Inhibitory potential of bioactive extracts from southern Brazil mushrooms on the pathogenic oomycete Pythium insidiosum

Potencial inibitório de extratos bioativos de cogumelos do sul do Brasil sobre o oomiceto patogênico Pythium insidiosum

ABSTRACT:

Pythium insidiosum is an important oomycete pathogen of mammals that causes pythiosis, an endemic disease in warm climates that stands out for its unfavorable prognosis, lethality in the affected species, and difficulties in treatment. This study evaluated in vitro anti-P. insidiosum potential of aqueous, hydroethanolic, and ethanolic extracts of indigenous wild mushrooms from southern Brazil. The extracts were prepared from Amanita gemmata, Amanita muscaria, Auricularia auricula, Gymnopilus junonius, Lactarius deliciosus, Laccaria laccata, Psilocybe cubensis, and Russula xerampelina. In vitro susceptibility assays employed the microdilution technique according to the M38-A2 protocol CLSI. The hydroethanolic and ethanolic extracts of R. xerampelina showed anti-P. insidiosum activity at minimum inhibitory concentrations ranging from 1.87-7.50 mg/mL. The other mushroom species extracts showed no inhibitory effects on growth of P. insidiosum. This is the first study to evaluate the antimicrobial activity of mushrooms on oomycetes, evidencing the antimicrobial potential of R. xerampelina on the pathogen P. insidiosum. So, the present study expands new perspectives, since the secondary metabolites produced by mushrooms can be potential targets for the development of new categories of medicines. However, considering the wide biodiversity of Brazilian mushrooms, we suggested that the search for other basidiomycetes species with anti-P. insidiosum action needs to be expanded.

Key words:
antimicrobial; susceptibility; Oomycota; pythiosis; fungi; basidiomycetes

RESUMO:

Pythium insidiosum é um importante oomiceto patógeno de mamíferos causador da pitiose, uma doença endêmica em climas quentes e que se destaca pelo prognóstico desfavorável, letalidade nas espécies afetadas e dificuldades no tratamento. Este estudo avaliou in vitro o potencial anti-P. insidiosum dos extratos aquosos, hidroetanólicos e etanólicos de cogumelos silvestres do sul do Brasil. Os extratos foram preparados a partir de Amanita gemmata, Amanita muscaria, Auricularia auricula, Gymnopilus junonius, Lactarius deliciosus, Laccaria laccata, Psilocybe cubensis e Russula xerampelina. Os ensaios de suscetibilidade in vitro empregaram a técnica de microdiluição em caldo de acordo com o protocolo M38-A2, CLSI. Os extratos hidroetanólico e etanólico de R. xerampelina apresentaram atividade anti-P. insidiosum em concentrações inibitórias mínimas que variaram de 1,87-7,50 mg/mL. Os extratos das demais espécies de cogumelos não apresentaram efeitos inibitórios sobre o crescimento de P. insidiosum. Este é o primeiro estudo a avaliar a atividade antimicrobiana de cogumelos sobre oomicetos, evidenciando o potencial antimicrobiano de R. xerampelina sobre o patógeno P. insidiosum. Assim, o presente estudo amplia novas perspectivas, uma vez que os metabólitos secundários produzidos pelos cogumelos podem ser alvos potenciais para o desenvolvimento de novas categorias de fármacos. Porém, levando em consideração a ampla biodiversidade de cogumelos brasileiros, sugere-se que a busca por outras espécies de basidiomicetos com atividade anti-P. insidiosum precisa ser ampliada.

Palavras-chave:
antimicrobiano; suscetibilidade; Oomycota; pitiose; fungos; basidiomicetos

Pythium insidiosum is an aquatic oomycete and the primary causative agent of pythiosis, a relevant disease with an unfavorable prognosis that affects domestic animals (horses, dogs, cattle, cats, goats, and sheep), wild animals (birds and wild mammals) and humans (YOLANDA & KRAJAEJUN, 2022). In the last decade, other Pythium species, including Pythium aphanidermatum and Pythium periculosum, have been described as affecting mammalian hosts (THONGSUK et al., 2021; MIRAGLIA et al., 2022). The disease has been mainly reported in the Americas, some European countries, Southeast Asia, Oceania, and Africa (YOLANDA & KRAJAEJUN, 2022). In Brazil and Thailand, pythiosis is endemic in horses and humans, respectively (SOUTO et al., 2021; YOLANDA & KRAJAEJUN, 2022).

