The endophytobiome of wild Rubiaceae as a source of antagonistic fungi against the American Leaf Spot of coffee (Mycena citricolor).

AIMS: The American leaf spot, caused by Mycena citricolor, is an important disease of coffee (Coffea arabica), mostly in Central America. Currently, there are limited pathogen control alternatives that are environment friendly and economically accessible. The use of fungi isolated from the plant endomycobiota in their native habitats is on the rise because studies show their great potential for biological control. To begin to generate a green alternative to control M. citricolor, the objectives of the present study were to (i) collect, identify, screen (in vitro and in planta), and select endophytic fungi from wild Rubiaceae collected in old-growth forests of Costa Rica; (ii) confirm endophytic colonization in coffee plantlets; (iii) evaluate the effects of the endophytes on plantlet development; and (iv) corroborate the antagonistic ability in planta. METHODS AND RESULTS: Through in vitro and in planta antagonism assays, we found that out of the selected isolates (i.e. Daldinia eschscholzii GU11N, Nectria pseudotrichia GUHN1, Purpureocillium aff. lilacinum CT24, Sarocladium aff. kiliense CT25, Trichoderma rifaii CT5, T. aff. crassum G1C, T. aff. atroviride G7T, T. aff. strigosellum GU12, and Xylaria multiplex GU14T), Trichoderma spp. produced the highest growth inhibition percentages in vitro. Trichoderma isolates CT5 and G1C were then tested in planta using Coffea arabica cv. caturra plantlets. Endophytic colonization was verified, followed by in planta growth promotion and antagonism assays. CONCLUSIONS: Results show that Trichoderma isolates CT5 and G1C have potential for plant growth promotion and antagonism against Mycena citricolor, reducing incidence and severity, and preventing plant mortality.

Virulence of native isolates of entomopathogenic fungi (Hypocreales) against the "sweetpotato whitefly" Bemisia tabaci (Hemiptera: Aleyrodidae), including the effects of temperature and fungicides.

Hypocrella, Moelleriella and related species in the Hypocreales (Ascomycota, Sordariomycetes) cause epizootics of whiteflies and scale insects in nature. However, studies on their host specificity, virulence, infection cycles, optimal development under laboratory conditions, and compatibility with other control methods, are unexplored for most species. Under laboratory conditions, the virulence of several isolates of field-collected hypocrealean fungi (Hypocrella, Moelleriella, Regiocrella, and Verticillium) was determined on Bemisia tabaci eggs and 4th instar nymphs. In addition to virulence, the effect of temperature and two commercial fungicides on growth rates and germination of the isolates was evaluated. None of the isolates infected the eggs, while M. libera, M. ochracea, and M. turbinata caused high nymphal mortality. Moelleriella libera was the most virulent isolate. At all temperatures, M. libera, Regiocrella sp. (P17H20), and Verticillium cf. pseudohemipterigenum had the highest germination and growth rates. The optimal growth temperature depended on the isolate, but at 23 °C and 25 °C, the probability of spore germination was higher for most isolates. Finally, the fungicides azoxystrobin and chlorothalonil inhibited growth rates and conidial germination at 24 and 48 h of exposure. This research produces vital knowledge on the virulence and infection cycles of poorly studied native species of entomopathogenic fungi. In addition, the results provide information on the optimal temperature for development in laboratory conditions and susceptibility to fungicides, which could contribute to future biological control strategies.

Purpureocillium roseum sp. nov. A new ocular pathogen for humans and mice resistant to antifungals.

