1. Bibliographic Information

1.1. Title

The central topic of the paper is the investigation of fungal species in soil samples from areas in Rio de Janeiro, Brazil, known for sporotrichosis transmission, specifically searching for Sporothrix species and evaluating the antagonistic activity of other isolated fungi against Sporothrix brasiliensis.

1.2. Authors

The authors are Gisela Lara da Costa, Isabella Escórcio Ferreira, Danielly Corrêa-Moreira, Anna Marinho, Adilson Benedito de Almeida, Sandro Antônio Pereira, Cintia Moraes Borba, and Manoel Marques Evangelista Oliveira. Their primary affiliations are with the Laboratory of Taxonomy, Biochemistry and Bioprospecting of Fungi, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil. Some authors also hold positions at the Evandro Chagas National Institute of Infectious Diseases, FIOCRUZ, emphasizing a focus on infectious diseases, mycology, and public health research.

1.3. Journal/Conference

The paper was published in Frontiers in Cellular and Infection Microbiology, specifically in the Fungal Pathogenesis section. Frontiers journals are generally well-regarded open-access journals, and this specific section indicates a focus on microbial infections, particularly those caused by fungi, suggesting a relevant and influential venue for this type of research.

1.4. Publication Year

The paper was published on December 1, 2022.

1.5. Abstract

Since 1998, sporotrichosis, a disease caused by Sporothrix spp. transmitted through contact with infected cats, has become a significant public health concern in Rio de Janeiro, Brazil. Despite efforts, isolating these fungal species from environmental sources often proves difficult. This study collected and cultured soil samples from residences in Rio de Janeiro cities where sporotrichosis-infected cats resided, aiming to isolate Sporothrix spp., but this attempt was unsuccessful. However, various other saprophytic fungal species were isolated and identified from the soil, with Purpureocillium lilacinum being the most frequent. Consequently, the researchers investigated the in vitro interaction between P. lilacinum and S. brasiliensis, the primary causative agent of sporotrichosis in the region. The results demonstrated that ten isolates of P. lilacinum effectively inhibited the radial mycelial growth of S. brasiliensis to varying degrees. The observed interaction pattern was categorized as overgrowth by antagonist. The study concludes that fast-growing fungal species capable of producing metabolites might impede the growth of Sporothrix spp., thereby opening avenues for identifying secondary metabolites with potential biological activity against pathogenic fungi.

1.6. Original Source Link

The original source link is /files/papers/691ac5c0110b75dcc59ae416/paper.pdf. This link points to the PDF file of the officially published article.

2. Executive Summary

2.1. Background & Motivation

The core problem the paper addresses is the hyperendemic nature of sporotrichosis in Rio de Janeiro, Brazil, primarily caused by Sporothrix brasiliensis and transmitted zoonotically from infected cats to humans. Despite its prevalence, environmental studies consistently face difficulties in isolating Sporothrix spp. from soil and other natural sources. This gap in understanding the environmental reservoir and ecology of Sporothrix makes it challenging to implement effective public health interventions.

This problem is important because understanding the environmental presence and dynamics of Sporothrix is crucial for controlling the epidemic and preventing new infections. The sporotrichosis transmission belt in Rio de Janeiro highlights a critical public health challenge. Previous research has suggested the environment could act as a reservoir, but successful environmental isolations are rare, and the reasons for this difficulty are unclear. The paper's entry point is to investigate not only the presence of Sporothrix but also the presence of other saprophytic fungi in endemic soil samples, hypothesizing that interactions within the soil microbiome could explain the low Sporothrix isolation rates.

2.2. Main Contributions / Findings

The paper's primary contributions and key findings are:

• Confirmation of Sporothrix Isolation Difficulty: The study reaffirmed the challenge of isolating Sporothrix spp. from environmental soil samples, even from hyperendemic areas where infected cats reside. This reinforces the need for alternative detection methods (e.g., molecular).

• Identification of Dominant Saprophytic Fungi: The researchers successfully isolated and identified various saprophytic fungal species from the soil, with Purpureocillium lilacinum being the most prevalent. This provides insight into the competing fungal microbiome in these environments.

• Demonstration of Antagonistic Activity: Ten isolates of P. lilacinum exhibited significant antagonistic activity against S. brasiliensis in vitro, inhibiting its radial mycelial growth by 8% to 25%. This is a novel finding concerning P. lilacinum's interaction with Sporothrix brasiliensis.

• Characterization of Interaction Pattern: The observed in vitro interaction between P. lilacinum and S. brasiliensis followed a pattern described as overgrowth by antagonist, where P. lilacinum eventually covered the S. brasiliensis colony.