The main clinical manifestations in affected animals are characterized by the development of cutaneous, subcutaneous and gastrointestinal lesions, and in humans, the disease occurs in ocular, subcutaneous and systemic forms (YOLANDA & KRAJAEJUN, 2022). Early detection of the disease and quick treatment are crucial for an effective clinical cure. The currently available treatments for pythiosis in animals and humans include surgery, immunotherapy and different classes of antimicrobial drugs (PEREIRA et al., 2013; YOLANDA & KRAJAEJUN, 2021; PERMPALUNG et al., 2020; YOLANDA & KRAJAEJUN, 2022; MEDHASI et al., 2022).

Nevertheless, treating pythiosis is challenging because conventional drugs are not usually effective, particularly antifungal drugs that act on the ergosterol of the cytoplasmic membrane of fungi, which is absent in oomycetes (MEDHASI et al., 2022). Thus, the perspectives of managing pythiosis, in most cases, are associated with combinations of different forms of treatment (PEREIRA et al., 2013); however, standardized therapeutic protocols are non-existent and the results of currently used treatments are not constantly satisfactory. In this sense, it is crucial to seek new compounds that can contribute for treating pythiosis.

The phylum Basidiomycota is a large group of fungi comprising more than 30,000 species, of which about 15,000 are known. Approximately 2,000 species of mushrooms are used for human consumption and around 700 have medicinal properties (SANTOS & CARVALHO, 2021). The secondary metabolites produced by Agaricomycetes mushrooms, such as terpenoids, flavonoids, tannins, alkaloids, and polysaccharides, are of great importance in the pharmaceutical industry, which may be potential targets for developing novel drug categories. Such bioactive compounds confer several biological activities, including antitumor, anti-inflammatory, antioxidant, immunomodulatory, antiparasitic, antimicrobial (antibacterial, antifungal, antiviral), neuroprotective, and cardioprotective effect (VAMANU, 2018; GEBREYOHANNES et al., 2019; ROSA et al., 2020; SANTOS & CARVALHO, 2021; VOLCÃO et al., 2021a; ABDELSHAFY et al., 2022).

Although, 22 mushroom taxa have been studied for different biological activities (SANTOS & CARVALHO, 2021), no research has been carried out to evaluate the activity of mushroom extracts on pathogenic oomycetes. Thus, this study investigated the potential of different extracts of the macrofungi Amanita gemmata, Amanita muscaria, Auricularia auricula, Gymnopilus junonius, Lactarius deliciosus, Laccaria laccata, Psilocybe cubensis, and Russula xerampelina on the pathogen oomycete P. insidiosum.

The reproductive structure, including pileus and stipe of macrofungi Amanita gemmata, Amanita muscaria, Auricularia auricula, Gymnopilus junonius, Lactarius deliciosus, Laccaria laccata, Psilocybe cubensis, and Russula xerampelina (SISGEN A487273) were collected in three vegetated areas in the cities of Capão do Leão (31º45’48”S 52º29’02”W) and Pelotas (31º46’19”S 52º20’33”W), located in Rio Grande do Sul State, in the south of Brazil, during autumn, winter and spring. Two areas were characterized by coniferous woodland and small trees, predominately Pinus spp. species, and one area contains physiognomic restinga vegetation types, including sandy restinga and peat restinga forests.

After collection, the macrofungi were identified according to macro- and micromorphological characteristics. Subsequently, the mushrooms were aliquoted, packed in brown paper envelopes, and transferred to an oven at 50 ºC for 96 h for basidiocarp dehydration. Subsequently, they were stored in hermetically sealed containers and protected from light and humidity until extract production (VOLCÃO et al., 2021a; VOLCÃO et al., 2021b).