BACKGROUND: Infectious keratitis is the main cause of preventable blindness worldwide, with about 1.5-2.0 million new cases occurring per year. This inflammatory response may be due to infections caused by bacteria, fungi, viruses or parasites. Fungal keratitis is a poorly studied health problem. OBJECTIVES: This study aimed to identify a new fungal species by molecular methods and to explore the possible efficacy of the three most common antifungals used in human keratitis in Mexico by performing in vitro analysis. The capacity of this pathogen to cause corneal infection in a murine model was also evaluated. METHODS: The fungal strain was isolated from a patient with a corneal ulcer. To identify the fungus, taxonomic and phylogenetic analyses (nrDNA ITS and LSU data set) were performed. An antifungal susceptibility assay for amphotericin B, itraconazole and voriconazole was carried out. The fungal isolate was used to develop a keratitis model in BALB/c mice; entire eyes and ocular tissues were preserved and processed for histopathologic examination. RESULTS AND CONCLUSION: This fungal genus has hitherto not been reported with human keratitis in Mexico. We described a new species Purpurecillium roseum isolated from corneal infection. P roseum showed resistance to amphotericin B and itraconazole and was sensitive to voriconazole. In vivo study demonstrated that P roseum had capacity to developed corneal infection and to penetrate deeper corneal tissue. The global change in fungal infections has emphasised the need to develop better diagnostic mycology laboratories and to recognise the group of potential fungal pathogens.

Richness and metallo-tolerance of cultivable fungi recovered from three high altitude glaciers from Citlaltépetl and Iztaccíhuatl volcanoes (Mexico).

In Mexico little is known about high-altitude glacial psychrotolerant or psychrophilic fungal species, with most glacial fungi isolated from polar environments or Alpine glaciers. It has been documented that some of these species may play an important role in bioremediation of contaminated environments with heavy metals. In the present study, 75 fungi were isolated from glaciers in Citlaltépetl (5675 masl) and Iztaccíhuatl (5286 masl) volcanoes. Combining morphological characteristics and molecular methods, based on ITS rDNA, 38 fungi were partially identified to genus level, 35 belonging to Ascomycota and three to Mucoromycota. The most abundant genera were Cladosporium, followed by Alternaria and Sordariomycetes order. All isolated fungi were psychrotolerant, pigmented and resistant to different concentrations of Cr(III) and Pb(II), while none tolerated Hg(II). Fungi most tolerant to Cr(III) and Pb(II) belong to the genera Stemphylium, Cladosporium and Penicillium and to a lesser extent Aureobasidium and Sordariomycetes. To our knowledge, this is the first report on cultivable mycobiota richness and their Cr and Pb tolerance. The results open new research possibilities about fungal diversity and heavy metals myco-remediation. Extremophilic fungal communities should be further investigated before global warming causes permanent changes and we miss the opportunity to describe these sites in Mexico.

microbeMASST: a taxonomically informed mass spectrometry search tool for microbial metabolomics data.

microbeMASST, a taxonomically informed mass spectrometry (MS) search tool, tackles limited microbial metabolite annotation in untargeted metabolomics experiments. Leveraging a curated database of >60,000 microbial monocultures, users can search known and unknown MS/MS spectra and link them to their respective microbial producers via MS/MS fragmentation patterns. Identification of microbe-derived metabolites and relative producers without a priori knowledge will vastly enhance the understanding of microorganisms' role in ecology and human health.

Fungi with history: Unveiling the mycobiota of historic documents of Costa Rica.

We studied the physicochemical characteristics and mycobiota associated to five key historic documents from Costa Rica, including the Independence Act of Costa Rica from 1821. We used nondestructive techniques (i.e., ATR-FTIR and XRF) to determine paper and ink composition. Results show that some documents are composed of cotton-based paper, whereas others were made of wood cellulose with an increased lignin content. We also determined that the ink employed in some of the documents is ferrogallic. Cultivation and molecular techniques were used to characterize the fungi inhabiting the documents. In total, 22 fungal isolates were obtained: 15 from the wood-cellulose-based documents and seven from the other three cotton-based. We also tested the cellulolytic activity of the recovered fungi; 95% of the fungi presented cellulolytic activity correlated to their ability to cause deterioration of the paper. Results suggest that cotton-based paper is the most resistant to fungal colonization and that most of the isolates have cellulolytic activity. This work increases the knowledge of the fungal diversity that inhabits historic documents and its relationship with paper composition and provides valuable information to develop strategies to conserve and restore these invaluable documents.

Differential Volatile Organic Compound Expression in the Interaction of Daldinia eschscholtzii and Mycena citricolor.