• Implication for Sporothrix Ecology and Bioprospecting: The findings suggest that competition from fast-growing, metabolite-producing fungi like P. lilacinum might contribute to the observed difficulty in isolating Sporothrix spp. from the environment. This opens new avenues for research into secondary metabolites produced by P. lilacinum as potential antifungal agents against pathogenic fungi.

  These findings address the problem by offering a plausible explanation for the elusive nature of environmental Sporothrix and by identifying a candidate biocontrol agent or source of antifungal compounds.

3. Prerequisite Knowledge & Related Work

3.1. Foundational Concepts

To fully understand this paper, a novice reader should be familiar with the following concepts:

• Sporotrichosis: A subacute or chronic mycosis (fungal infection) caused by Sporothrix spp.. It commonly affects the skin and subcutaneous tissue, but can spread to other parts of the body. In Brazil, it has become a significant public health problem due to zoonotic transmission from infected cats to humans.

• Sporothrix spp. and Sporothrix brasiliensis: Sporothrix is a genus of thermally dimorphic fungi. Thermally dimorphic fungi are fungi that can exist in two distinct morphological forms depending on the temperature: a filamentous mold form (or mycelial form) at ambient temperatures (e.g., in the environment) and a yeast-like form at body temperature (e.g., within an infected host). Sporothrix brasiliensis is one species within the Sporothrix complex that has emerged as the primary cause of the zoonotic sporotrichosis epidemic in Brazil, particularly in Rio de Janeiro.

• Saprophytic Fungi: Fungi that obtain their nutrients from dead organic matter (e.g., decaying plants, soil components). They play a crucial role in decomposition and nutrient cycling in ecosystems. In the context of this paper, these are the environmental fungi that live in the soil alongside potentially pathogenic fungi.

• Antagonism: In microbiology, antagonism refers to an interaction between two or more organisms where one organism inhibits the growth or activity of another. This can occur through various mechanisms, such as competition for resources, antibiosis (production of inhibitory metabolites), or mycoparasitism (one fungus parasitizing another).

• Biocontrol Agent: An organism used to suppress or control the population of another harmful organism (e.g., a pest or pathogen). Purpureocillium lilacinum is known as a biocontrol agent against nematodes and insects. The paper explores its potential as a biocontrol agent against pathogenic fungi.

• Mycelial Growth / Radial Mycelial Growth: The mycelium is the vegetative part of a fungus, consisting of a network of fine white filaments called hyphae. Mycelial growth refers to the expansion of this fungal network. Radial mycelial growth specifically describes the outward expansion of a fungal colony in a circular pattern on a flat surface (like an agar plate), typically measured by its radius or diameter over time.

• Colony Forming Unit (CFU): A unit used in microbiology to estimate the number of viable bacterial or fungal cells in a sample. It represents the number of cells that are capable of multiplying to form a visible colony on a culture medium. In this study, CFU refers to fungal colonies.

• Serial Dilution Technique: A laboratory method used to decrease the concentration of a sample (e.g., soil suspension) by a specific, known factor. This technique is often used to obtain a manageable number of CFU on agar plates for isolation and counting, allowing for the isolation of individual colonies.

• Potato Dextrose Agar (PDA): A common culture medium used in mycology for the growth and isolation of fungi. It contains potato infusion (providing nutrients), dextrose (a sugar for energy), and agar (a gelling agent). Chloramphenicol is often added to PDA to inhibit bacterial growth, allowing fungi to grow more selectively.

• Microcultures: A technique used in mycology to observe the micromorphological characteristics of fungi (e.g., shape of conidia, arrangement of hyphae). A small piece of agar with the fungus is placed on a microscope slide and covered with a coverslip, allowing for direct microscopic observation of growth and spore formation. The Riddell method is a common microculture technique.

3.2. Previous Works

The paper contextualizes its research by referencing several prior studies:

• Sporotrichosis Epidemiology in Rio de Janeiro:

  • Orofino-Costa et al., 2017; Schubach et al., 2008; Gremião et al., 2020: These works establish that sporotrichosis has been a significant public health problem in Rio de Janeiro since 1998, with a notable increase in human and feline cases. They highlight the zoonotic transmission from infected cats to humans in the metropolitan area.
  • Silva et al., 2012; Brasil, 2021: These studies identified a transmission belt of sporotrichosis along the Rio de Janeiro city border and adjacent municipalities, demonstrating the spatial distribution of the disease and its widespread presence across administrative regions of the state.
  • Oliveira et al., 2011; Gremião et al., 2020: Molecular analyses confirmed Sporothrix brasiliensis as the primary cause of this zoonotic disease in the region.

