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The Plant Genome Nov 2020Brown stem rot (BSR) reduces soybean [Glycine max (L.) Merr.] yield by up to 38%. The BSR causal agent is Phialophora gregata f. sp. sojae, a slow-growing, necrotrophic...
Brown stem rot (BSR) reduces soybean [Glycine max (L.) Merr.] yield by up to 38%. The BSR causal agent is Phialophora gregata f. sp. sojae, a slow-growing, necrotrophic fungus whose life cycle includes latent and pathogenic phases, each lasting several weeks. Brown stem rot foliar symptoms are often misdiagnosed as other soybean diseases or nutrient stress, making BSR resistance especially difficult to phenotype. To shed light on the genes and networks contributing to P. gregata resistance, we conducted RNA sequencing (RNA-seq) of a resistant genotype (PI 437970, Rbs3). Leaf, stem, and root tissues were collected 12, 24, and 36 h after stab inoculation with P. gregata, or mock infection, in the plant stem. By using multiple tissues and time points, we could see that leaves, stems, and roots use the same defense pathways. Our analyses suggest that P. gregata induces a biphasic defense response, with pathogen-associated molecular pattern (PAMP) triggered immunity observed in leaves at 12 and 24 h after infection (HAI) and effector triggered immunity detected at 36 h after infection in the stems. Gene networks associated with defense, photosynthesis, nutrient homeostasis, DNA replication, and growth are the hallmarks of resistance to P. gregata. While P. gregata is a slow-growing pathogen, our results demonstrate that pathogen recognition occurs hours after infection. By exploiting the genes and networks described here, we will be able to develop novel diagnostic tools to facilitate breeding and screening for BSR resistance.
Topics: Ascomycota; Disease Resistance; Humans; Plant Diseases; Plant Stems; Glycine max
PubMed: 33217212
DOI: 10.1002/tpg2.20037 -
Plant Disease Oct 2020Maize (Zea mays L.) is one of the most important commodities, and Brazil is the second-largest maize exporter country in the world. In April 2019, the period of the...
Maize (Zea mays L.) is one of the most important commodities, and Brazil is the second-largest maize exporter country in the world. In April 2019, the period of the second crop maize (safrinha), it was observed black decayed lesions on roots and wilting of some maize plants, causing a "sudden death" in a commercial area in the west of Paraná state, Brazil (Figure 1A-C). Symptomatic root and stalk were collected, and tissues surface disinfected with 70% ethanol for 30 s, 1.5% NaOCl for 1 min and rinsed three times in sterile distilled water, slices of necrotic tissues were transferred to potato dextrose agar (PDA) medium and grown for 7 days at 27 ± 1ºC with a photoperiod of 12 h. Pure cultures were obtained through monosporic isolation. The fungal morphology is alike Gaeumannomyces radicicola, which is a synonym of Phialophora radicicola var. radicicola, Harpophora radicicola, P. zeicola, H. zeicola and G. graminis var. maydis (Hernández-Restrepo et al. 2016). Colonies on PDA showed flat, white to light gray at first (Fig. 1D), turning gray to black with age (Fig. 1E). Colony diameter approximately 5.2 cm on PDA in the dark after 7 days at 27ºC. Conidiophores with slightly thickened wall, mostly branched, varying in dimensions, with a range of 57.5-166.5 (avg. 128.7 μm) × 2.9-5.9 (avg. 4.2 μm) n = 25 (Fig. 1H-J). The conidia showed lunate-shaped with rounded ends, produced successively at the apex of phialide, 3.3-9.7 (avg. 6.6 μm) × 1.5-3.6 μm (avg. 2.5 μm), n = 100 (Fig. 1G-J). Morphological characteristics were comparable to the description of this specie (Cain 1952; Gams 2000; McKeen 1952). The total genomic DNA of a representative isolate, LEMIDPRZm 19-01 was extracted and the partial large subunit (28S nrDNA; LSU), internal transcribed spacer nrDNA including the intervening 5.8S nrDNA (ITS), and part of the largest subunit of the RNA polymerase II gene (RPB1) were amplified and sequenced, as following by Hernández-Restrepo et al. (2016) and Klaubauf et al. (2014). The primers to LSU - NL1 (O'Donnel, 1993) and LR5 (Vilgalys; Hester, 1990); ITS - ITS5 and ITS4 (White et al., 1990); and RPB1 - RPB1F and RPB1R (Klaubauf et al., 2014) were used in this study. The gene sequences of LSU (MT123866), ITS (MT114427), and RPB1 (MT123867) were deposited in GenBank and showed 99.67%, 99.75%, and 100% identity with type material G. radicicola CBS 296.53 (KM484962, KM484845, and KM485061). A multi-locus phylogenetic analysis based on Bayesian Inference showed the isolate LEMIDPRZm 19-01 in the G. radicicola clade (Fig. 2). To confirm pathogenicity, ex vivo assays were performed with mycelial PDA discs of 5 mm from a 