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.

Genome size variation within Crithmum maritimum: Clues on the colonization of insular environments.

Angiosperms present an astonishing diversity of genome sizes that can vary intra- or interspecifically. The remarkable new cytogenomic data shed some light on our understanding of evolution, but few studies were performed with insular and mainland populations to test possible correlations with dispersal, speciation, and adaptations to insular environments. Here, patterns of cytogenomic diversity were assessed among geographic samples (ca. 114) of Crithmum maritimum (Apiaceae), collected across the Azores and Madeira archipelagos, as well as in adjacent continental areas of Portugal. Using flow cytometry, the results indicated a significant intraspecific genome size variation, spanning from reduced sizes in the insular populations to larger ones in the mainland populations. Moreover, there was a tendency for an increase in genome size along the mainland populations, associated with lower temperatures, higher precipitation, and lower precipitation seasonality. However, this gradient might be the result of historic phylogeographical events associated with previous dispersal and extinction of local populations. Overall, our findings provided evidence that smaller genome sizes might play a critical role in the colonization of islands, corroborating other studies that argue that organisms with smaller genomes use fewer resources, having a selective advantage under insular environments. Although further studies are needed to improve our understanding of the mechanisms underlying genome size evolution on islands, conservation strategies must be promoted to protect the rich cytogenomic diversity found among C. maritimum populations, which occur in coastal areas that are particularly threatened by human activity, pollution, invasive species, and climate changes.

Subcuticular−Intracellular Hemibiotrophy of Colletotrichum lupini in Lupinus mutabilis

Anthracnose caused by Colletotrichum lupini is the most important disease affecting lupin cultivation worldwide. Lupinus mutabilis has been widely studied due to its high protein and oil content. However, it has proved to be sensitive to anthracnose, which limits the expansion of its cultivation. In this work, we seek to unveil the strategy that is used by C. lupini to infect and colonize L. mutabilis tissues using light and transmission electron microscopy (TEM). On petioles, pathogen penetration occurred from melanized appressoria, subcuticular intramural hyphae were seen 2 days after inoculation (dai), and the adjacent host cells remained intact. The switch to necrotrophy was observed 3 dai. At this time, the hyphae extended their colonization to the epidermal, cortex, and vascular cells. Wall degradation was more evident in the epidermal cells. TEM observations also revealed a loss of plasma membrane integrity and different levels of cytoplasm disorganization in the infected epidermal cells and in those of the first layers of the cortex. The disintegration of organelles occurred and was particularly visible in the chloroplasts. The necrotrophic phase culminated with the development of acervuli 6 dai. C. lupini used the same infection strategy on stems, but there was a delay in the penetration of host tissues and the appearance of the first symptoms.

Diversity and Cytogenomic Characterization of Wild Carrots in the Macaronesian Islands.

The Macaronesian islands constitute an enormous reservoir of genetic variation of wild carrots (subtribe Daucinae; Apiaceae), including 10 endemic species, but an accurate understanding of the diversification processes within these islands is still lacking. We conducted a review of the morphology, ecology, and conservation status of the Daucinae species and, on the basis of a comprehensive dataset, we estimated the genome size variation for 16 taxa (around 320 samples) occurring in different habitats across the Macaronesian islands in comparison to mainland specimens. Results showed that taxa with larger genomes (e.g., Daucus crinitus: 2.544 pg) were generally found in mainland regions, while the insular endemic taxa from Azores and Cabo Verde have smaller genomes. Melanoselinum decipiens and Monizia edulis, both endemic to Madeira Island, showed intermediate values. Positive correlations were found between mean genome size and some morphological traits (e.g., spiny or winged fruits) and also with habit (herbaceous or woody). Despite the great morphological variation found within the Cabo Verde endemic species, the 2C-values obtained were quite homogeneous between these taxa and the subspecies of Daucus carota, supporting the close relationship among these taxa. Overall, this study improved the global knowledge of DNA content for Macaronesian endemics and shed light into the mechanisms underpinning diversity patterns of wild carrots in the western Mediterranean region.

Exploring physicochemical and cytogenomic diversity of African cowpea and common bean.

