The Toxic Mechanism of Gliotoxins and Biosynthetic Strategies for Toxicity Prevention.

Gliotoxin is a kind of epipolythiodioxopiperazine derived from different fungi that is characterized by a disulfide bridge. Gliotoxins can be biosynthesized by a gli gene cluster and regulated by a positive GliZ regulator. Gliotoxins show cytotoxic effects via the suppression the function of macrophage immune function, inflammation, antiangiogenesis, DNA damage by ROS production, peroxide damage by the inhibition of various enzymes, and apoptosis through different signal pathways. In the other hand, gliotoxins can also be beneficial with different doses. Low doses of gliotoxin can be used as an antioxidant, in the diagnosis and treatment of HIV, and as an anti-tumor agent in the future. Gliotoxins have also been used in the control of plant pathogens, including Pythium ultimum and Sclerotinia sclerotiorum. Thus, it is important to elucidate the toxic mechanism of gliotoxins. The toxic mechanism of gliotoxins and biosynthetic strategies to reduce the toxicity of gliotoxins and their producing strains are summarized in this review.

Vascular pythiosis caused by Pythium aphanidermatum: the first case report in Asia.

BACKGROUND: Pythium, soil-borne plant pathogens, are in the class Oomycetes. They are not true fungi, but are related to diatom and algae. There are two human pathogens including P. insidiosum and P. aphanidermatum. To date, only one case of pythiosis caused by P. aphanidermatum has been reported. We present herein the first case of P. aphanidermatum vascular pythiosis in Asia. CASE PRESENTATION: A 47-year-old Thai woman, living in North Thailand, with ß thalassemia/hemoglobin E presented with acute recurrent arterial insufficiency of both legs. Emergent embolectomy with clot removal was performed. The pathology of the clot exhibited noncaseous granulomatous inflammation with many fungal hyphal elements. PCR identified P. aphanidermatum with 100% identity. Final diagnosis is vascular pythiosis. Unfortunately, the patient eventually expired after treatment with itraconazole, terbinafine, azithromycin, and doxycycline. CONCLUSIONS: To date, only one case of pythiosis caused by P. aphanidermatum has been reported. We present herein the first case of P. aphanidermatum vascular pythiosis in Asia.

Enhancing the Potentiality of Trichoderma harzianum against Pythium Pathogen of Beans Using Chamomile (Matricaria chamomilla, L.) Flower Extract.

Our present study was designed to investigate the role of both Trichoderma harzianum and chamomile (Matricaria chamomilla L.) flower extract in mutual reaction against growth of Pythium ultimum. In vitro, the activity of chamomile extract was found to reduce the radial growth of Pythium ultimum up to 30% compared to the control. Whereas, the radial growth reduction effect of T. harzianum against P. ultimum reached 81.6% after 120 h. Data also showed the productivity of total phenolics and total flavonoids by T. harzianum, was 12.18 and 6.33 mg QE/100 mL culture filtrate, respectively. However, these compounds were determined in chamomile flower extract at concentrations of 75.33 and 24.29 mg QE/100 mL, respectively. The fractionation of aqueous extract of chamomile flower using HPLC provided several polyphenolic compounds such as pyrogallol, myricetin, rosemarinic acid, catechol, p-coumaric acid, benzoic acid, chlorogenic acid and other minor compounds. In vivo, the potentiality of T. harzianum with chamomile flower extract against Pythium pathogen of bean was investigated. Data obtained showed a reduction in the percentage of rotted seed and infected seedling up to 28 and 8%, respectively. Whereas, the survival increased up to 64% compared to other ones. There was also a significant promotion in growth features, total chlorophyll, carotenoids, total polyphenols and flavonoids, polyphenol-oxidase and peroxidase enzymes compared to other ones. To the best of our knowledge, there are no reported studies that included the mutual association of fungus, T. harzianum with the extract taken from the chamomile flower against P. ultimum, either in vitro or in vivo. In conclusion, the application of both T. harzianum and/or M. chamomilla extracts in the control of bean Pythium pathogen showed significant results.

Antimicrobial peptide (AMP) from Zingiber zerumbet rhizomes with inhibitory effect on Pythium myriotylum secretory proteases and zoospore viability.

