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Genome assembly and annotation using short-paired reads is challenging for eukaryotic organisms due to their large size, variable ploidy and large number of repetitive elements. However, the use of single-molecule long reads improves assembly quality (completeness and contiguity), but haplotype duplications still pose assembly challenges. To address the effect of read length on genome assembly quality, gene prediction and annotation, we compared genome assemblers and sequencing technologies with four strains of the ectomycorrhizal fungus Laccaria trichodermophora. By analysing the predicted repertoire of carbohydrate enzymes, we investigated the effects of assembly quality on functional inferences. Libraries were generated using three different sequencing platforms (Illumina Next-Seq, Mi-Seq and PacBio Sequel), and genomes were assembled using single and hybrid assemblies/libraries. Long reads or hybrid assemby resolved the collapsing of repeated regions, but the nuclear heterozygous versions remained unresolved. In dikaryotic fungi, each cell includes two nuclei and each nucleus has differences not only in allelic gene version but also in gene composition and synteny. These heterokaryotic cells produce fragmentation and size overestimation of the genome assembly of each nucleus. Hybrid assembly revealed a wider functional diversity of genomes. Here, several predicted oxidizing activities on glycosyl residues of oligosaccharides and several chitooligosaccharide acetylase activities would have passed unnoticed in short-read assemblies. Also, the size and fragmentation of the genome assembly, in combination with heterozygosity analysis, allowed us to distinguish homokaryotic and heterokaryotic strains isolated from L. trichodermophora fruit bodies.

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Two Cerrado rust fungi, Phakopsora rossmaniae and Aplopsora hennenii, described in 1993 and 1995 and originally assigned to families Phakopsoraceae and Ochropsoraceae, respectively, were subjected to molecular phylogenetic analyses using fragments of the nuc 28S and 18S rDNA and mitochondrial cytochrome c oxidase subunit 3 (CO3) gene. Although both taxa were morphologically well placed in their original genera, they were shown to belong in a strongly supported new lineage within the Raveneliineae distant from the Phakopsoraceae and Ochropsoraceae. Therefore, we properly treated this lineage as the new genus Cerradopsora now harboring C. rossmaniae (type species) and C. hennenii. However, this novel phakopsoroid genus remains in uncertain familial position without support to be included in any of the families that share space within the Raveneliineae.

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Background: The leaves of Serjania erecta Radlk (Sapindaceae) are renowned in ethnobotany for their medicinal properties and are significant as a medicinal resource for traditional Brazilian communities. As necrotic spots are common on these leaves, indicating interaction with phytopathogenic fungi, it was hypothesized that biotrophic fungal species colonize the leaf tissues of S. erecta. Methods: To test this hypothesis, we employed standard techniques in plant anatomy, which enabled us to investigate the interaction of fungal structures with plant tissues and describe the morphoanatomical and histochemical characteristics of the epidermis and limbus of S. erecta. Results: The anatomical analysis showed the existence of leaf teeth on the leaf tips. Additionally, hyphae, conidiospores, and spores of Bipolaris/Curvularia species were detected on the adaxial epidermis. Moreover, melanized microsclerotia were found in glandular areas of the leaf teeth and the phloem, providing evidence of biotrophic behavior. The hypothesis that biotrophic phytopathogenic fungi interact with S. erecta leaf tissues was confirmed, despite the presence of many bioactive compounds (such as flavonoids, alkaloids, and essential oils), as evidenced by histochemical analyses. The presence of tector, glandular, and scabiform trichomes on the leaf teeth and epidermis was also revealed. This study presents, for the first time, the synthesis of essential oils and alkaloids in the leaves of S. erecta. Additionally, it investigates previously unexplained aspects of the anatomy and histochemistry of the species, as well as its interaction with resident microorganisms. Therefore, it is recommended that future research focus on extracting and characterizing the oils and alkaloids of S. erecta, as well as exploring other aspects related to its microbiome and its relationship.

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Plants are at risk of attack by various pathogenic organisms. During pathogenesis, microorganisms produce molecules with conserved structures that are recognized by plants that then initiate a defense response. Plants also experience iron deficiency. To address problems caused by iron deficiency, plants use two strategies focused on iron absorption from the rhizosphere. Strategy I is based on rhizosphere acidification and iron reduction, whereas Strategy II is based on iron chelation. Pathogenic defense and iron uptake are not isolated phenomena: the antimicrobial phenols are produced by the plant during defense, chelate and solubilize iron; therefore, the production and secretion of these molecules also increase in response to iron deficiency. In contrast, phytohormone jasmonic acid and salicylic acid that induce pathogen-resistant genes also modulate the expression of genes related to iron uptake. Iron deficiency also induces the expression of defense-related genes. Therefore, in the present review, we address the cross-talk that exists between the defense mechanisms of both Systemic Resistance and Systemic Acquired Resistance pathways and the response to iron deficiency in plants, with particular emphasis on the regulation genetic expression.