In sub-Saharan Africa, grain legumes (pulses) are essential food sources and play an important role in sustainable agriculture. Among the major pulse crops, the native cowpea (Vigna unguiculata) and introduced common bean (Phaseolus vulgaris) stand out. This paper has two main goals. First, we provide a comprehensive view of the available genetic resources of these genera in Africa, including data on germplasm collections and mapping biodiversity-rich areas. Second, we investigate patterns of physicochemical and cytogenomic variation across Africa to explore the geographical structuring of variation between native and introduced beans. Our results revealed that 73 Vigna and 5 Phaseolus species occur in tropical regions of Africa, with 8 countries accounting for more than 20 native species. Conversely, germplasm collections are poorly represented when compared to the worldwide collections. Regarding the nuclear DNA content, on average, V. unguiculata presents significantly higher values than P. vulgaris. Also, V. unguiculata is enriched in B, Mg, S, and Zn, while P. vulgaris has more Fe, Ca, and Cu. Overall, our study suggests that the physicochemical and cytogenomic diversity of native Vigna species is higher than previously thought, representing valuable food resources to reduce food insecurity and hunger, particularly of people living in African developing countries.

The use of flow cytometry for fungal nuclear DNA quantification.

Genome size information is sparse across fungi, with information being available for less than 2000 species. So far, most records have been obtained using static, microscope-based cytometry methods or derived from genome sequencing projects. Flow cytometry is now considered the state-of-the-art method for obtaining genome size measurements, and appropriate methods and DNA standards are available, enabling the analysis of most genome size ranges in a rapid, robust and inexpensive way. The average fungal genome size is 60 Mbp, but sizes vary across phylogeny, ranging from 2.2 (Encephalitozoon romaleae) to 3706 Mbp (Jafnea semitosta). In several fungal clades, genome size expansion seems to accompany evolution either to plant mutualism or to plant parasitism (particularly biotrophy), and fungi that interact with plants seem to have larger genomes than saprobes and those that interact with animals. Whereas flow cytometry for nuclear DNA quantification is routinely employed in plant sciences for genome size and ploidy studies, its use in fungal biology is still infrequent. Appropriate standards, methods and best practices are described here, with the aim of stimulating a more generalized and widespread use of flow cytometry for fungal genome size measurement.

The genetic legacy of fragmentation and overexploitation in the threatened medicinal African pepper-bark tree, Warburgia salutaris.

The pepper-bark tree (Warburgia salutaris) is one of the most highly valued medicinal plant species worldwide. Native to southern Africa, this species has been extensively harvested for the bark, which is widely used in traditional health practices. Illegal harvesting coupled with habitat degradation has contributed to fragmentation of populations and a severe decline in its distribution. Even though the species is included in the IUCN Red List as Endangered, genetic data that would help conservation efforts and future re-introductions are absent. We therefore developed new molecular markers to understand patterns of genetic diversity, structure, and gene flow of W. salutaris in one of its most important areas of occurrence (Mozambique). In this study, we have shown that, despite fragmentation and overexploitation, this species maintains a relatively high level of genetic diversity supporting the existence of random mating. Two genetic groups were found corresponding to the northern and southern locations. Our study suggests that, if local extinctions occurred in Mozambique, the pepper-bark tree persisted in sufficient numbers to retain a large proportion of genetic diversity. Management plans should concentrate on maintaining this high level of genetic variability through both in and ex-situ conservation actions.

Response to Anthracnose in a Tarwi (Lupinus mutabilis) Collection Is Influenced by Anthocyanin Pigmentation.

Anthracnose, caused by Colletotrichum lupini, is a major limiting factor for lupin production. Tarwi or Andean Lupin (Lupinus mutabilis) is generally regarded as susceptible to anthracnose, but the high protein and oil content of its seeds raise interest in promoting its cultivation in Europe. In this study we evaluated the response to anthracnose of 10 tarwi accessions contrasting in anthocyanin pigmentation, by comparison to white lupin (Lupinus albus), using a contemporary Portuguese fungal isolate. A severity rating scale was optimized, including weighted parameters considering the type of symptoms and organs affected. All tarwi accessions were classified as susceptible, exhibiting sporulating necroses on the main stem from seven days after inoculation. Anthracnose severity was lower on anthocyanin-rich tarwi plants, with accession LM34/LIB209 standing out as the less susceptible. Accession I82/LIB201 better combines anthracnose response and yield. In global terms, disease severity was lower on white lupin than on tarwi. Although based on a limited collection, the results of the study show the existence of genetic variability among L. mutabilis towards anthracnose response relatable with anthocyanin pigmentation, providing insights for more detailed and thorough characterization of tarwi resistance to anthracnose.