Protease mediated proteolysis has been widely implicated in virulence of necrotrophic fungal pathogens. This is counteracted in plants by evolving new and effective antimicrobial peptides (AMP) that constitute important components of innate immune system. Peptide extraction from rhizome of Zingiber zerumbet was optimized using ammonium sulphate (50-80% w/v) and acetone (60 and 100% v/v) with maximal protein recovery of 1.2 ± 0.4 mg/g obtained using 100% acetone. Evaluation of inhibitory potential of Z. zerumbet rhizome protein extract to prominent hydrolases of necrotrophic Pythium myriotylum revealed maximal inhibition of proteases (75.8%) compared to other hydrolytic enzymes. Protein was purified by Sephacryl S200HR resin resulting in twofold purification and protease inhibition of 84.4%. Non-reducing polyacrylamide gel electrophoresis (PAGE) of the fractions yielded two bands of 75 kDa and 25 kDa molecular size. Peptide mass fingerprint of the protein bands using matrix assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectroscopy (MS) and subsequent MASCOT searches revealed peptide match to methylesterase from Arabidopsis thaliana (15%) and to hypothetical protein from Oryza sativa (98%) respectively. Further centrifugal filter purification using Amicon Ultra (10,000 MW cut-off) filter, yielded a prominent band of 25 kDa size. Concentration dependent inhibition of zoospore viability by Z. zerumbet AMP designated as ZzAMP was observed with maximal inhibition of 89.5% at 4 µg protein and an IC50 value of 0.59 µg. Studies are of particular relevance in the context of identifying the molecules involved in imparting below ground defense in Z. zerumbet as well in development of AMPs as potential candidate molecules for control of necrotrophic pathogens of agricultural relevance.

In Vitro Susceptibility of Thai Pythium insidiosum Isolates to Antibacterial Agents.

Human pythiosis is a life-threatening human disease caused by Pythium insidiosum In Thailand, vascular pythiosis is the most common form and carries a mortality rate of 10 to 40%, despite aggressive treatment with radical surgery, antifungal agents, and immunotherapy. Itraconazole and terbinafine have been the mainstay of treatment, until recently, based on case report data showing potential synergistic effects against Brazilian P. insidiosum isolates. However, the synergistic effects of itraconazole and terbinafine against Thai P. insidiosum isolates were not observed. This study tested the in vitro susceptibilities of 27 Thai human P. insidiosum isolates (clade II, n = 17; clade IV, n = 10), 12 Thai environmental P. insidiosum isolates (clade II, n = 4; clade IV, n = 8), and 11 non-Thai animal P. insidiosum isolates (clade I, n = 9; clade II, n = 2) to antibiotics in eight antibacterial classes to evaluate alternative effective treatments. Tetracycline and macrolide antibiotics demonstrated in vitro activity against Thai P. insidiosum isolates, with doxycycline MICs (1 to 16 µg/ml), minocycline MICs (1 to 4 µg/ml), tigecycline MICs (1 to 4 µg/ml), azithromycin MICs (1 to 16 µg/ml), and clarithromycin MICs (0.125 to 8 µg/ml) being the lowest, on average. Synergistic effects of tetracyclines and macrolides were also observed.

Biogenic silver nanoparticles in the treatment of experimental pythiosis Bio-AgNP in pythiosis therapy.

Pythiosis is a rapidly progressing disease that can be lethal to affected individuals due to resistance to available therapeutic protocols. The disease affects mammals, with the largest number of reports in horses and humans. The present study investigated the activity of biogenic silver nanoparticles (bioAgNP) in the treatment of experimental pythiosis. The disease was reproduced in nine female 90-day-old New Zealand rabbits. Animals were divided into three groups: group1 (control, n = 3) daily and topically treated with a nonionized gel-based formulation and 1 ml of sterile distilled water intralesion administered every 48 hours; group 2 (n = 3), daily and topically treated with gel-based formulation containing 1 µg/ml bio-AgNP; group 3 (n = 3), treated with 1 ml bio-AgNP in 1 µg/ml aqueous solution intralesion administered every 48 hours. Animals were treated for 45 days, and the area of subcutaneous lesions was measured every 5 days. Results showed that groups 2 and 3 differed from control group (P < .05) in the lesion area, as well as the amount of hyphae within the lesions. It was observed that lesions of treated animals (groups 2 and 3) did not differ from each other, showing that the application route did not influence the regression of lesions. However, it was observed that one animal from group 2 reached clinical cure at 35 days of treatment. This research is pioneer in the application of nanocomposites for the treatment of experimental pythiosis and showed that bio-AgNP can be powerful allies of integrative medicine and can be included in pythiosis therapeutic protocols.