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We assembled a dual-layered biological network to study the roles of resistance gene analogs (RGAs) in the resistance of sugarcane to infection by the biotrophic fungus causing smut disease. Based on sugarcane-Arabidopsis orthology, the modeling used metabolic and protein-protein interaction (PPI) data from Arabidopsis thaliana (from Kyoto Encyclopedia of Genes and Genomes (KEGG) and BioGRID databases) and plant resistance curated knowledge for Viridiplantae obtained through text mining of the UniProt/SwissProt database. With the network, we integrated functional annotations and transcriptome data from two sugarcane genotypes that differ significantly in resistance to smut and applied a series of analyses to compare the transcriptomes and understand both signal perception and transduction in plant resistance. We show that the smut-resistant sugarcane has a larger arsenal of RGAs encompassing transcriptionally modulated subnetworks with other resistance elements, reaching hub proteins of primary metabolism. This approach may benefit molecular breeders in search of markers associated with quantitative resistance to diseases in non-model systems.

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Although asterinaceous fungi have been studied for many years, all previous attempts to isolate, cultivate, and propagate these fungi in vitro have failed. This paper provides the first reports of in vitro isolation of representative strains of species belonging to five fungi from different genera belonging to Asterinales. To confirm if the sequences of DNA obtained from the mycelia are the same obtained in the direct extraction, a phylogenetic analysis of nuc LSU rDNA was performed. This paper reports for the first time the success of in vitro culturing of asterinaceous fungi using the ascospores ejection technique, opening perspectives of studies of genetics, physiology, among other aspects of the biology for this very understudied group of fungi.

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The fungal pathogen Sporisorium scitamineum causes sugarcane smut disease. We have previously shown that resistant sugarcane plants induce ROS, coinciding with a delay in fungal colonization. Here, we investigated whether the fungus modifies the enzymatic antioxidant system in vitro and when colonizing sugarcane tissues in response to ROS. In vitro, the exposure to ROS did not affect cell integrity, and a combination of superoxide dismutases (SOD) and catalases (CAT) were active. In vitro, the fungus did not alter the expression of the transcriptional regulator Yap1 and the effector Pep1. The fungus activated distinct enzymes when colonizing plant tissues. Instead of CAT, S. scitamineum induced glutathione peroxidase (Gpx) expression only when colonizing smut-resistant plants. Yap1 had an earlier expression in both smut-susceptible and -resistant plants, with no apparent correlation with the expression of antioxidant genes sod, cat, gpx, or external redox imbalance. The expression of the effector pep1 was induced only in smut-resistant plants, potentially in response to ROS. These results collectively suggest that S. scitamineum copes with oxidative stress by inducing different mechanisms depending on the conditions (in vitro/in planta) and intensity of ROS. Moreover, the effector Pep1 is responsive to the stress imposed only by the sugarcane resistant genotype.

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Postbloom fruit drop (PFD), caused mainly by Colletotrichum abscissum, is one of the most severe citrus diseases and can causes up to 80% fruit loss in favorable climatic conditions. According to the literature, other Colletotrichum species colonize hosts using distinct strategies: intracellular hemibiotrophic or subcuticular intramural necrotrophic colonization. However, so far, for C. abscissum only the necrotrophic stage has been described and some aspects remain unclear in PFD disease cycle. To better understand the disease cycle, microscopy studies could be applied. However, even using eGFP strains (expressing green fluorescent protein), the results are unclear due to the autofluorescence of citrus leaves. To eliminate this problem and to study the interaction between C. abscissum-citrus we used a destaining and staining methodologies, and we observed that in leaves, even applying injury before inoculation, C. abscissum does not colonize adjacent tissues. Apparently, in the leaves the fungus only uses the nutrients exposed in the artificial lesions for growth, and then produces large amount of spores. However, in flowers, C. abscissum penetrated and colonized the tissues of the petals 12 h after inoculation. In the early stages of infection, we observed the development of primary biotrophic hyphae, suggesting this species as a hemibiotrophic fungus, with a short biotrophic phase during flower colonization followed by dominant necrotrophic colonization. In conclusion, the use of an eGFP strain of C. abscissum and a different methodology of destaining and staining allowed a better understanding of the morphology and mechanisms used by this citrus pathogen to colonize the host.