Pathological, Morphological, Cytogenomic, Biochemical and Molecular Data Support the Distinction between Colletotrichum cigarro comb. et stat. nov. and Colletotrichum kahawae.

The genus Colletotrichum has witnessed tremendous variations over the years in the number of species recognized, ranging from 11 to several hundreds. Host-specific fungal species, once the rule, are now the exception, with polyphagous behavior regarded as normal in this genus. The species Colletotrichum kahawae was created to accommodate the pathogens that have the unique ability to infect green developing coffee berries causing the devastating Coffee Berry Disease in Africa, but its close phylogenetic relationship to a polyphagous group of fungi in the C. gloeosporioides species complex led some researchers to regard these pathogens as members of a wider species. In this work we combine pathological, morphological, cytogenomic, biochemical, and molecular data of a comprehensive set of phylogenetically-related isolates to show that the Coffee Berry Disease pathogen forms a separate species, C. kahawae, and also to assign the closely related fungi, previously in C. kahawae subsp. cigarro, to a new species, C. cigarro comb. et stat. nov. This taxonomic clarification provides an opportunity to link phylogeny and functional biology, and additionally enables a much-needed tool for plant pathology and agronomy, associating exclusively C. kahawae to the Coffee Berry Disease pathogen.

Pathological and Epidemiological Characterization of First Outbreak of Daylily Rust in Europe and Evaluation of Puccinia hemerocallidis Resistance in Hemerocallis Cultivars.

Daylily rust-caused by Puccinia hemerocallidis-was confined to Eastern Asia until the disease was reported in Oceania, Africa, the Americas and Portugal in the 21st century. Although information on rust resistance of American cultivars is available, little is known about the resistance of European bred cultivars, threating the ornamental sector if the fungus spreads to other European countries. Aiming to provide tools to address this, we analyzed the Portuguese pathogens and characterized rust resistance in a selection of cultivars, while optimizing disease rating scales. Morphologic, genetic and cytogenomic characterization of four isolates reveals narrow diversity and raises the question whether the pathogen may have originated in North- or Central America. Daily records of multiple symptomatologic parameters enabled a detailed disease progress analysis, discriminating cultivars according to their resistance levels and revealing susceptibility as the most common state. Among the tested cultivars, 12 out of 17 began to show symptoms between 6-8 dai and were classified as susceptible. Cultivars 'Stella d'Oro', 'Bitsy' and 'Cherry Tiger' behaved as moderately resistant although the occurrence of late sporulation on leaves suggests incomplete resistance and challenges common rating scales. The identification of resistance sources in European breeding lines is crucial for the sustainable future of daylilies.

Olive anthracnose: a yield- and oil quality-degrading disease caused by several species of Colletotrichum that differ in virulence, host preference and geographical distribution.

Olive anthracnose causes fruit rot leading to its drop or mummification, resulting in yield losses and the degradation of oil quality. TAXONOMY AND DISTRIBUTION: The disease is caused by diverse species of Colletotrichum, mostly clustering in the C. acutatum species complex. Colletotrichum nymphaeae and C. godetiae are the prevalent species in the Northern Hemisphere, whereas C. acutatum sensu stricto is the most frequent species in the Southern Hemisphere, although it is recently and quickly emerging in the Northern Hemisphere. The disease has been reported from all continents, but it attains higher incidence and severity in the west of the Mediterranean Basin, where it is endemic in traditional orchards of susceptible cultivars. LIFE CYCLE: The pathogens are able to survive on vegetative organs. On the fruit surface, infections remain quiescent until fruit maturity, when typical anthracnose symptoms develop. Under severe epidemics, defoliation and death of branches can also occur. Pathogen species differ in virulence, although this depends on the cultivar. CONTROL: The selection of resistant cultivars depends strongly on pathogen diversity and environmental conditions, posing added difficulties to breeding efforts. Chemical disease control is normally achieved with copper-based fungicides, although this may be insufficient under highly favourable disease conditions and causes concern because of the presence of fungicide residues in the oil. In areas in which the incidence is high, farmers tend to anticipate harvest, with consequences in yield and oil characteristics. CHALLENGES: Olive production systems, harvest and post-harvest processing have experienced profound changes in recent years, namely new training systems using specific cultivars, new harvest and processing techniques and new organoleptic market requests. Changes are also occurring in both the geographical distribution of pathogen populations and the taxonomic framework. In addition, stricter rules concerning pesticide use are likely to have a strong impact on control strategies. A detailed knowledge of pathogen diversity, population dynamics and host-pathogen interactions is basal for the deployment of durable and effective disease control strategies, whether based on resistance breeding, agronomic practices or biological or chemical control.