• Environmental Sporothrix Isolation Difficulties:

  • Poester et al., 2018: Suggested that the environment might serve as a reservoir for S. brasiliensis in hyperendemic areas, with potential transfer from sick animals (lesions, feces, buried bodies). However, they also noted the general difficulty and infrequency of successful environmental Sporothrix isolations.
  • Howard and Orr, 1963; Mackinnon et al., 1969; Kenyon et al., 1984; Dixon et al., 1991; Sanchez-Aleman et al., 2004; Mendoza et al., 2007; Metha et al., 2007; Criseo and Romeo, 2010; Cruz Choappa et al., 2014; Rodrigues et al., 2014: These numerous studies collectively underscore the long-standing challenge of isolating Sporothrix spp. from environmental sources, often reporting low success rates or unsuccessful isolations.
  • Rabello et al., 2022: A very recent study that attempted to isolate Sporothrix spp. from demolition wood in a sporotrichosis-affected house in Petrópolis, Rio de Janeiro. They only succeeded in isolating S. brasiliensis directly from the cat, not the environment, further supporting the difficulty.
  • Almeida-Silva et al., 2022: Another recent work that collected soil samples from endemic areas in Rio de Janeiro. While they found S. brasiliensis DNA using molecular techniques, they observed no fungal growth compatible with Sporothrix spp., highlighting the discrepancy between molecular detection and culture isolation.

• Fungal Antagonism and Biocontrol:

  • Boddy and Hiscox, 2016: This review provides a foundational understanding that saprophytic species can exert antagonistic actions on each other's growth, suggesting a mechanism for the difficulty in isolating Sporothrix. It also explains that fungi compete via rapid growth, sporulation, stress recovery, and the use/negation of inhibitors (secondary metabolites).
  • Porter, 1924 apud Skidmore and Dickinson, 1976: This work provides the classification of fungal interaction patterns, including overgrowth by antagonist, which is used to categorize the P. lilacinum - S. brasiliensis interaction in this paper.
  • Rahman et al., 2009; Sonkar, 2018: These studies demonstrate antagonistic potential and varied inhibition percentages among different Trichoderma strains against various fungal pathogens, showing that antagonistic activity is strain-dependent.
  • Barra et al., 2015; Hotaka et al., 2015; Singh et al., 2013; Liu et al., 2014: These studies highlight P. lilacinum's known role as a biocontrol agent against insects and nematodes.
  • Elsherbiny et al., 2021; Lan et al., 2017: These works report P. lilacinum's ability to inhibit the growth of other fungi like Penicillium digitatum and Verticillium dahliae.
  • Liu et al., 2020: This study discovered a new antifungal lipopeptaibol (leucinostatin Z) from P. lilacinum against Botrytis cinerea after co-culturing, supporting the hypothesis that P. lilacinum produces antifungal metabolites.
  • Mikami et al., 1989; Khan et al., 2003; Park et al., 2004; Sharma and Sharma, 2016: These works mention P. lilacinum's production of various metabolites, including leucinostatins, paecilotoxin, and mycotoxins.

3.3. Technological Evolution

The field of environmental mycology has evolved from purely culture-based methods to incorporate molecular techniques for detecting fungal DNA in samples. Historically, isolating specific fungi like Sporothrix spp. from complex environmental matrices like soil has been challenging due to several factors, including:

1. Low abundance: The target fungus might be present in very low concentrations.

2. Competition: Other faster-growing saprophytic fungi and bacteria can outcompete and overgrow the target fungus on culture media.

3. Specific growth requirements: Sporothrix spp. may have specific or fastidious growth requirements that are not met by standard isolation media or conditions.

4. Viability issues: The fungus might be present but non-viable, or in a dormant state, making culture isolation difficult.

   This paper's work fits within this technological timeline by acknowledging the limitations of culture-based methods for Sporothrix and then shifting focus to understanding the microbial interactions that might explain these limitations. It bridges classical mycology (culture, phenotypic identification) with ecological interaction studies, implicitly laying groundwork for future molecular studies on metabolite production and antagonism.

3.4. Differentiation Analysis

Compared to previous studies that primarily focused on attempts to isolate Sporothrix spp. from the environment (often with limited success), this paper introduces a crucial differentiation:

• Focus on Antagonistic Fungi: Instead of solely reporting the failure to isolate Sporothrix, this study actively investigated why such isolation might be difficult by identifying other prevalent saprophytic fungi in the soil and assessing their antagonistic activity against S. brasiliensis. This shifts the research paradigm from mere detection to understanding ecological interactions.

