the evaluation of the host distribution, plant families such as Asteraceae and Poaceae were found to be highly infected with different species of Puccinia. The analyses of the trends in the published literature on rust showed that researchers from Asian countries are among those who have published the highest numbers of papers on all the continents. The NCBI search showed 292,000 hits of repeated and whole-genome sequences. While some sequences are too short, others are too lengthy, and some are incomplete. It was also observed that the taxonomic statuses of a number of Puccinia spp. are still unclear, as only morpho-taxonomic traits have been used to identify the majority of them. Moreover, molecular data on most Puccinia spp. are not available so far, and their taxonomic placement is still doubtful; hence, they are classified as incertae sedis. This generates a potential area of research interest for both current and future mycologists. Similarly, the generic names of many Puccinia spp. have been changed or transferred to different genera; however, the incorporation of this revision is still required in their original collections (types or records). Furthermore, the phylogenetic analyses exposed the polyphyletic nature of this genus, which requires further DNA-based analyses of the rust disease caused by Puccinia to develop a better understanding of its taxonomic placement. A study carried out by the authors of [6,31,32] also confirmed the polyphyletic nature of the rust genus Puccinia. Therefore, fresh collections of Puccinia species and their molecular characterizations to generate molecular data are highly recommended so that their phylogenetic relationships can be explained more precisely. All these limitations generate excellent opportunities for mycologists to explore this rust genus based on morphotaxonomy and molecular data to determine and confirm the taxonomic positions of its species. Additionally, the development of a universal digital platform exclusively for global rust fungi is also recommended in the present study so that researchers who are working on this specific group of fungi can take advantage of this information in one place.