The Colletotrichum acutatum Species Complex as a Model System to Study Evolution and Host Specialization in Plant Pathogens.

Colletotrichum spp. infect a wide diversity of hosts, causing plant diseases on many economically important crops worldwide. The genus contains approximately 189 species organized into at least 11 major phylogenetic lineages, also known as species complexes. The Colletotrichum acutatum species complex is a diverse yet relatively closely related group of plant pathogenic fungi within this genus. Within the species complex we find a wide diversity of important traits such as host range and host preference, mode of reproduction and differences in the strategy used to infect their hosts. Research on fungal comparative genomics have attempted to find correlations in these traits and patterns of gene family evolution but such studies typically compare fungi from different genera or even different fungal Orders. The C. acutatum species complex contains most of this diversity within a group of relatively closely related species. This Perspective article presents a review of the current knowledge on C. acutatum phylogeny, biology, and pathology. It also demonstrates the suitability of C. acutatum for the study of gene family evolution on a fine scale to uncover evolutionary events in the genome that are associated with the evolution of phenotypic characters important for host interactions.

Validation of standards suitable for genome size estimation of fungi.

Genome size information is fundamental to genome sequencing and may also uncover genomic aspects of evolution. Flow Cytometry, the preferred method for genome size estimation, requires suitable standards. Here we validate Inonotus hispidus, Colletotrichum acutatum and Cenococcum geophilum (41, 68 and 203Mbp), as standards for fungal genome size estimation.

The coffee leaf rust pathogen Hemileia vastatrix: one and a half centuries around the tropics.

TAXONOMY AND HISTORY: Hemileia vastatrix Berk. and Broome (Basidiomycota, Pucciniales) was described in 1869 in eastern Africa and Ceylon as the agent of coffee leaf rust and has spread to all coffee cultivation areas worldwide. Major disease outbreaks in Asia, Africa and America caused and continue to cause severe yield losses, making this the most important disease of Arabica coffee, a cash crop for many tropical and sub-tropical countries. LIFE CYCLE AND DISEASE SYMPTOMS: Hemileia vastatrix is a hemicyclic fungus with the urediniosporic life cycle as its most important (if not only) source of inoculum. Chlorotic spots are the first macroscopic symptoms, preceding the differentiation of suprastomatal, bouquet-shaped, orange-coloured uredinia. The disease can cause yield losses of up to 35% and have a polyetic epidemiological impact on subsequent years. DISEASE CONTROL: Although the use of fungicides is one of the preferred immediate control measures, the use of resistant cultivars is considered to be the most effective and durable disease control strategy. The discovery of 'Híbrido de Timor' provided sources of resistance that have been used in several breeding programmes and that have been proven to be effective and durable, as some have been in use for more than 30 years. GENETIC DIVERSITY AND MOLECULAR PATHOGENICITY: Although exhibiting limited genetic polymorphism, the very large genome of H. vastatrix (c. 797 Mbp) conceals great pathological diversity, with more than 50 physiological races. Gene expression studies have revealed a very precocious activation of signalling pathways and production of putative effectors, suggesting that the plant-fungus dialogue starts as early as at the germ tube stage, and have provided clues for the identification of avr genes.

Comparative Validation of Conventional and RNA-Seq Data-Derived Reference Genes for qPCR Expression Studies of Colletotrichum kahawae.

Colletotrichum kahawae is an emergent fungal pathogen causing severe epidemics of Coffee Berry Disease on Arabica coffee crops in Africa. Currently, the molecular mechanisms underlying the Coffea arabica-C. kahawae interaction are still poorly understood, as well as the differences in pathogen aggressiveness, which makes the development of functional studies for this pathosystem a crucial step. Quantitative real time PCR (qPCR) has been one of the most promising approaches to perform gene expression analyses. However, proper data normalization with suitable reference genes is an absolute requirement. In this study, a set of 8 candidate reference genes were selected based on two different approaches (literature and Illumina RNA-seq datasets) to assess the best normalization factor for qPCR expression analysis of C. kahawae samples. The gene expression stability of candidate reference genes was evaluated for four isolates of C. kahawae bearing different aggressiveness patterns (Ang29, Ang67, Zim12 and Que2), at different stages of fungal development and key time points of the plant-fungus interaction process. Gene expression stability was assessed using the pairwise method incorporated in geNorm and the model-based method used by NormFinder software. For C. arabica-C. kahawae interaction samples, the best normalization factor included the combination of PP1, Act and ck34620 genes, while for C. kahawae samples the combination of PP1, Act and ck20430 revealed to be the most appropriate choice. These results suggest that RNA-seq analyses can provide alternative sources of reference genes in addition to classical reference genes. The analysis of expression profiles of bifunctional catalase-peroxidase (cat2) and trihydroxynaphthalene reductase (thr1) genes further enabled the validation of the selected reference genes. This study provides, for the first time, the tools required to conduct accurate qPCR studies in C. kahawae considering its aggressiveness pattern, developmental stage and host interaction.

