Diversity of fungi obtained from bats captured in urban forest fragments in Sinop, Mato Grosso, Brazil

Bats are important for the homeostasis of ecosystems and serve as hosts of various microorganisms including bacteria, viruses, and fungi with pathogenic potential. This study aimed to isolate fungi from biological samples obtained from bats captured in the city of Sinop (state of Mato Grosso, Brazil), where large areas of deforestation exist due to urbanization and agriculture. On the basis of the flow of people and domestic animals, 48 bats were captured in eleven urban forest fragments. The samples were processed and submitted to microbiological cultures, to isolate and to identify the fungal genera. Thirty-four (70.83%) of the captured bats were positive for fungi; 18 (37.5%) and 16 (33.33%) of these bats were female and male, respectively. Penicillium sp., Scopulariopsis sp., Fusarium sp., Aspergillus sp., Alternaria sp., Cryptococcus sp., Trichosporon sp., and Candida sp., which may cause opportunistic infections, were isolated. The bat species with the highest number of fungal isolates was Molossus molossus: 21 isolates (43.8%). According to our results, bats captured in urban forest fragments in Sinop harbor pathogenic fungi, increasing the risk of opportunistic fungal infections in humans and domestic animals.

Evaluation of metal nano-particles as growth promoters and fungi inhibitors for cereal crops

Background Nano-particles of metals can be routinely synthesized. The cereal seeds treatment with the particles can improve early growth and crop production. Moreover, the treatment is robust and economical. Methods Metal (Fe0, Cu0, Co0), zinc oxide (ZnO) and chitosan-stabilized silver nano-particles were synthesized and applied to cereal seeds. The germination rate, early plant development and inhibition effects on pathogenic fungi were quantified. Results It was found that all nano-particles had a positive effect on the development of healthy cereal seedlings. In particular, the length of the above-ground part of the seedlings was increased by 8–22%. The highest inhibition effect was observed on Helminthosporium teres with the application of Co0 and chitosan-Ag. Pre-sowing treatment with metal nano-particles reduced the number of infected grains by two times for wheat and 3.6 times for barley. The application also increases the chlorophylls and carotenoids in both uninfected and infected seedlings. Conclusions The results demonstrated a robust application of nano-particles in improving cereal production. Graphical Abstract

Discovery of microRNA-like Small RNAs in Pathogenic Plant Fungus Verticillium nonalfalfae Using High-Throughput Sequencing and qPCR and RLM-RACE Validation

Verticillium nonalfalfae (V. nonalfalfae) is one of the most problematic hop (Humulus lupulus L.) pathogens, as the highly virulent fungal pathotypes cause severe annual yield losses due to infections of entire hop fields. In recent years, the RNA interference (RNAi) mechanism has become one of the main areas of focus in plant—fungal pathogen interaction studies and has been implicated as one of the major contributors to fungal pathogenicity. MicroRNA-like RNAs (milRNAs) have been identified in several important plant pathogenic fungi; however, to date, no milRNA has been reported in the V. nonalfalfae species. In the present study, using a high-throughput sequencing approach and extensive bioinformatics analysis, a total of 156 milRNA precursors were identified in the annotated V. nonalfalfae genome, and 27 of these milRNA precursors were selected as true milRNA candidates, with appropriate microRNA hairpin secondary structures. The stem-loop RT-qPCR assay was used for milRNA validation; a total of nine V. nonalfalfae milRNAs were detected, and their expression was confirmed. The milRNA expression patterns, determined by the absolute quantification approach, imply that milRNAs play an important role in the pathogenicity of highly virulent V. nonalfalfae pathotypes. Computational analysis predicted milRNA targets in the V. nonalfalfae genome and in the host hop transcriptome, and the activity of milRNA-mediated RNAi target cleavage was subsequently confirmed for two selected endogenous fungal target gene models using the 5′ RLM-RACE approach.

Morphological and Molecular Diversity of Ginger (Zingiber officinale Roscoe) Pathogenic Fungi in Chilga District, North Gondar, Ethiopia

Ginger diseases caused by fungal pathogens have become one of the most serious problems causing reduced production around the world. It has also caused a major problem among farmers in different parts of Ethiopia resulting in a huge decline in rhizome yield. However, the exact causative agents of this disease have not been identified in the state. Although there are few studies related to pathogenic fungus identification, molecular level identification of fungal pathogen was not done in the area. Therefore, this study was undertaken to isolate and characterized the fungal causative agent of ginger disease from the diseased plant and the soil samples collected around the diseased plant from Chilga district, Gondar, Ethiopia. Samples from infected ginger plants and the soil around the infected plant were collected. Culturing and purification of isolates were made using Potato Dextrose Agar supplemented with antibacterial agent chloramphenicol. The morphological characterization was done by structural identification of the isolates under the microscope using lactophenol cotton blue stains. Isolated fungi were cultured and molecular identification was done using an internal transcribed spacer (ITS) of ribosomal DNA (rDNA). A total of 15 fungal morphotypes including 11 Aspergillus spp. (73.3%), 2 Penicillium spp. (13.3%), and single uncultured fungus clone S23 were isolated from the samples representing all the plant organs and the soil. Aspergillus spp. (73.3%) was the most common and seems to be the major causative agent. To the best of our knowledge, this is the first report of ginger pathogenic fungi in Ethiopia identified using ITS rDNA molecular techniques. This study will lay foundation for the development of management strategies for fungal diseases infecting ginger.