Fungi exhibit a wide range of ecological guilds, but those that live within the inner tissues of plants (also known as endophytes) are particularly relevant due to the benefits they sometimes provide to their hosts, such as herbivory deterrence, disease protection, and growth promotion. Recently, endophytes have gained interest as potential biocontrol agents against crop pathogens, for example, coffee plants (Coffea arabica). Published results from research performed in our laboratory showed that endophytic fungi isolated from wild Rubiaceae plants were effective in reducing the effects of the American leaf spot of coffee (Mycena citricolor). One of these isolates (GU11N) from the plant Randia grandifolia was identified as Daldinia eschscholtzii (Xylariales). Its antagonism mechanisms, effects, and chemistry against M. citricolor were investigated by analyzing its volatile profile alone and in the presence of the pathogen in contactless and dual culture assays. The experimental design involved direct sampling of agar plugs in vials for headspace (HS) and headspace solid-phase microextraction (HS-SPME) gas chromatography-mass spectrometry (GC-MS) analysis. Additionally, we used ultrahigh-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS/MS) to identify nonvolatile compounds from organic extracts of the mycelia involved in the interaction. Results showed that more volatile compounds were identified using HS-SPME (39 components) than those by the HS technique (13 components), sharing only 12 compounds. Statistical tests suggest that D. eschscholtzii inhibited the growth of M. citricolor through the release of VOCs containing a combination of 1,8-dimethoxynapththalene and terpene compounds affecting M. citricolor pseudopilei. The damaging effects of 1,8-dimethoxynaphthalene were corroborated in an in vitro test against M. citricolor pseudopilei; scanning electron microscopy (SEM) photographs confirmed structural damage. After analyzing the UHPLC-HRMS/MS data, a predominance of fatty acid derivatives was found among the putatively identified compounds. However, a considerable proportion of features (37.3%) remained unannotated. In conclusion, our study suggests that D. eschscholtzii has potential as a biocontrol agent against M. citricolor and that 1,8-dimethoxynaphthalene contributes to the observed damage to the pathogen's reproductive structures.

A Taxonomically-informed Mass Spectrometry Search Tool for Microbial Metabolomics Data.

MicrobeMASST, a taxonomically-informed mass spectrometry (MS) search tool, tackles limited microbial metabolite annotation in untargeted metabolomics experiments. Leveraging a curated database of >60,000 microbial monocultures, users can search known and unknown MS/MS spectra and link them to their respective microbial producers via MS/MS fragmentation patterns. Identification of microbial-derived metabolites and relative producers, without a priori knowledge, will vastly enhance the understanding of microorganisms' role in ecology and human health.

Biodeterioration and cellulolytic activity by fungi isolated from a nineteenth-century painting at the National Theatre of Costa Rica.

Living organisms can induce deterioration of cultural heritage. Conservation strategies aimed at avoiding damage and aiding restoration, require a comprehensive knowledge of structure, chemical composition, and identity of microorganisms that colonize artworks. The National Theatre of Costa Rica (NTCR), a building with historic architecture, houses several oil paintings from the nineteenth century, some with visible signs of biodeterioration. One of them is a large format painting on canvas called La Danza (size 9.83 × 5.13 m) from 1896 by Italian artist Vespasiano Bignami, located on the ceiling of the theatre's foyer. In the present study, we undertook a physicochemical and microbiological study of La Danza to identify the fungal species that inhabit the artwork and are responsible for the damage observed. Scanning electron microscope (SEM) images and attenuated total reflectance - Fourier transform infrared (ATR-FTIR) spectroscopic data indicated that the canvas material is made of hemp, the binder contains linseed oil and lead white, and a material in the inner face of the canvas is mainly composed of beeswax. Fungi were isolated onto potato dextrose agar (PDA) and carboxymethyl cellulose (CMC) agar, and then identified with molecular (BTUB, nrDNA ITS, and TEF1 regions) and morphological methods. Four isolates belonging to the genera Myxospora, Pestalotiopsis, Ustilago, and aff. Penicillium, were obtained. Qualitative tests showed cellulolytic activity in all isolated specimens, confirming their possible role in biodeterioration of the canvas. Phylogenetic and morphological data revealed a new species of Myxospora we name here as Myxospora theatro sp. nov., in reference to NTCR. The findings broaden the knowledge of fungi capable of inhabiting and damaging cultural heritage. They also provide valuable information to develop strategies for conservation and restoration of oil paintings on canvas.