• Identification of Purpureocillium lilacinum as a key antagonist: The paper specifically identifies P. lilacinum as the most frequent saprophytic fungus and systematically tests its antagonistic potential, providing concrete evidence for fungal competition as a factor in Sporothrix ecology.

• Implication for Bioprospecting: By demonstrating in vitro inhibition and overgrowth, the study opens a new line of inquiry into P. lilacinum as a source of novel antifungal secondary metabolites, a direction less explored in the context of Sporothrix environmental ecology.

  The core innovation lies in leveraging the failure to isolate Sporothrix to investigate the success of other microbial communities in the same environment and their potential antagonistic effects, thus providing a mechanistic hypothesis for the observed isolation difficulties.

4. Methodology

4.1. Principles

The core idea behind this study's methodology is to explore the fungal diversity in soil from sporotrichosis endemic areas where Sporothrix spp. isolation is notoriously difficult, with a specific focus on identifying saprophytic fungi that might exert antagonistic effects on Sporothrix brasiliensis. The theoretical basis relies on the principle of microbial competition in complex environments like soil, where various fungi compete for resources and space, often employing antagonistic strategies such as rapid growth or the production of inhibitory secondary metabolites. By isolating prevalent soil fungi and testing their in vitro interaction with S. brasiliensis, the researchers aimed to identify potential antagonists and understand their role in shaping the environmental ecology of Sporothrix.

4.2. Core Methodology In-depth (Layer by Layer)

4.2.1. Soil Samples Collection

Soil samples were collected from residential areas within the sporotrichosis transmission belt in the state of Rio de Janeiro, Brazil. Specifically, samples were taken from cities known for sporotrichosis cases in cats: Petrópolis (located in the metropolitan region) and Vassouras (located in the central-south region). These areas are characterized by Atlantic Forest environments. Approximately 50 grams of soil were collected from each site, specifically 10 cm below the topsoil layer. The collected samples were then stored in Falcon tubes for transportation to the laboratory, maintaining conditions suitable for preserving fungal viability.

4.2.2. Isolation of Fungi from Soil Samples

The fungi were isolated from the collected soil samples using a serial dilution technique, followed by plate inoculation. This method is adapted from Clark (1965).

1. Preparation of Soil Suspension:

   • One gram of the collected soil was accurately weighed.
   • This soil was then placed into a 28 x 150 mm test tube.
   • 10 mL of sterile distilled water was added to the test tube.
   • The mixture was homogenized using a vortex mixer for 3 minutes to disperse soil particles and release fungal spores/hyphae into the water.

2. Serial Dilutions:

   • From the initial 10 mL soil solution, 1 mL was transferred to a new test tube containing 9 mL of sterile distilled water. This created a $10^{-1}$ dilution.
   • This process was repeated serially to create dilutions ranging from $10^{-1}$ to $10^{-5}$. For example, transferring 1 mL from the $10^{-1}$ tube to another 9 mL sterile distilled water tube creates a $10^{-2}$ dilution, and so on.

3. Inoculation and Incubation:

   • From the $10^{-3}$ dilution, aliquots of 0.1 mL were transferred to Petri dishes.
   • Each Petri dish contained Potato Dextrose Agar (PDA) (from Difco, Becton-Dickinson and Company, New Jersey, USA) supplemented with chloramphenicol at a concentration of 0.05 g.mL$^{-1}$. Chloramphenicol is an antibiotic added to inhibit the growth of bacteria, thereby promoting the selective growth of fungi.
   • The inoculated Petri dishes were placed in a BOD-type temperature chamber (an incubator with controlled biological oxygen demand) and incubated at a controlled temperature of $28^\circ\text{C} \pm 1^\circ\text{C}$ for seven days.
   • Daily monitoring was performed to observe fungal growth and emergence of colony forming units (CFU).

4. Isolation and Storage:

   • The most representative CFU (individual fungal colonies) that appeared on the plates were isolated.
   • These isolated colonies were then transferred to test tubes containing the same PDA medium.
   • The test tubes were kept in a BOD-type air-conditioned chamber at $28^\circ\text{C}$ for future identification and characterization.

4.2.3. Identification by Phenotypic Characteristics of the Fungal Isolates

The isolated fungal colonies were identified based on their macroscopic and micromorphological characteristics.

1. Macroscopic Analysis:

   • Fungal colonies were grown on PDA medium and incubated at $28^\circ\text{C}$.
   • The colony diameter was measured using a Mitutoyo digital caliper (Mitutoyo America Corporation, Aurora, Illinois, USA) with a resolution of 0.01 mm.
   • Other relevant macroscopic characteristics observed for identification included:
     • Texture of the colony (e.g., cottony, powdery, velvety).
     • Coloration of conidia (asexual spores).
     • Coloration of the mycelium (the fungal mass) and the reverse side of the colony (bottom of the plate).
     • Presence and characterization of exudate (liquid droplets on the colony surface).
     • Presence of soluble pigments (pigments diffused into the agar medium).
   • These observations were compared against established taxonomic keys and descriptions (Barron, 1972; Barnett and Hunter, 1998).