The CfMK1 Gene Regulates Reproduction, Appressorium Formation, and Pathogenesis in a Pear Anthracnose-Causing Fungus

Colletotrichum fructicola, the causal agent of pear anthracnose, causes significant annual economic losses. Mitogen-activated protein kinase (MAPK) cascades are highly conserved signal transduction pathways that play a crucial role in mediating cellular responses to environmental and host signals in plant pathogenic fungi. In this study, we identified an ortholog of the FUS3/KSS1-related MAPK gene, CfMK1, and characterized its function in C. fructicola. The Cfmk1 deletion mutants exhibited poorly developed aerial hyphae, autolysis, no conidial mass or perithecia on solid plates. However, the conidiation of the Cfmk1 mutant in PDB liquid medium was normal compared with that of the wild type (WT). Conidia of the Cfmk1 mutant exhibited a reduced germination rate on glass slides or plant surfaces. The Cfmk1 deletion mutants were unable to form appressoria and lost the capacity to penetrate plant epidermal cells. The ability of the Cfmk1 mutants to infect pear leaves and fruit was severely reduced. Moreover, RNA sequencing (RNA-seq) analysis of the WT and Cfmk1 mutant was performed, and the results revealed 1886 upregulated and 1554 downregulated differentially expressed genes (DEGs) in the mutant. The DEGs were significantly enriched in cell wall and pathogenesis terms, which was consistent with the defects of the Cfmk1 mutant in cell wall integrity and plant infection. Overall, our data demonstrate that CfMK1 plays critical roles in the regulation of aerial hyphal growth, asexual and sexual reproduction, autolysis, appressorium formation, and pathogenicity.

Deletion of YLR358C Contributes to Reduce Cell Wall Integrity in Saccharomyces Cerevisiae

The mechanism of fungal cell wall synthesis and assembly is still unclear. Saccharomyces cerevisiae (S. cerevisiae) and pathogenic fungi are conserved in cell wall construction and response to stress signals, and often respond to cell wall stress through activated cell wall integrity (CWI) pathways. Whether the YLR358C open reading frame regulates CWI remains unclear. This study found that the growth of S. cerevisiae with YLR358C knockout was significantly inhibited on the medium containing different concentrations of cell wall interfering agents Calcofluor White (CFW), Congo Red (CR) and sodium dodecyl sulfate (SDS). CFW staining showed that the cell wall chitin was down-regulated, and transmission electron microscopy also observed a decrease in cell wall thickness. Transcriptome sequencing and analysis showed that YLR358C gene may be involved in the regulation of CWI signaling pathway. It was found by qRT-PCR that WSC3, SWI4 and HSP12 were differentially expressed after YLR358C was knocked out. The above results suggest that YLR358C may regulate the integrity of the yeast cell walls and has some potential for application in fermentation.

Phenolic degradation by catechol dioxygenases is associated with pathogenic fungi with a necrotrophic lifestyle in the Ceratocystidaceae

Fungal species of the Ceratocystidaceae grow on their host plants using a variety of different lifestyles, from saprophytic to highly pathogenic. Although many genomes of fungi in the Ceratocystidaceae are publicly available, it is not known how the genes that encode catechol dioxygenases (CDOs), enzymes involved in the degradation of phenolic plant defence compounds, differ among members of the Ceratocystidaceae. The aim of this study was therefore to identify and characterize the genes encoding CDOs in the genomes of Ceratocystidaceae representatives. We found that genes encoding CDOs are more abundant in pathogenic necrotrophic species of the Ceratocystidaceae and less abundant in saprophytic species. The loss of the CDO genes and the associated 3-oxoadipate catabolic pathway appears to have occurred in a lineage-specific manner. Taken together, this study revealed a positive association between CDO gene copy number and fungal lifestyle in Ceratocystidaceae representatives.

In Vitro and In Vivo Interactions of TOR Inhibitor AZD8055 and Azoles against Pathogenic Fungi

Limited options of antifungals and the emergence of drug resistance in fungal pathogens has been a multifaceted clinical challenge. Combination therapy represents a valuable alternative to antifungal monotherapy.

Biological Control of Fusarium culmorum, Fusarium graminearum and Fusarium poae by Antagonistic Yeasts

The genus Fusarium is considered to be one of the most pathogenic, phytotoxic and toxin-producing group of microorganisms in the world. Plants infected by these fungi are characterized by a reduced consumer and commercial value, mainly due to the contamination of crops with mycotoxins. Therefore, effective methods of reducing fungi of the genus Fusarium must be implemented already in the field before harvesting, especially with alternative methods to pesticides such as biocontrol. In this study we identified yeasts that inhibit the growth of the pathogenic fungi Fusarium culmorum, F. graminearum and F. poae. Tested yeasts came from different culture collections, or were obtained from organic and conventional cereals. The greater number of yeast isolates from organic cereals showed antagonistic activity against fungi of the genus Fusarium compared to isolates from the conventional cultivation system. Cryptococcus carnescens (E22) isolated from organic wheat was the only isolate that limited the mycelial growth of all three tested fungi and was the best antagonist against F. poae. Selected yeasts showed various mechanisms of action against fungi, including competition for nutrients and space, production of volatile metabolites, reduction of spore germination, production of siderophores or production of extracellular lytic enzymes: chitinase and β-1,3-glucanase. Of all the investigated mechanisms of yeast antagonism against Fusarium, competition for nutrients and the ability to inhibit spore germination prevailed.