A metabolomic platform to identify and quantify polyphenols in coffee and related species using liquid chromatography mass spectrometry.

Introduction: Products of plant secondary metabolism, such as phenolic compounds, flavonoids, alkaloids, and hormones, play an important role in plant growth, development, stress resistance. The plant family Rubiaceae is extremely diverse and abundant in Central America and contains several economically important genera, e.g. Coffea and other medicinal plants. These are known for the production of bioactive polyphenols (e.g. caffeine and quinine), which have had major impacts on human society. The overall goal of this study was to develop a high-throughput workflow to identify and quantify plant polyphenols. Methods: First, a method was optimized to extract over 40 families of phytochemicals. Then, a high-throughput metabolomic platform has been developed to identify and quantify 184 polyphenols in 15 min. Results: The current metabolomics study of secondary metabolites was conducted on leaves from one commercial coffee variety and two wild species that also belong to the Rubiaceae family. Global profiling was performed using liquid chromatography high-resolution time-of-flight mass spectrometry. Features whose abundance was significantly different between coffee species were discriminated using statistical analysis and annotated using spectral databases. The identified features were validated by commercially available standards using our newly developed liquid chromatography tandem mass spectrometry method. Discussion: Caffeine, trigonelline and theobromine were highly abundant in coffee leaves, as expected. Interestingly, wild Rubiaceae leaves had a higher diversity of phytochemicals in comparison to commercial coffee: defense-related molecules, such as phenylpropanoids (e.g., cinnamic acid), the terpenoid gibberellic acid, and the monolignol sinapaldehyde were found more abundantly in wild Rubiaceae leaves.

Step-by-Step Pipeline for the Ecological Analysis of Endophytic Fungi using ITS nrDNA Data.

The nuclear ribosomal DNA internal transcribed spacer (ITS) is accepted as the genetic marker or barcode of choice for the identification of fungal samples. Here, we present a protocol to analyze fungal ITS data, from quality preprocessing of raw sequences to identification of operational taxonomic units (OTUs), taxonomic classification, and assignment of functional traits. The pipeline relies on well-established and manually curated data collections, namely the UNITE database and the FUNGuild script. As an example, real ITS data from culturable endophytic fungi were analyzed, providing detailed descriptions for every step, parameter, and downstream analysis, and finishing with a phylogenetic analysis of the sequences and assigned ecological roles. This article constitutes a comprehensive guide for researchers that have little familiarity with bioinformatic analysis of essential steps required in further ecological studies of fungal communities. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Raw sequencing data processing Support Protocol: Building a BLAST database Basic Protocol 2: Obtaining information from databases Basic Protocol 3: Phylogenetic analysis.

Two new cellulolytic fungal species isolated from a 19th-century art collection.

The archive of the Universidad de Costa Rica maintains a nineteenth-century French collection of drawings and lithographs in which the biodeterioration by fungi is rampant. Because of nutritional conditions in which these fungi grew, we suspected that they possessed an ability to degrade cellulose. In this work our goal was to isolate and identify the fungal species responsible for the biodegradation of a nineteenth-century art collection and determine their cellulolytic activity. Fungi were isolated using potato-dextrose-agar (PDA) and water-agar with carboxymethyl cellulose (CMC). The identification of the fungi was assessed through DNA sequencing (nrDNA ITS and α-actin regions) complemented with morphological analyses. Assays for cellulolytic activity were conducted with Gram's iodine as dye. Nineteen isolates were obtained, of which seventeen were identified through DNA sequencing to species level, belonging mainly to genera Arthrinium, Aspergillus, Chaetomium, Cladosporium, Colletotrichum, Penicillium and Trichoderma. For two samples that could not be identified through their ITS and α-actin sequences, a morphological analysis was conducted; they were identified as new species, named Periconia epilithographicola sp. nov. and Coniochaeta cipronana sp. nov. Qualitative tests showed that the fungal collection presents important cellulolytic activity.