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KEY MESSAGE: We provide a transcriptional profile of coffee rust interaction and identified putative up regulated resistant genes Coffee rust disease, caused by the fungus Hemileia vastatrix, is one of the major diseases in coffee throughout the world. The use of resistant cultivars is considered to be the most effective control strategy for this disease. To identify candidate genes related to different mechanism defense in coffee, we present a time-course comparative gene expression profile of Caturra (susceptible) and Híbrido de Timor (HdT, resistant) in response to H. vastatrix race XXXIII infection. The main objectives were to obtain a global overview of transcriptome in both interaction, compatible and incompatible, and, specially, analyze up-regulated HdT specific genes with inducible resistant and defense signaling pathways. Using both Coffea canephora as a reference genome and de novo assembly, we obtained 43,159 transcripts. At early infection events (12 and 24 h after infection), HdT responded to the attack of H. vastatrix with a larger number of up-regulated genes than Caturra, which was related to prehaustorial resistance. The genes found in HdT at early hours were involved in receptor-like kinases, response ion fluxes, production of reactive oxygen species, protein phosphorylation, ethylene biosynthesis and callose deposition. We selected 13 up-regulated HdT-exclusive genes to validate by real-time qPCR, which most of them confirmed their higher expression in HdT than in Caturra at early stage of infection. These genes have the potential to assist the development of new coffee rust control strategies. Collectively, our results provide understanding of expression profiles in coffee-H. vastatrix interaction over a time course in susceptible and resistant coffee plants.

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Leaf peach curl is a devastating disease affecting leaves, flowers and fruits, caused by the dimorphic fungus Taphrina deformans. To gain insight into the mechanisms of fungus pathogenesis and plant responses, leaves of a resistant and two susceptible Prunus persica genotypes were inoculated with blastospores (yeast), and the infection was monitored during 120 h post inoculation (h.p.i.). Fungal dimorphism to the filamentous form and induction of reactive oxygen species (ROS), callose synthesis, cell death and defence compound production were observed independently of the genotype. Fungal load significantly decreased after 120 h.p.i. in the resistant genotype, while the pathogen tended to grow in the susceptible genotypes. Metabolic profiling revealed a biphasic re-programming of plant tissue in susceptible genotypes, with an initial stage co-incident with the yeast form of the fungus and a second when the hypha is developed. Transcriptional analysis of PRs and plant hormone-related genes indicated that pathogenesis-related (PR) proteins are involved in P. persica defence responses against T. deformans and that salicylic acid is induced in the resistant genotype. Conducted experiments allowed the elucidation of common and differential responses in susceptible versus resistant genotypes and thus allow us to construct a picture of early events during T. deformans infection.

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Four new Asterolibertia species and a new variety of Cirsosia splendida, all found on native Cerrado plants, belonging to three host families are described, illustrated and named as: A. bahiensis sp. nov. on Erythroxylum sp. (Erythroxylaceae); A. barrinhensis sp. nov. on Diospyros burchellii (Ebenaceae); A. campograndensis sp. nov. on Hirtella glandulosa (Chrysobalanaceae); A. parinaricola sp. nov. on Parinari obtusifolia (Chrysobalanaceae); and Cirsosia splendida var. laevigata var. nov., showing both sexual and asexual morphs, on H. glandulosa and H. gracilipes (Chrysobalanaceae). Finally, A. licaniae is reported on a new host, H. gracilipes. Keys to all the known species of Asterolibertia and Cirsosia are included.

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Tropical rust caused by the biotrophic fungus Phakopsora gossypii is an emerging disease in cotton that has caused significant yield losses of crop/cotton cultivated in Brazil. Considering the current importance of tropical rust and the need to obtain additional basic information about its causal agent to better control this disease, the present study aimed to determine the infection process of P. gossypii in cotton leaves using scanning electron microscopy (SEM). Thirty-day-old plants were inoculated with a suspension of P. gossypii uredospores, and leaf fragments were collected 42 h after inoculation (hai) as well as 20, 25 and 35 days after inoculation (dai) for SEM observations. By 42 hai, the uredospores of P. gossypii had germinated and produced a germ tube and an appressorium that may directly penetrate the leaf cuticle. At 20 dai, closed uredia containing uredospores were observed on the abaxial leaf surface. At 25 dai, the uredia started to open and became fully open by 35 dai and contained many uredospores. By 25 dai, fungal hyphae were growing abundantly in the mesophyll next to the uredia that formed in the leaf fragments with total or partial removal of the epidermis. The results of the present study provide novel information regarding the infection process of P. gossypii in cotton leaves, which might be useful for the development of new and more effective strategies for tropical rust control.(AU)