The aqueous, ethanolic and hydroethanolic extracts were produced from the macrofungi dehydrated and triturated by maceration. The aqueous extracts (AQE) were produced for all macrofungi evaluated by diluting each crushed mushroom (25 g) in distilled water (100 mL). The hydroethanolic extracts (HEE) were prepared for A. auricula, L. deliciosus, L. laccata, and R. xerampelina by adding the crushed mushrooms (25 g) to 50% ethanol (100 mL). To prepare the ethanolic extracts (EE), 25 g of crushed mushrooms, including A. gemmata, A. auricula, L. deliciosus, L. laccata, and R. xerampelina, were added to 95% ethanol solution (100 mL). The solutions were then incubated in an ultrasonic bath (SB-5200 DTDN Ultrasonic Cleaner) at 40 ºC for 120 min to extract the compounds present in the macrofungi. The suspensions were filtered on Whatman^® filter paper no. 1 to eliminate the particulate matter. The suspensions were stored in Falcon tubes in a freezer at -20 ºC (VOLCÃO et al., 2021a, 2021b). All extracts from mushrooms evaluated were produced by the Laboratório de Biologia, Ecologia e Aplicação de Fungos at Universidade Federal de Pelotas (UFPel).

Eleven P. insidiosum isolates (SISGEN A139392), including nine from pythiosis in horses and dogs and two standard strains (CBS702.83 and CBS777.84) belonging to the Laboratório de Micologia (LABMICO), Departamento de Microbiologia and Parasitologia at UFPel, were evaluated. The clinical isolates were identified by their macro and micromorphological characteristics and molecularly confirmed, as previously described by AZEVEDO et al. (2012).

All P. insidiosum inocula were obtained using the zoosporogenesis process described by Ianiski et al. (2021). In brief, P. insidiosum isolates previously grown on yeast extract agar were transferred to Petri plates containing V8 agar with fragments of sterile grass (Paspalum notatum) and incubated at 37 ºC for 3 days. The grass fragments containing P. insidiosum mycelium were then transferred to Petri plates containing induction medium (20 mL) and incubated at 37 ºC for 12-24 h. After this period, the plates were incubated at 37 ºC at 3000 rpm for 5 min. The free zoospores in the induction medium were counted in a Neubauer chamber under light microscopy (100 and 400 ×). The induction medium, initially containing 30,000 zoospores/mL for each P. insidiosum isolate, underwent a dilution (1:10) in RPMI 1640 broth, adjusted to a pH of 7.0. The susceptibility profile was assessed following the CLSI M38-A2 broth microdilution method (CLSI, 2008).

For testing, the extracts were diluted in RPMI 1640 glucose buffered at pH 7.0 with 0.165 M MOPS in a 1:1 ratio to form the stock solutions. The concentrations of the extracts in the wells ranged from 0.01 to 7.50 mg/mL. Aliquots of 100μL of these dilutions were dispensed sequentially into the microplates, filling the wells belonging to columns numbered one to ten. A volume of 100 μL of the inoculum was dispensed to these columns. Positive (100 µL of RPMI and 100 µL of inoculum) and negative (100 µL of RPMI and 100 µL of the extract) control columns were used for each test. Additionally, a plate containing ethanol + inoculum was prepared as control for all tests. The plates were incubated at 40 rpm shaking at 37 °C for 48 h; all tests were performed in quadruplicate. Readings were performed visually and considered the growth of hyphae. The lowest concentration of the extracts capable of inhibiting P. insidiosum growth was identified as the minimum inhibitory concentration (MIC). The concentrations capable of inhibiting 50% and 90% of the isolates were called MIC₅₀ and MIC₉₀, respectively. Concentrations above the MIC were used to determine the minimum oomicidal concentration (MOC). For this purpose, 100 μL of the dilution was transferred to tubes containing 900 μL of Sabouraud broth and incubated at 37°C for 48h. The lowest concentration of the extract that did not show hyphae growth was considered the MOC.

Among the different mushroom extracts evaluated, only the HEE and EE of R. xerampelina showed anti-P. insidiosum activity, with the MIC ranging from 1.87 to 7.50 mg/mL for both extracts. The values obtained in the MIC₅₀ and MIC₉₀ were 7.50 and 1.87 mg/mL, respectively. The MOC of the HEE and EE was equal to the MIC. The other macrofungal extracts showed no inhibitory effect on P. insidiosum at the concentrations evaluated (Table 1).