Inhibition of Rhizoctonia solani RhCh-14 and Pythium ultimum PyFr-14 by Paenibacillus polymyxa NMA1017 and Burkholderia cenocepacia CACua-24: A proposal for biocontrol of phytopathogenic fungi.

Biocontrol has emerged in recent years as an alternative to pesticides. Given the importance of environmental preservation using biocontrol, in this study two antagonistic bacteria against phytopathogenic fungi were isolated and evaluated. These bacterial strains, identified as Paenibacillus polymyxa NMA1017 and Burkholderia cenocepacia CACua-24, inhibited (70 to 80%) the development of two phytopathogens of economic importance: the fungus Rhizoctonia solani RhCh-14, isolated from chili pepper, and the oomycete Pythium ultimum PyFr-14, isolated from tomato. The spectrum was not limited to the previous pathogens, but also to other phytopathogenic fungus, some bacteria and other oomycetes. Fungi-bacteria microcultures observed with optical and scanning electron microscopy revealed hyphae disintegration and pores formation. The antifungal activity was found also in the supernatant, suggesting a diffusible compound is present. Innocuous tests on tobacco leaves, blood agar, bean seed germination and in Galleria mellonella larvae showed that strain NMA1017 has the potential to be a biocontrol agent. Greenhouse experiments with bean plants inoculated with P. polymyxa exhibited the efficacy to inhibit the growth of R. solani and P. ultimum. Furthermore, P. polymyxa NMA1017 showed plant growth promotion activities, such as siderophore synthesis and nitrogen fixation which can contribute to the crop development.

Human Pythiosis: Emergence of Fungal-Like Organism.

Pythiosis is an emerging infectious disease caused by the aquatic oomycete Pythium insidiosum, a fungal-like organism. It is believed that P. insidiosum's zoospores, its infected form, play major role in pathogenesis. Vascular and ocular infections are the most common clinical manifestation in humans. It is difficult to establish the diagnosis given its relatively rarity and difficulty to distinguish P. insidiosum from other molds. Delay in diagnosis and treatment has been associated with poor outcomes. High index of suspicion is the key, particularly in thalassemia patients with arterial insufficiency and patients with fungal keratitis/endophthalmitis without improvement on antifungal therapy. Tissue culture and zoospore induction remain gold standard for diagnosis; however, DNA-based method should be performed simultaneously. The combination of radical surgery, antifungal agents, and immunotherapy has been recommended. It was previously believed that surgery with negative surgical margins was the essential to survive in vascular pythiosis; however, it was recently found that patients could have residual disease despite documented negative surgical margins as infected clot may be dislodged to proximal arterial sites prior to surgery. Serum ß-D-glucan (BG) has been used to monitor disease response after treatment initiation in vascular pythiosis. A significant decrease in BG levels within 2 weeks after surgery is indicative of the absence of residual infection. Unfortunately, monitoring tools for ocular pythiosis are not yet available. Itraconazole plus terbinafine have generally been used in P. insidiosum-infected patients; however, antibacterial agents, including azithromycin and linezolid, have also been used with favorable outcomes in ocular disease. Recently, azithromycin or clarithromycin plus doxycyclin were used in two relapsed vascular pythiosis patients with good outcomes.

Adjunctive antibacterial agents as a salvage therapy in relapsed vascular pythiosis patients.

Human vascular pythiosis is a life-threatening condition caused by Pythium insidiosum. Patients with unresectable intra-abdominal artery involvement have not previously survived, despite being treated with antifungal agents and immunotherapy. We report two novel cases of intra-abdominal pythiosis in patients for whom surgery could not be performed, who were successfully treated with adjunctive antibacterial agents.