7-day-old culture using detached roots (adapted method by Degani et al., 2019), on wounded and unwounded stalk and leaves, each treatment consisted of five replications. PDA discs without fungal were used in negative tissue controls. Pathogenicity tests were also conducted in vivo, two experiments performed: i) the stalk tissue was inoculated by sterilized toothpick grown on PDA with fungal mycelium and the leaves inoculated as ex vivo assay, and toothpick without fungal mycelium was used to stalk negative control, whereas PDA discs without fungal were used in the tested leaves; ii) 6 mycelial PDA discs/500 mL were placed on potato dextrose broth (PDB) media and it remained in agitation for 10 days to obtain a mycelial suspension. Subsequently, the mycelial was crushed to soil infestation, and 50 mL from this suspension were dropped in each 2 L maize pot with soil sterilization 10 days after emergence. Maize pots with soil sterilization without mycelium fungal were used as negative controls. Four replications (maize pots), for each treatment, were used in both tests. Experiments were repeated twice. In the ex vivo assay, all inoculated tissues with and without wounds showed necrotic lesions (Fig. 1K-N). In the first in vivo assay, stalk rot symptoms, including wilting of the inoculated plants causing premature plant death, were observed within 6 days (Fig. 1O-Q). In the second in vivo assay, inoculated plants had inferior growth than compared with plant control. Sixty days after inoculation, the plants were removed from the pots and it was observed a roots degeneration with symptoms of necrosis (Fig. 1R-U). No symptoms were detected in the control treatments and the pathogen was re-isolated from symptomatic tissues confirming Koch's postulate for all assays. So far, to our knowledge, the pathogen distribution was reported solely in the west area of Paraná state, but it may become a potential threat to Brazilian maize production. Further monitoring is necessary to better understand the epidemiology of this pathogen to address a strategy for disease control. The pathogen has already been detected in Canada, South Africa, and China. To our knowledge, this is the first report of G. radicicola in Brazil, as well as in South America.
PubMed: 33026305
DOI: 10.1094/PDIS-03-20-0556-PDN -
PloS One 2020Chromoblastomycosis (CBM) is a chronic subcutaneous mycosis caused by traumatic implantation of many species of black fungi. Due to the refractoriness of some cases and...
Chromoblastomycosis (CBM) is a chronic subcutaneous mycosis caused by traumatic implantation of many species of black fungi. Due to the refractoriness of some cases and common recurrence of CBM, a more effective and less time-consuming treatment is mandatory. The aim of this study was to identify compounds with in vitro antifungal activity in the Pathogen Box® compound collection against different CBM agents. Synergism of these compounds with drugs currently used to treat CBM was also assessed. An initial screening of the drugs present in this collection at 1 μM was performed with a Fonsecaea pedrosoi clinical strain according to the EUCAST protocol. The compounds with activity against this fungus were also tested against other seven etiologic agents of CBM (Cladophialophora carrionii, Phialophora verrucosa, Exophiala jeanselmei, Exophiala dermatitidis, Fonsecaea monophora, Fonsecaea nubica, and Rhinocladiella similis) at concentrations ranging from 0.039 to 10 μM. The analysis of potential synergism of these compounds with itraconazole and terbinafine was performed by the checkerboard method. Eight compounds inhibited more than 60% of the F. pedrosoi growth: difenoconazole, bitertanol, iodoquinol, azoxystrobin, MMV688179, MMV021013, trifloxystrobin, and auranofin. Iodoquinol produced the lowest MIC values (1.25-2.5 μM) and MMV688179 showed MICs that were higher than all compounds tested (5 - >10 μM). When auranofin and itraconazole were tested in combination, a synergistic interaction (FICI = 0.37) was observed against the C. carrionii isolate. Toxicity analysis revealed that MMV021013 showed high selectivity indices (SI ≥ 10) against the fungi tested. In summary, auranofin, iodoquinol, and MMV021013 were identified as promising compounds to be tested in CBM models of infection.
Topics: Acetates; Antifungal Agents; Ascomycota; Auranofin; Biphenyl Compounds; Chromoblastomycosis; Dioxolanes; Drug Synergism; Exophiala; Fungi; Humans; Imines; Iodoquinol; Pyrimidines; Strobilurins; Triazoles
PubMed: 32401759
DOI: 10.1371/journal.pone.0229630 -
Microorganisms Apr 2020Dark septate endophytes (DSEs) are often trace element (TE)-tolerant fungi and are abundant in TE-polluted environments. The production of melanin, a black polymer found...