Genomic Patterns of Positive Selection at the Origin of Rust Fungi.

Understanding the origin and evolution of pathogenicity and biotrophic life-style of rust fungi has remained a conundrum for decades. Research on the molecular mechanisms responsible for rust fungi evolution has been hampered by their biotrophic life-style until the sequencing of some rust fungi genomes. With the availability of multiple whole genomes and EST data for this group, it is now possible to employ genome-wide surveys and investigate how natural selection shaped their evolution. In this work, we employed a phylogenomics approach to search for positive selection and genes undergoing accelerated evolution at the origin of rust fungi on an assembly of single copy genes conserved across a broad range of basidiomycetes. Up to 985 genes were screened for positive selection on the phylogenetic branch leading to rusts, revealing a pervasive signal of positive selection throughout the data set with the proportion of positively selected genes ranging between 19.6-33.3%. Additionally, 30 genes were found to be under accelerated evolution at the origin of rust fungi, probably due to a mixture of positive selection and relaxation of purifying selection. Functional annotation of the positively selected genes revealed an enrichment in genes involved in the biosynthesis of secondary metabolites and several metabolism and transporter classes.

A method for obtaining RNA from Hemileia vastatrix appressoria produced in planta, suitable for transcriptomic analyses.

Appressoria are the first infection structures developed by rust fungi and require specific topographic signals from the host for their differentiation. The ease in obtaining appressoria in vitro for these biotrophic fungi led to studies concerning gene expression and gene discovery at appressorial level, avoiding the need to distinguish plant and fungal transcripts. However, in some pathosystems, it was observed that gene expression in appressoria seems to be influenced by host-derived signals, suggesting that transcriptomic analyses performed from in planta differentiated appressoria would be potentially more informative than those from in vitro differentiated appressoria. Nevertheless analysing appressorial RNA obtained from in planta samples is often hampered by an excessive dilution of fungal RNA within plant RNA, besides uncertainty regarding the fungal or plant origin of RNA from highly conserved genes. To circumvent these difficulties, we have recovered Hemileia vastatrix appressoria from Arabica coffee leaf surface using a film of nitrocellulose dissolved in butyl and ethyl acetates (nail polish), and extracted fungal RNA from the polish peel. RNA thus obtained is of good quality and usable for cDNA synthesis and transcriptomic (quantitative PCR) studies. This method could provide the means to investigate specific host-induced appressoria-related fungal pathogenicity factors.

Flow cytometry reveals that the rust fungus, Uromyces bidentis (Pucciniales), possesses the largest fungal genome reported--2489 Mbp.

Among the Eukaryotes, Fungi have relatively small genomes (average of 44.2 Mbp across 1850 species). The order Pucciniales (Basidiomycota) has the largest average genome size among fungi (305 Mbp), and includes the two largest fungal genomes reported so far (Puccinia chrysanthemi and Gymnosporangium confusum, with 806.5 and 893.2 Mbp, respectively). In this work, flow cytometry was employed to determine the genome size of the Bidens pilosa rust pathogen, Uromyces bidentis. The results obtained revealed that U. bidentis presents a surprisingly large haploid genome size of 2489 Mbp. This value is almost three times larger than the previous largest fungal genome reported and over 50 times larger than the average fungal genome size. Microscopic examination of U. bidentis nuclei also showed that they are not as different in size from the B. pilosa nuclei when compared with the differences between other rusts and their host plants. This result further reinforces the position of the Pucciniales as the fungal group with the largest genomes, prompting studies addressing the role of repetitive elements and polyploidy in the evolution, pathological specialization and diversity of fungal species.