2. Micromorphological Evaluation (Microcultures):

   • Microcultures were prepared using the Riddell method (Riddell, 1950). This involves placing a small block of PDA agar inoculated with the fungus on a microscope slide and covering it with a coverslip, allowing for fungal growth in a confined space suitable for direct microscopic observation.
   • The microcultures were incubated for 7 days at $28^\circ\text{C}$.
   • After incubation, the micromorphology of the fungi was observed under a Nikon light microscope, E400 model (Nikon Instruments Inc., Melville, NY, United States).
   • To enhance visibility, the fungal structures were stained with Amann lactophenol plus cotton blue solution, which stains fungal chitin blue, making hyphae and spores more discernible.
   • Observations focused on characteristics such as hyphal morphology, conidiophore structure, conidia shape and arrangement, and presence of other specialized fungal structures, crucial for species-level identification.

4.2.4. Screening by Dual Culture Method

To evaluate the antagonistic activity of the isolated soil fungi against Sporothrix brasiliensis, a dual culture assay was performed (adapted from Liu et al., 2020).

1. Fungi Preparation:

   • A constant quantity of the putative antagonist fungus (isolated from soil) and the Sporothrix brasiliensis type strain CBS120339 were prepared using a platinum loop.

2. Inoculation:

   • Both fungi were separately point inoculated onto PDA medium in Petri dishes.
   • They were placed 2 cm from the edge of the Petri dish, at opposite ends, ensuring an initial distance for independent growth before interaction.

3. Control and Replicates:

   • As a control, S. brasiliensis was inoculated in a similar manner (single point, 2 cm from edge) on a fresh PDA plate without any antagonist. This control allowed for measuring the uninhibited growth of S. brasiliensis.
   • All pairings (antagonist + S. brasiliensis) and control plates were carried out in triplicate to ensure reproducibility and statistical robustness.

4. Incubation:

   • All plates were incubated at $28^\circ\text{C}$ for seven days.
   • Antagonistic activity was initially evaluated after seven days.
   • The interaction between the species was continuously monitored until the $28^\text{th}$ day of incubation at the same temperature, to observe long-term interaction patterns.

5. Evaluation of Antagonistic Activity:

   • The antagonistic activity was quantified by measuring the radial growth of both fungi in the test plates and compared to the S. brasiliensis control plate.
   • The Percentage Inhibition of Radial Growth (PIRG) was calculated using the formula developed by Skidmore and Dickinson (1976), as presented in the paper: $ \mathrm{PIRG} = \frac{(\mathrm{R1} - \mathrm{R2})}{\mathrm{R1}} \times 100 $ Where:
     • PIRG represents the percentage inhibition of radial growth. This metric quantifies how much the growth of S. brasiliensis was reduced due to the presence of the antagonist.
     • R1 is the radius (half of the diameter) of the S. brasiliensis colony when grown alone in the control plate. This serves as the maximum potential growth without inhibition.
     • R2 is the radius of the S. brasiliensis colony when grown in the presence of the putative antagonist in the dual culture plate. This represents the actual growth under competitive conditions.
   • The formula effectively calculates the proportion of S. brasiliensis growth that was inhibited by the antagonist, expressed as a percentage.

5. Experimental Setup

5.1. Datasets

The primary dataset for this study consisted of soil samples collected from specific geographical locations:

• Source: Soil from residences situated in cities within the state of Rio de Janeiro, Brazil. These residences were specifically chosen because they housed cats diagnosed with sporotrichosis, indicating an active transmission belt for the disease. The cities mentioned are Petrópolis (in the metropolitan region) and Vassouras (in the central-south region), both known to be hyperendemic areas for sporotrichosis.

• Scale and Characteristics: Approximately 50 grams of soil were collected from each site, 10 cm below the topsoil. The samples originated from Atlantic Forest areas, suggesting a particular environmental microbiome context. The soil samples themselves represent a complex microbial community including various saprophytic fungi, bacteria, and other microorganisms.

• Target Organism: The Sporothrix brasiliensis type strain CBS120339 was used as the target pathogen against which the antagonistic activity of isolated soil fungi was evaluated in vitro. This is a well-characterized strain used for research purposes.