Table 1
In vitro susceptibility of Pythium insidiosum (n = 11) to aqueous (AQE), hydroethanolic (HEE) and ethanolic extracts (EE) of southern Brazil mushrooms Amanita gemmata, Amanita muscaria, Auricularia auricula, Gymnopilus junonius, Lactarius deliciosus, Laccaria laccata, Psilocybe cubensis and Russula xerampelina.

--------------------------------------------Number and percentages (%) of isolates in each extract concentration-------------------------------------
Mushrooms Extracts	7.50	3.75	1.87	0.93-0.01	MIC50	MIC90
R. xerampelina HEE	5 (45.45%)	4 (36.37%)	2 (18.18%)	+	3.75	1.87
R. xerampelina EE	7 (63.63%)	2 (18.18%)	2 (18.18%)	+	7.50	1.87
R. xerampelina AQE	+	+	+	+	ND	ND
A. gemmata AQE, EE	+	+	+	+	ND	ND
A. muscaria AQE	+	+	+	+	ND	ND
A. auriculata AQE, HEE, EE	+	+	+	+	ND	ND
G. junonius AQE	+	+	+	+	ND	ND
L. deliciosus AQE, HEE, EE	+	+	+	+	ND	ND
L. laccata AQE, HEE, EE	+	+	+	+	ND	ND
P. cubensis AQE	+	+	+	+	ND	ND

• MIC: minimum inhibitory concentration; MIC₅₀: minimum concentration for 50% growth inhibition of the isolates; MIC₉₀: minimum concentration for 90% growth inhibition of the isolates; (+) All P. insidiosum isolates (n = 11) grew at this concentration. ND: not determined.

The severity and lethality of infections caused by P. insidiosum in humans and animals and challenges in treating the disease have driven research to expand the therapeutic alternatives and arsenal of antimicrobial compounds with anti-P. insidiosum activity (PERMPALUNG et al, 2020; VALENTE et al., 2020; IANISKI et al., 2021; SILVEIRA et al., 2022; MEDHASI et al., 2022; BRAGA et al., 2023). In this sense, this research contributes with these previous studies by demonstrating for the first time the anti-P. insidiosum action of the Brazilian mushroom R. xerampelina.

Although, interest in food and medicinal effects of mushrooms has grown in Americas in last decades, it is noted that few species of mushrooms from Rio Grande do Sul, the southernmost state in Brazil, have been collected, as well as their biological properties have not been fully evaluated (VOLCÃO et al., 2021a). Interestingly, in this region the pampa biome predominates, a very old natural ecosystem, where there are natural fields, composed of grasses, fragmented and gallery forests. In this biome, some Pinus spp. and Eucalyptus spp. constitute important associations with different species of basidiomycetes (VOLCÃO et al., 2021a). Thus, research is required to study the diversity and medicinal potential of wild mushrooms in this region of Brazil. In order to fill this gap, the present study sought to evaluate the potential of mushrooms from the pampa biome on an oomycete species.

Few studies have evaluated the antimicrobial activity of R. xerampelina. However, VOLCÃO et al. (2021b) studied the antioxidant activity of the HEE of R. xerampelina utilized in this study and determined the total flavonoid and phenol contents. They found that the phenolic acid concentration was 3.0 mg GAE/g (microgram of gallic acid equivalent per gram of extract), while the flavonoid concentration was 1098.33 mg CAE/g (microgram of catechin equivalent per gram of extract). Additionally, the authors found that this extract had antimicrobial effects on Pseudomonas aeruginosa, with MIC of 2.5 mg/mL, without cytotoxic effects on Vero cells. It is believed that the probable anti-P. insidiosum action of R. xerampelina is due to the presence of these bioactive compounds. Nevertheless, further research is required to verify which compounds present antimicrobial activity on this oomycete.

According previous studies, the antimicrobial activity of macrofungi is closely related to the antioxidant effects of bioactive substances, such as phenolic compounds (VAMANU, 2018; ABDELSHAFY et al., 2022). However, the chemical composition of macrofungal extracts and the content of bioactive substances can be influenced by various factors, including solvent type and temperature used for extraction, the structure (pileus, stipe, or both) used for extraction, mushroom maturity and geographical location of the collected mushroom specimen (VOLCÃO et al., 2021b).