Dark septate endophytes (DSEs) are often trace element (TE)-tolerant fungi and are abundant in TE-polluted environments. The production of melanin, a black polymer found in cell walls, was hypothesized by several authors to play a role in the TE tolerance of DSEs. To test this hypothesis, we established a series of experiments using albino strains and melanin inhibitors and examined the responses to Cd and Zn. Six DSEs belonging to genera sp., sp. and , were evaluated. The strains mainly produced 1,8-dihydroxynaphthalene (DHN) melanin whereas 3,4-dihydroxyphenylalanin melanin was also synthetized. Cd and Zn decreased melanin synthesis in most of the strains. A reduction in melanin concentration in hyphae through the use of tricyclazole, an inhibitor of DHN-melanin synthesis, did not reduce the tolerance of the strains to Cd and Zn. Similarly, albino mutants of sp. were not more sensitive to Cd and Zn than the WT strain. Moreover, tricyclazole-treated colonies accumulated less Cd but more Zn compared to untreated colonies. The Cd and Zn contents of albino strains were variable and similar to that of the WT. The results suggest that melanin production is not an important functional trait that contributes to Cd and Zn tolerance, but might contribute to Cd accumulation.
PubMed: 32276491
DOI: 10.3390/microorganisms8040537 -
Acta Cytologica 2020Phaeohyphomycosis caused by phaeoid fungi is a type of mycosis emerging worldwide which causes a wide variety of clinical manifestations.
OBJECTIVE
Phaeohyphomycosis caused by phaeoid fungi is a type of mycosis emerging worldwide which causes a wide variety of clinical manifestations.
STUDY DESIGN
A retrospective analysis of 11 cases diagnosed with fungal inflammation on cytology over a period of 6 years (2013-2018) was done along with culture/histopathologic confirmation.
RESULTS
Of the total of 11 cases, 9 cases presented with subcutaneous swellings and 1 case each with brain and lung lesions. The age range was 30-83 years (mean: 53.6); 8 patients were male and 3 were female. Cytologic smears showed fungal profiles with septate tortuous hyphae, as well as swollen and narrow, yeast-like swellings with an irregular breadth of the hyphae in all cases. The fungal profiles were visualized on a Masson-Fontana stain. The background showed inflammatory cells, giant cells, and necrosis in variable proportions. Five cases were diagnosed as phaeohyphomycosis on cytology, whereas 3 cases were misdiagnosed as aspergillus and 2 as candida. In 1 case, typing of the fungus was not done. Histopathology was available in 5 cases, and in all these a diagnosis of phaeohyphomycosis was reached. Ten of the 11 cases had confirmation on fungal culture.
CONCLUSIONS
Phaeoid fungi are rarely seen in routine cytologic practice. Careful evaluation of cytologic smears and an awareness of the characteristic morphologic features of phaeohyphomycosis are helpful in arriving at a correct diagnosis. Fine needle aspiration cytology provides a rapid diagnosis, enabling prompt therapy.
Topics: Adult; Aged; Aged, 80 and over; Cytodiagnosis; Female; Follow-Up Studies; Humans; Male; Middle Aged; Phaeohyphomycosis; Phialophora; Retrospective Studies
PubMed: 32203955
DOI: 10.1159/000506432 -
MycoKeys 2020During a survey on fungi associated with wood necroses of trees in Germany, strains belonging to the Leotiomycetes and Eurotiomycetes were detected by preliminary...
During a survey on fungi associated with wood necroses of trees in Germany, strains belonging to the Leotiomycetes and Eurotiomycetes were detected by preliminary analyses of ITS sequences. Multi-locus phylogenetic analyses (LSU, ITS, , , depending on genus) of 31 of the 45 strains from and reference strains revealed several new taxa, including , a new genus in the Helotiales (Leotiomycetes) with a collophorina-like asexual morph. Seven species (Helotiales, Leotiomycetes) were treated. The 29 strains from belonged to five species, of which and were dominating; , and were revealed as new species. The genus was reported from for the first time. was combined in and differentiated from , which was resurrected. Asexual morphs of two species (Helotiales, Leotiomycetes) were described, including one new species, . Two species (Phaeomoniellales, Eurotiomycetes) were detected, including the new species . and are reported as host plants of .