  These datasets were chosen because they represent the actual environmental context where sporotrichosis is prevalent, allowing for the direct investigation of the fungal communities that interact with Sporothrix spp. in its natural habitat. The use of a type strain of S. brasiliensis ensures a consistent and reliable target for antagonism assays.

5.2. Evaluation Metrics

The primary evaluation metric used in this study is the Percentage Inhibition of Radial Growth (PIRG).

1. Conceptual Definition: Percentage Inhibition of Radial Growth (PIRG) quantifies the extent to which the mycelial growth of a target fungus (Sporothrix brasiliensis in this study) is reduced when grown in the presence of an antagonist fungus (e.g., Purpureocillium lilacinum), compared to its growth when cultured alone. It measures the effectiveness of the antagonist in suppressing the radial expansion of the target fungal colony on an agar plate. A higher PIRG value indicates stronger inhibitory activity.

2. Mathematical Formula: The PIRG is calculated using the following formula: $ \mathrm{PIRG} = \frac{(\mathrm{R1} - \mathrm{R2})}{\mathrm{R1}} \times 100 $

3. Symbol Explanation:

   • PIRG: Percentage Inhibition of Radial Growth, expressed as a percentage.
   • R1: The radius of the Sporothrix brasiliensis colony (in mm) when grown alone in the control plate (i.e., without the antagonist). This represents the maximum potential growth under the experimental conditions.
   • R2: The radius of the Sporothrix brasiliensis colony (in mm) when grown in the dual culture plate in the presence of the putative antagonist. This represents the actual growth achieved under antagonistic conditions.

5.3. Baselines

The baseline used in this study for evaluating antagonistic activity is the uninhibited growth of Sporothrix brasiliensis.

• Description: For each dual culture assay involving an antagonist and S. brasiliensis, a control plate was set up where S. brasiliensis was inoculated alone on PDA medium under identical incubation conditions. The radial growth of S. brasiliensis on this control plate (measured as R1) serves as the baseline.
• Representativeness: This baseline is representative because it provides the maximum growth potential of S. brasiliensis in the absence of any antagonistic pressure, allowing for a direct and quantitative comparison to determine the inhibitory effect (PIRG) of the putative antagonists.

6. Results & Analysis

6.1. Fungal Species from Soil Samples

The study aimed to isolate Sporothrix spp. from 143 Colony Forming Units (CFU) obtained from soil samples, with 46 CFU from Petrópolis (Pe-46) and 97 from Vassouras (Va-97). However, Sporothrix sp. was not isolated from any of the soil samples.

Of the total CFU, 137 were filamentous fungi and 6 were yeast-like colonies (which were not further evaluated). Eight genera of filamentous fungi were identified using classical taxonomy. Purpureocillium sp. was the most frequently isolated genus, accounting for 35 isolates (6 from Petrópolis and 29 from Vassouras). It was followed by Penicillium sp. with 16 isolates (7 from Petrópolis and 9 from Vassouras), Aspergillus sp. with 7 isolates (1 from Petrópolis and 6 from Vassouras), Beauveria sp. with 5 isolates (all from Petrópolis), Trichoderma sp. with 5 isolates (4 from Petrópolis and 1 from Vassouras), Fusarium sp. with 5 isolates (3 from Petrópolis and 2 from Vassouras), Metarhizium sp. with 4 isolates (3 from Petrópolis and 1 from Vassouras), and Scopulariopsis sp. with 1 isolate (from Petrópolis). A group of 25 isolates (4 from Petrópolis and 21 from Vassouras) did not produce any identifiable spores and were classified as Mycelia sterilia.

The species-level identification of 63 selected isolates revealed:

• Purpureocillium lilacinum: 35 isolates (Pe-6/Va-29)

• Penicillium species: Penicillium citrinum (Pe-2/Va-4), Penicillium decumbens (Va-5), Penicillium expansum (Pe-2), Penicillium pinophilum (Pe-2), Penicillium oxalicum (Pe-1)

• Aspergillus species: Aspergillus versicolor (Pe-1/Va-3), Aspergillus fumigatus (Va-1), Aspergillus clavatus (Va-1), Aspergillus sydiwii (Va-1)

• Metarhizium anisoplae: 4 isolates (Pe-3/Va-1)

• Scopulariopsis brevicaulis: 1 isolate (Pe-1)

  The following figure from the original paper (Figure 1) illustrates the distribution of identified fungal genera:

  该图像是饼图，展示了从土壤样本中分离出的不同真菌种类的数量分布。各部分颜色代表不同的真菌属，最大类别为 Aspergillus sp.（35个分离物），其次是 Purpureocillium sp.（16个分离物），以及其他真菌种类如 Beauveria sp. 和 Fusarium sp. 等。

FIGURE 1

Analysis: The results demonstrate a diverse fungal community in the soil samples from sporotrichosis endemic areas. The predominant presence of Purpureocillium lilacinum is a key finding, as this species was subsequently chosen for antagonistic activity evaluation. The failure to isolate Sporothrix spp. directly from the environment, despite molecular evidence of its presence in similar studies (Almeida-Silva et al., 2022), strongly supports the paper's central hypothesis that environmental factors or microbial interactions may hinder its culturable isolation. The high frequency of Mycelia sterilia suggests that some fungi may be difficult to induce sporulation in vitro or are not easily identifiable by phenotypic methods alone.