Nonetheless, the other species of mushrooms evaluated in this study, except for R. xerampelina, did not show any anti-P. insidiosum activity. However, a previous study evaluated the antimicrobial action of 35 different macrofungi and reported that only three genera had antibacterial and antifungal activity (GEBREYOHANNES et al., 2019). In this sense, our study evaluated three extracts of eight indigenous wild mushrooms species and we reported one species with anti-P. insidiosum activity. It is important to point out that previous studies evaluating the antimicrobial potential of mushroom extracts were carried out on bacteria and fungi (VAMANU, 2018; GEBREYOHANNES et al., 2019; ROSA et al., 2020; VOLCÃO et al., 2021a, 2021b). The differential of our study was the pioneering research into the inhibitory action of basidiomycetes against the mammalian pathogen oomycete. However, considering the wide biodiversity of Brazilian mushrooms, we suggest that the search for other basidiomycetes with potential to inhibit the growth this important fungal-like microorganism needs to be augmented.

This research represents a preliminary and pioneering investigation into the anti-P. insidiosum potential of indigenous wild mushrooms from southern Brazil. The antimicrobial activity of ethanolic and hydroethanolic extracts of R. xerampelina was demonstrated. Taking into account the wide biodiversity of Brazilian mushrooms, as future perspectives we proposed to expand the search for other species of basidiomycetes with anti-P. insidisoum activity. Furthermore, the bioactive compounds produced by Agaricomycetes mushrooms are potential targets for developing novel drug categories.

ACKNOWLEDGEMENTS

This research was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) - Finance Code 001 and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for student and researcher scholarships.