PubMed: 32189979
DOI: 10.3897/mycokeys.63.46836 -
Journal of Enzyme Inhibition and... Dec 2020causes several fungal human diseases, mainly chromoblastomycosis, which is extremely difficult to treat. Several studies have shown that human immunodeficiency virus...
causes several fungal human diseases, mainly chromoblastomycosis, which is extremely difficult to treat. Several studies have shown that human immunodeficiency virus peptidase inhibitors (HIV-PIs) are attractive candidates for antifungal therapies. This work focused on studying the action of HIV-PIs on peptidase activity secreted by and their effects on fungal proliferation and macrophage interaction. We detected a peptidase activity from able to cleave albumin, sensitive to pepstatin A and HIV-PIs, especially lopinavir, ritonavir and amprenavir, showing for the first time that this fungus secretes aspartic-type peptidase. Furthermore, lopinavir, ritonavir and nelfinavir reduced the fungal growth, causing remarkable ultrastructural alterations. Lopinavir and ritonavir also affected the conidia-macrophage adhesion and macrophage killing. Interestingly, had its growth inhibited by ritonavir combined with either itraconazole or ketoconazole. Collectively, our results support the antifungal action of HIV-PIs and their relevance as a possible alternative therapy for fungal infections.
Topics: Antifungal Agents; Aspartic Acid Proteases; Carbamates; Dose-Response Relationship, Drug; Furans; HIV Protease Inhibitors; Humans; Lopinavir; Macrophages; Microbial Sensitivity Tests; Molecular Structure; Phialophora; Ritonavir; Structure-Activity Relationship; Sulfonamides
PubMed: 32037904
DOI: 10.1080/14756366.2020.1724994 -
Journal of Clinical Immunology Apr 2020Autosomal recessive (AR) CARD9 deficiency is an inherited immune disorder which results in impaired innate immunity against various fungi. Superficial and invasive... (Review)
Review
PURPOSE
Autosomal recessive (AR) CARD9 deficiency is an inherited immune disorder which results in impaired innate immunity against various fungi. Superficial and invasive fungal infections, mainly caused by Candida or Trichophyton species, are the hallmark of CARD9 deficiency. Together with the increasing number of CARD9-deficient patients reported, different pathogenic fungal species have been described such as Phialophora, Exophiala, Corynespora, Aureobasidium, and Ochroconis. Saprochaete capitata is an opportunistic infectious agent in immunocompromised patients and is a common cause of invasive fungal disease in patients with hematological malignancies. In this study, we investigated the causative genetic defect in a patient with S. capitata fungal infection which disseminated to lymph nodes and common bile duct.
METHODS
The identification of the isolated yeast strain was made by direct microscopic examination and confirmed by internal transcribed spacer (ITS) sequencing. We applied whole exome sequencing to search for the disease-causing mutation. Sanger sequencing was used to validate the mutation in the patient and his parents.
RESULTS
S. capitata was isolated from the biopsy specimen as the causative microorganism responsible for the invasive fungal disease in the patient. Whole exome sequencing revealed a homozygous c.883C > T, (p.Q295*) mutation in CARD9, confirmed by Sanger sequencing.
CONCLUSIONS
This is the first report of invasive Saprochaete infection associated with autosomal recessive (AR) CARD9 deficiency in the literature and thereby further extends the spectrum of fungal diseases seen in these patients.
Topics: Adolescent; CARD Signaling Adaptor Proteins; Candidiasis, Chronic Mucocutaneous; Cholestasis; Chromosome Disorders; Genes, Recessive; Humans; Immunocompromised Host; Invasive Fungal Infections; Iraq; Male; Saccharomycetales; Sequence Deletion; Exome Sequencing
PubMed: 32020378
DOI: 10.1007/s10875-020-00759-w -
Journal of Clinical Microbiology Jan 2020
PubMed: 31992652
DOI: 10.1128/JCM.01657-18 -
Journal of Agricultural and Food... Feb 2020The mycotoxin zearalenone (ZEN) is a secondary metabolite produced mainly by Fusarium species. ZEN poses health hazards both for humans and animals, as a major...
The mycotoxin zearalenone (ZEN) is a secondary metabolite produced mainly by Fusarium species. ZEN poses health hazards both for humans and animals, as a major contaminant in the food and feed industries. Currently, there is no effective technique for degrading ZEN during industrial processes. In this study, we isolated and biochemically characterized a novel lactone hydrolase, ZHD607, isolated from , cloned, and exogenously expressed in . ZHD607 was characterized as a mesophilic lactone hydrolase having a neutral pH and showing optimal activity at 35 °C and pH 8.0. Two mutants, ZHDM1 and I160Y, generated from ZHD607 based on structure and sequence alignment analyses, exhibited 2.9- and 3.4-fold higher activity towards ZEN than did ZHD607. Molecular dynamics simulation revealed diverse mechanisms driving this improved catalytic activity. These findings enrich our knowledge about ZHD enzyme family and represent an important step toward industrialization of ZEN-detoxifying lactone hydrolases.
Topics: Biocatalysis; DNA Mutational Analysis; Enzyme Stability; Fungal Proteins; Hydrogen-Ion Concentration; Hydrolases; Lactones; Molecular Dynamics Simulation; Mutation; Phialophora; Zearalenone
PubMed: 31760747
DOI: 10.1021/acs.jafc.9b05853