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Tropical rust caused by the biotrophic fungus Phakopsora gossypii is an emerging disease in cotton that has caused significant yield losses of crop/cotton cultivated in Brazil. Considering the current importance of tropical rust and the need to obtain additional basic information about its causal agent to better control this disease, the present study aimed to determine the infection process of P. gossypii in cotton leaves using scanning electron microscopy (SEM). Thirty-day-old plants were inoculated with a suspension of P. gossypii uredospores, and leaf fragments were collected 42 h after inoculation (hai) as well as 20, 25 and 35 days after inoculation (dai) for SEM observations. By 42 hai, the uredospores of P. gossypii had germinated and produced a germ tube and an appressorium that may directly penetrate the leaf cuticle. At 20 dai, closed uredia containing uredospores were observed on the abaxial leaf surface. At 25 dai, the uredia started to open and became fully open by 35 dai and contained many uredospores. By 25 dai, fungal hyphae were growing abundantly in the mesophyll next to the uredia that formed in the leaf fragments with total or partial removal of the epidermis. The results of the present study provide novel information regarding the infection process of P. gossypii in cotton leaves, which might be useful for the development of new and more effective strategies for tropical rust control.

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Diaporthe(telomorfo) - Phomopsis(anamorfo) (DP) constituye un grupo fúngico de amplia diversidad genética con más de 900 especies distribuidas en un amplio rango de hospedantes que incluye especies cultivadas y no cultivadas, forestales, frutales y malezas. Los aislamientos de DP son hemi-biótrofos y disponen de diferentes fuentes de inóculo primario, como el rastrojo y las semillas, para reiniciar sus ciclos de parasitismo-saprofitismo. Ellos colonizan los tejidos del hospedante desde los estadios tempranos del desarrollo y establecen relaciones nutricionales de endofitia y necrotrofia fúngica. La plasticidad del género Phomopsisha favorecido su expansión a diferentes agro-ecosistemas y hospedantes constituyendo un importante riesgo epidemiológico. El objetivo fue validar la identidad y evaluar las relaciones biológicas de 12 aislamientos de P. longicollay D. phaseolorumvar. sojaeobtenidos en distintos agro-ambientes templados y subtropicales de Argentina, para analizar la variabilidad y estrategias de conservación de la bio-diversidad fúngica. Las cualidades macro-morfo-lógicas(textura y color de colonias, forma y distribución de estromas, desarrollo, forma y distribución de cuerpos fructíferos), y los caracteres micro-morfológicos(tamaño y forma de conidios, ascos y ascosporas) permitieron identificar a tres nuevos aislamientos de P. longicollaincluidos en el complejo D/P. El análisis molecular complementario corrigió las limitaciones derivadas de la caracterización basada sólo en marcadores morfológicos y logró reubicar al aislamiento AFP.8413 de identidad dudosa, en el nodo correspondiente a P. longicolla.De esta manera, la caracterización molecular definió la identidad de los aislamientos y los ubicó en los 4 taxones del complejo DP: diez aislamientos fueron incluidos en Plo(AFP.Gpo 4.4, AFP.Gpo 3.5, AFP.Gpo 4.3, AFP.Gpo 3.4, AFP.CaA, AFP. CaB, AFP.B5L16, AFP.B4L17, AFP.227B2, AFP.8413), un aislamiento incluido en Dps(AFP.Qcol7), un aislamiento en Dpc(AFP.Dpc16) y dos aislamientos incluidos en Dpm(AFP.Dpm109 y AFP.Dpm112). La adecuada identificación de P. longicollay el avance en el conocimiento de las relaciones biológicas (hibridaciones homo o heterotálicas) entre variedades de D. phaseolorum (P. phaseoli)y especies de Diaporthe- Phomopsispermiten comprender la plasticidad para colonizar un amplio rango de hospedantes, los mecanismos de variabilidad genética y la preservación de la diversidad fúngica.