REFERENCES

• ABDELSHAFY, A. M. et al. A comprehensive review on phenolic compounds from edible mushrooms: Occurrence, biological activity, application and future prospective. Critical Reviews in Food Science and Nutrition, v.62, n.22, p.6204-6224, 2022. Available from: <Available from: https://pubmed.ncbi.nlm.nih.gov/33729055 >. Accessed: Mar. 20, 2023. doi: 10.3390/jof8020182.
  » https://doi.org/10.3390/jof8020182.» https://pubmed.ncbi.nlm.nih.gov/33729055
• AZEVEDO, M. I. et al. Phylogenetic relationships of Brazilian isolates of Pythium insidiosum based on ITS rDNA and cytochrome oxidase II gene sequences. Veterinary Microbiology, v.159, n.1-2, p.141-148, 2012. Available from <Available from https://pubmed.ncbi.nlm.nih.gov/22483240/ >. Accessed: Mar. 21, 2023. doi: 10.1016/j.vetmic.2012.03.030.
  » https://doi.org/10.1016/j.vetmic.2012.03.030.» https://pubmed.ncbi.nlm.nih.gov/22483240/
• BRAGA, C. Q. et al. In vitro and ex vivo anti-Pythium insidiosum potential of ozonated sunfower oil. Brazilian Journal of Microbiology, v.55, n.1, p.867-873. 2023. Available from: <Available from: https://link.springer.com/article/10.1007/s42770-023-01173-1 >. Accessed: Dec. 10, 2023. doi: 10.1007/s42770-023-01173.
  » https://doi.org/10.1007/s42770-023-01173.» https://link.springer.com/article/10.1007/s42770-023-01173-1
• CLINICAL AND LABORATORY STANDARDS INSTITUTE (CLSI). Reference method for broth dilution antifungal susceptibility testing of filamentous fungi: Approved standard. Vol. 28. 2nd ed. M38-A2 CLSI.Wayne: Clinical and Laboratory Standards Institute, 2008, 1-35.
• GEBREYOHANNES, G. et al. Determination of antimicrobial activity of extracts of indigenous wild mushrooms against pathogenic organisms. Evidence-Based Complementary and Alternative Medicine, e6212673, 2019. Available from: <Available from: https://pubmed.ncbi.nlm.nih.gov/30906415/ >. Accessed: Mar. 10, 2023. doi: 10.1155/2019/6212673.
  » https://doi.org/10.1155/2019/6212673.» https://pubmed.ncbi.nlm.nih.gov/30906415/
• IANISKI, L. B. et al. In vitro anti-Pythium insidiosum activity of amorolfine hydrochloride and azithromycin, alone and in combination. Medical Mycology, v.59, n.1, p.67-73. 2021. Available from: <Available from: https://pubmed.ncbi.nlm.nih.gov/32400872/ >. Accessed: Nov. 28, 2023. doi: 10.1093/mmy/myaa032.
  » https://doi.org/10.1093/mmy/myaa032.» https://pubmed.ncbi.nlm.nih.gov/32400872/
• MEDHASI, S. et al. Review: Antimicrobial therapy for human pythiosis. Antibiotics, v.11, n.4, p.450, 2022. Available from: <Available from: https://pubmed.ncbi.nlm.nih.gov/35453202/ >. Accessed: Jan. 08, 2023. doi: 0.3390/antibiotics11040450.
  » https://doi.org/0.3390/antibiotics11040450.» https://pubmed.ncbi.nlm.nih.gov/35453202/
• MIRAGLIA, B. M. et al. Pythium insidiosum complex hides a cryptic novel species: Pythium periculosum Fungal Biology, v.126, n.5, p.366-374, 2022. Available from: <Available from: https://pubmed.ncbi.nlm.nih.gov/35501032 >. Accessed: Jun. 08, 2022. doi: 10.1016/j.funbio.2022.03.002.
  » https://doi.org/10.1016/j.funbio.2022.03.002.» https://pubmed.ncbi.nlm.nih.gov/35501032
• PEREIRA, D. I. B. et al. Canine gastrointestinal pythiosis treatment by combined antifungal and immunotherapy and review of published studies. Mycopathologia, v.176, n.3, p.309-315, 2013. Available from: <Available from: https://pubmed.ncbi.nlm.nih.gov/23918089/ >. Accessed: Mar. 29, 2023. doi: 10.1007/s11046-013-9683-7.
  » https://doi.org/10.1007/s11046-013-9683-7.» https://pubmed.ncbi.nlm.nih.gov/23918089/
• PERMPALUNG, N. et al. Human pythiosis: emergence of fungal-like organism. Mycopathologia, v.185, n.5, p.801-812, 2020. Available from: <Available from: https://pubmed.ncbi.nlm.nih.gov/31845178/ >. Accessed: Mar. 10, 2023. doi: 10.1007/s11046-019-00412-0.
  » https://doi.org/10.1007/s11046-019-00412-0.» https://pubmed.ncbi.nlm.nih.gov/31845178/
• ROSA, G. B. et al. Investigation of nutritional composition, antioxidant compounds, and antimicrobial activity of wild culinary-medicinal mushrooms Boletus edulis and Lactarius deliciosus (Agaricomycetes) from Brazil. International Journal of Medicinal Mushrooms, v.22, n.10, p.931-942, 2020. Available from: <Available from: https://pubmed.ncbi.nlm.nih.gov/33426823/ >. Accessed: Mar. 16, 2023. doi: 10.1615/IntJMedMushrooms.2020036347.