6.2. Antagonistic Activity of Fungal Species Against Sporothrix brasiliensis

From the identified species, three genera found in both municipalities (P. lilacinum, A. versicolor, M. anisoplae) were selected for antagonistic screening. However, it was not possible to quantify the inhibition potential of A. versicolor and M. anisoplae isolates because their colonies exhibited very fast overgrowth, completely covering the Petri dish in less than 7 days, making radial growth measurements inaccurate for the target S. brasiliensis.

In contrast, ten isolates of P. lilacinum were tested and successfully inhibited the radial mycelial growth of S. brasiliensis. The mean Percentage Inhibition of Radial Growth (PIRG) values varied among the P. lilacinum isolates, ranging from 8% to 25%.

The following are the results from Table 1 of the original paper:

The following figure from the original paper (Figure 2) illustrates the growth inhibition of S. brasiliensis by two P. lilacinum isolates on the $7^\text{th}$ day of incubation:

该图像是图表，展示了在第7天培养后的两组样本。左侧为LTBBF-PL4，抑制率为 $21.73\\%$；右侧为LTBBF-PL9，抑制率为 $18.18\\%$。

FIGURE 2 $7 ^ { \mathrm { t h } }$ day of incubation. Control (A) LTBBF-PL4 $( 2 1 . 7 3 \\%$ inhibition); (B) LTBBF-PL9 $( 1 8 . 1 8 \\%$ inhibition).

The visual representation in Figure 2 clearly shows a reduced colony size of S. brasiliensis when co-cultured with P. lilacinum isolates (LTBBF-PL4 and LTBBF-PL9) compared to the S. brasiliensis control plate (A), confirming the inhibitory effect.

The interaction between P. lilacinum and S. brasiliensis was monitored for up to 28 days. This extended observation revealed a pattern classified as overgrowth by antagonist (Porter, 1924 apud Skidmore and Dickinson, 1976). This means that while S. brasiliensis growth was inhibited, the P. lilacinum colony eventually grew over and engulfed the S. brasiliensis colony, especially by the $28^\text{th}$ day of incubation. On day 7, no physical overlap was observed, suggesting that initial inhibition might be due to diffusible metabolites rather than direct mycoparasitism.

The following figure from the original paper (Figure 3) further illustrates this overgrowth phenomenon:

该图像是图表，展示了 S. brasiliensis 的对照组与在 P. lilacinum 存在下的成长情况。图中箭头标示出两组样本中 S. brasiliensis 的生长差异，右侧样本显示出 P. lilacinum 对 S. brasiliensis 的抑制作用。

FIGURE 3 on the $2 8 ^ { \mathrm { t h } }$

Analysis: The results strongly support the hypothesis that saprophytic fungi present in the soil can exert antagonistic effects on pathogenic fungi like S. brasiliensis. The inability to measure PIRG for A. versicolor and M. anisoplae due to their extremely rapid growth suggests they are also strong competitors, possibly even more so than P. lilacinum under the tested conditions. The consistent inhibition by P. lilacinum isolates, despite variations in PIRG percentage, confirms its role as a potential biocontrol agent or source of antifungal compounds. The overgrowth by antagonist pattern indicates a robust competitive strategy, where P. lilacinum not only inhibits but also physically dominates S. brasiliensis over time. This antagonism could be mediated by rapid nutrient consumption, space occupation, or the production of secondary metabolites (as suggested by Liu et al., 2020, and others who identified leucinostatins and paecilotoxins from P. lilacinum). This finding offers a plausible explanation for the difficulty in isolating Sporothrix spp. from environmental samples; faster-growing antagonistic fungi may simply outcompete and suppress Sporothrix on culture media.