Diaporthe(teleomorpho)-Phomopsis (anamorph) (DP) is a fungal group of great genetic diversity with over 900 species associated to a wide host range that includes cultivated and uncultivated species, forest, fruit trees and weeds. DP isolates are hemi-biotrophs and have different sources of primary inoculum as stubble and seeds to restart cycles of parasitism -saprophytism. They colonize host tissues from early plant stages and establish different nutritional relationships, acting as endophytic and necrotrophic fungi. The plasticity of the Phomopsisgenus has favored its expansion to different agro-ecosystems and various hosts constituting an epidemiological risk. The objective was to validate the identity and evaluate the biological relationships among 12 isolates of P. longicollaand D. phaseolorumvar. sojae(anamorph P. phaseolivar. sojae)obtained in different tempered and subtropical agro-environments of Argentina, in order to analyze the variability and strategies for preserving fungal biodiversity. Macro-morphological attributes (such as texture and color of colonies, stroma shape and distribution, pycnidia and perythecia shape and distribution) and micro-morphological characteristics (such as size and shape of conidia, asci and ascospores) allowed identifying three new isolates as P. longicolla.A complementary molecular analysis was also made to overcome the limitations derived from the morphological analysis, thus the AFP.8413 isolate was finally identified as P. longicolla.The molecular characterization was useful to identify the evaluated isolates and to group them in four taxa of the Diaporthe-Phomopsiscomplex: ten isolates were included in P. longicolla,one isolate was included in D. phaseolorumvar. sojae(anamorph P. phaseolivar. sojae),one isolate was identified as D. phaseolorumvar. caulivoraand two isolates were included in D. phaseolorumvar. meridi-onalis.The use of phenotipic and molecular tools have contributed to an accurate identification of P. longicolla,and comprehension about the biological relationships (homo or heterothallic hibridizations) among D. phaseolo-rumvarieties (P. phaseoli)and species of Diaporthe-Phomopsis.This allowed also a better understanding of the mechanisms of fungic plasticity, to colonize and expand their host range and genetic variability, promoting thus their biodiversity conservation.

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Fungi belonging to the genus Trichoderma, commonly found in soil or colonizing plant roots, exert beneficial effects on plants, including the promotion of growth and the induction of resistance to disease. T. virens and T. atroviride secrete the proteins Sm1 and Epl1, respectively, which elicit local and systemic disease resistance in plants. In this work, we show that these fungi promote growth in tomato (Solanum lycopersicum) plants. T. virens was more effective than T. atroviride in promoting biomass gain, and both fungi were capable of inducing systemic protection in tomato against Alternaria solani, Botrytis cinerea, and Pseudomonas syringae pv. tomato (Pst DC3000). Deletion (KO) of epl1 in T. atroviride resulted in diminished systemic protection against A. solani and B. cinerea, whereas the T. virens sm1 KO strain was less effective in protecting tomato against Pst DC3000 and B. cinerea. Importantly, overexpression (OE) of epl1 and sm1 led to an increase in disease resistance against all tested pathogens. Although the Trichoderma WT strains induced both systemic acquired resistance (SAR)- and induced systemic resistance (ISR)-related genes in tomato, inoculation of plants with OE and KO strains revealed that Epl1 and Sm1 play a minor role in the induction of these genes. However, we found that Epl1 and Sm1 induce the expression of a peroxidase and an α-dioxygenase encoding genes, respectively, which could be important for tomato protection by Trichoderma spp. Altogether, these observations indicate that colonization by beneficial and/or infection by pathogenic microorganisms dictates many of the outcomes in plants, which are more complex than previously thought.

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Se describe un caso de micoparasitismo biotrófico de ocurrencia natural en el suelo, entre las hifas de una cepa de Fusarium oxysporum complex y Cunninghamella sp. Las hifas de F. oxysporum se desarrollaron sobre las células vivas del hospedador, mostrando 2 tipos de efectos parasíticos: uno de enrollamiento y otro de contacto con penetración de las hifas, sin la aparente eliminación del hospedador. Esta situación poco común en la literatura, demuestra las capacidades adaptativas de esta especie al micoparasitismo en grupos filogenéticamente distantes.

This paper describes a case of mycoparasitism naturally occurring, where Fusarium oxysporum parasitizes hyphae of Cunninghamella sp, to show mycoparasitism between the two fungi. This is a case of biotrophic mycoparasitism by contact. The hyphae of F. oxysporum developed closely along the living cells of the host showing mycoparasitic effect, some for a loop, and other contact with penetration of the hyphae. This situation is rare in the literature, demonstrates the adaptive capacities of this species to mycoparasitism in phylogenetically distant groups.

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