  » https://doi.org/10.1615/IntJMedMushrooms.2020036347.» https://pubmed.ncbi.nlm.nih.gov/33426823/
• SANTOS, G. S.; CARVALHO, C. M. A. Systematic review on bioactivity of Brazilian mushrooms (Agaricomycetes). International Journal of Medicinal Mushrooms, v.23, n.11, p.27-36, 2021. Available from: <Available from: https://pubmed.ncbi.nlm.nih.gov/34936306/ >. Accessed: Mar. 18, 2023. doi: 10.1615/IntJMedMushrooms.2021040410.
  » https://doi.org/10.1615/IntJMedMushrooms.2021040410.» https://pubmed.ncbi.nlm.nih.gov/34936306/
• SILVEIRA, J. S. S. et al. Melaleuca alternifolia formulations in the treatment of experimental pythiosis. Brazilian Journal of Microbiology, v.53, n.2, p.1011-1017, 2022. Available from: <Available from: https://pubmed.ncbi.nlm.nih.gov/35239152/ >. Accessed: Mar. 12, 2023. doi: 10.1007/s42770-022-00720-6.
  » https://doi.org/10.1007/s42770-022-00720-6.» https://pubmed.ncbi.nlm.nih.gov/35239152/
• SOUTO, E. P. F. et al. Pythiosis in Equidae in Northeastern Brazil: 1985-2020. Journal of Equine Veterinary Science, v.105, p.103726, 2021. Available from: <Available from: https://pubmed.ncbi.nlm.nih.gov/34607686/ >. Accessed: Mar. 14, 2023. doi: 10.1016/j.jevs.2021.103726.
  » https://doi.org/10.1016/j.jevs.2021.103726.» https://pubmed.ncbi.nlm.nih.gov/34607686/
• THONGSUK, P. et al. Vascular pythiosis caused by Pythium aphanidermatum: the first case report in Asia. European Journal of Medical Research, v.26, n.1, p.132, 2021. Available from: <Available from: https://pubmed.ncbi.nlm.nih.gov/34775999/ >. Accessed: Mar. 15, 2023. doi: 10.1186/s40001-021-00603-w.
  » https://doi.org/10.1186/s40001-021-00603-w.» https://pubmed.ncbi.nlm.nih.gov/34775999/
• VALENTE, J. S. S. et al. Biogenic silver nanoparticles in the treatment of experimental pythiosis. Medical Mycology, v.58, n.7, p.913-918, 2020. Available from: <Available from: https://pubmed.ncbi.nlm.nih.gov/32030424/ >. Accessed: Mar. 08, 2023. doi: 10.1093/mmy/myz141.
  » https://doi.org/10.1093/mmy/myz141.» https://pubmed.ncbi.nlm.nih.gov/32030424/
• VAMANU, E. Bioactive capacity of some Romanian wild edible mushrooms consumed mainly by local communities. Natural Product Research, v.32, n.4, p.440-443, 2018. Available from: <Available from: https://www.tandfonline.com/doi/full/10.1080/14786419.2017.1308365 >. Accessed: Mar. 03, 2023. doi: 10.1080/14786419.2017.1308365.
  » https://doi.org/10.1080/14786419.2017.1308365.» https://www.tandfonline.com/doi/full/10.1080/14786419.2017.1308365
• VOLCÃO, L. M. et al. Biological activity of aqueous extracts of Southern Brazilian mushrooms. International Journal of Environmental Health Research, v.31, n.2, p.148-159, 2021a. Available from: <Available from: https://pubmed.ncbi.nlm.nih.gov/31257910/ >. Accessed: Mar. 10, 2023. doi: 10.1080/09603123.2019.1634798.
  » https://doi.org/10.1080/09603123.2019.1634798.» https://pubmed.ncbi.nlm.nih.gov/31257910/
• VOLCÃO, L. M. et al. Bioactive extracts of Russula xerampelina and Suillus granulatus in the in vitro control of Pseudomonas aeruginosa phytopathogenic. South African Journal of Botany, v.140, p.218-225, 2021b. Available from: <Available from: https://linkinghub.elsevier.com/retrieve/pii/S0254629921001290 >. Accessed: Mar. 10, 2023. doi: 10.1016/j.sajb.2021.03.043.
  » https://doi.org/10.1016/j.sajb.2021.03.043.» https://linkinghub.elsevier.com/retrieve/pii/S0254629921001290
• YOLANDA, H.; KRAJAEJUN, T. History and perspective of immunotherapy for pythiosis. Vaccines, v.9, p.1080, 2021. Available from: <Available from: https://doi.org/10.3390/vaccines9101080 >. Accessed: Nov. 08, 2023. doi: 10.3390/vaccines9101080.
  » https://doi.org/10.3390/vaccines9101080.» https://doi.org/10.3390/vaccines9101080
• YOLANDA, H.; KRAJAEJUN, T. Global distribution and clinical features of pythiosis in humans and animals. Journal of Fungi, v.8, n.2, p.182, 2022. Available from: <Available from: https://www.mdpi.com/2309-608X/8/2/182 >. Accessed: Mar. 08, 2023. doi: 10.3390/jof8020182.
  » https://doi.org/10.3390/jof8020182.» https://www.mdpi.com/2309-608X/8/2/182

• CR-2023-0528.R1

Edited by

Editor: Rudi Weiblen (0000-0002-1737-9817)

Publication Dates

• Publication in this collection
  08 July 2024
• Date of issue
  2024

History

• Received
  27 Sept 2023
• Accepted
  22 Jan 2024
• Reviewed
  13 May 2024