7. Conclusion & Reflections

7.1. Conclusion Summary

This study successfully confirmed the persistent challenge of isolating Sporothrix spp. from environmental soil samples in sporotrichosis endemic areas of Rio de Janeiro, Brazil. Despite extensive efforts, no Sporothrix isolates were obtained. However, the research provided valuable insights by identifying other prevalent saprophytic fungal species in these soils, with Purpureocillium lilacinum being the most frequently isolated. A significant finding was the demonstration of in vitro antagonistic activity of ten P. lilacinum isolates against Sporothrix brasiliensis, the primary causative agent of sporotrichosis in the region. This antagonism was quantified by Percentage Inhibition of Radial Growth (PIRG), which ranged from 8% to 25%, and characterized by an overgrowth by antagonist pattern. The findings suggest that fast-growing fungal species capable of producing metabolites likely play a role in hindering the growth and culturable isolation of Sporothrix spp. in the environment. This work not only sheds light on the environmental ecology of Sporothrix but also opens a promising avenue for bioprospecting for secondary metabolites from P. lilacinum with potential antifungal activity against pathogenic fungi.

7.2. Limitations & Future Work

The authors acknowledged a key limitation of their study:

• Lack of Molecular Tools: The initial aim was to isolate Sporothrix spp., but the lack of molecular tools to detect Sporothrix DNA in the soil samples (even if not culturable) constitutes a deficiency. This means they could not confirm the presence of Sporothrix at a molecular level in the same samples from which they failed to culture it, which would have strengthened the argument for antagonism.

  Based on their findings and limitations, the authors suggested a clear direction for future research:

• Identification of Secondary Metabolites: The study "opens the way for the identification of secondary metabolites with biological activity that could be tested against pathogenic fungi." This involves the isolation, purification, and chemical characterization of compounds produced by P. lilacinum that are responsible for its antagonistic effect.

7.3. Personal Insights & Critique

This paper provides valuable insights into the complex microbial interactions occurring in natural environments and offers a compelling hypothesis for the long-standing difficulty in isolating Sporothrix spp..

• Inspiration for Ecological Understanding: The shift from simply reporting a failure to isolate Sporothrix to actively investigating the reasons for this failure by studying antagonistic interactions is a commendable methodological approach. It highlights the importance of understanding ecological dynamics rather than focusing solely on the target pathogen. This holistic view of the microbiome is crucial for understanding disease ecology.

• Potential for Bioprospecting: The identification of P. lilacinum as a significant antagonist is particularly inspiring. Given P. lilacinum's known capacity to produce diverse secondary metabolites (e.g., leucinostatins, paecilotoxins) and its established use as a biocontrol agent against nematodes and insects, its potential as a source of novel antifungal agents against Sporothrix is very promising. This could lead to new therapeutic strategies or environmental control methods for sporotrichosis.

• Complementary Methodologies: The paper implicitly reinforces the need for complementary methodologies in environmental mycology. While culture-based methods are essential for isolating viable organisms and studying phenotypic characteristics and interactions, molecular techniques (e.g., PCR, metagenomics) are crucial for detecting the presence of non-culturable or low-abundance organisms like Sporothrix spp. in complex environments. Integrating both would provide a more complete picture.

• Areas for Improvement / Unverified Assumptions:

  • In Vitro vs. In Situ: The study's in vitro nature is a limitation. While dual culture assays are excellent for screening antagonistic potential, environmental conditions are far more complex. Factors like soil composition, moisture, temperature fluctuations, pH, and the presence of a wider array of microorganisms can significantly alter fungal interactions in situ. Further studies should explore these interactions in more complex, environmentally relevant models (e.g., soil microcosms).
  • Specificity of Antagonism: While P. lilacinum showed antagonism against S. brasiliensis type strain CBS120339, it would be beneficial to test a wider range of Sporothrix brasiliensis clinical and environmental isolates to confirm the generalizability of this antagonistic effect.
  • Mechanisms of Antagonism: The paper suggests metabolite production and fast growth as mechanisms. Future work could directly investigate these:
    • Volatile and Diffusible Compounds: Performing assays with cell-free filtrates or volatile organic compounds (VOCs) from P. lilacinum cultures could isolate the effects of metabolites.
    • Nutrient Competition: More detailed studies on resource utilization by both fungi could elucidate the role of competitive exclusion.
    • Enzymatic Activity: Mycoparasitism could involve enzymes that degrade Sporothrix cell walls; investigating these could provide further insights.

• Transferability: The methods and conclusions could be transferred to other domains facing similar challenges in isolating specific pathogens from complex environments. For instance, understanding antagonistic fungal interactions could be relevant for plant pathology (e.g., biocontrol of soil-borne plant pathogens) or veterinary mycology in other environmental reservoirs.

  Overall, this paper provides a robust foundation for future research, shifting the narrative from Sporothrix's elusive environmental presence to the intriguing ecological forces that might govern it.