The Repurposed Drug Disulfiram Inhibits Urease and Aldehyde Dehydrogenase and Prevents In Vitro Growth of the Oomycete Pythium insidiosum.

Pythium insidiosum is an oomycete microorganism that causes a life-threatening infectious disease, called pythiosis, in humans and animals. The disease has been increasingly reported worldwide. Conventional antifungal drugs are ineffective against P. insidiosum Treatment of pythiosis requires the extensive removal of infected tissue (i.e., eye and leg), but inadequate surgery and recurrent infection often occur. A more effective treatment is needed for pythiosis patients. Drug repurposing is a promising strategy for the identification of a U.S. Food and Drug Administration-approved drug for the control of P. insidiosum Disulfiram has been approved to treat alcoholism, but it exhibits antimicrobial activity against various pathogens. In this study, we explored whether disulfiram possesses an anti-P. insidiosum activity. A total of 27 P. insidiosum strains, isolated from various hosts and geographic areas, were susceptible to disulfiram in a dose-dependent manner. The MIC range of disulfiram against P. insidiosum (8 to 32 mg/liter) was in line with that of other pathogens. Proteogenomic analysis indicated that several potential targets of disulfiram (i.e., aldehyde dehydrogenase and urease) were present in P. insidiosum By homology modeling and molecular docking, disulfiram can bind the putative aldehyde dehydrogenase and urease of P. insidiosum at low energies (i.e., -6.1 and -4.0 Kcal/mol, respectively). Disulfiram diminished the biochemical activities of these enzymes. In conclusion, disulfiram can inhibit the growth of many pathogenic microorganisms, including P. insidiosum The drug can bind and inactivate multiple proteins of P. insidiosum, which may contribute to its broad antimicrobial property. Drug repurposing of disulfiram could be a new treatment option for pythiosis.

Convergent Evolution of C239S Mutation in Pythium spp. ß-Tubulin Coincides with Inherent Insensitivity to Ethaboxam and Implications for Other Peronosporalean Oomycetes.

Ethaboxam is a benzamide antioomycete chemical (oomicide) used in corn and soybean seed treatments. Benzamides are hypothesized to bind to ß-tubulin, thus disrupting microtubule assembly. Recently, there have been reports of corn- and soybean-associated oomycetes that are insensitive to ethaboxam despite never having been exposed. Here, we investigate the evolutionary history and molecular mechanism of ethaboxam insensitivity. We tested the sensitivity of 194 isolates representing 83 species across four oomycete genera in the Peronosporalean lineage that were never exposed to ethaboxam. In all, 84% of isolates were sensitive to ethaboxam (effective concentration to reduce optical density at 600 nm by 50% when compared with the nonamended control [EC50] < 5 µg ml-1), whereas 16% were insensitive (EC50 > 11 µg ml-1). Of the insensitive isolates, two different transversion mutations were present in the 239th codon in ß-tubulin within three monophyletic groups of Pythium spp. The transversion mutations lead to the same amino acid change from an ancestral cysteine to serine (C239S), which coincides with ethaboxam insensitivity. In a treated soybean seed virulence assay, disease severity was not reduced on ethaboxam-treated seed for an isolate of Pythium aphanidermatum containing a S239 but was reduced for an isolate of P. irregulare containing a C239. We queried publicly available ß-tubulin sequences from other oomycetes in the Peronosporalean lineage to search for C239S mutations from other species not represented in our collection. This search resulted in other taxa that were either homozygous or heterozygous for C239S, including all available species within the genus Peronospora. Evidence presented herein supports the hypothesis that the convergent evolution of C239S within Peronosporalean oomycetes occurred without selection from ethaboxam yet confers insensitivity. We propose several evolutionary hypotheses for the repeated evolution of the C239S mutation.

Monitoring Anti-Pythium insidiosum IgG Antibodies and (1→3)-ß-d-Glucan in Vascular Pythiosis.

Despite aggressive treatment, vascular pythiosis has a mortality rate of 40%. This is due to delays in diagnosis and a lack of effective monitoring tools. To overcome this drawback, serum beta-d-glucan (BG) and P. insidiosum-specific antibody (Pi-Ab) were examined as potential monitoring markers in vascular pythiosis. A prospective cohort study of vascular pythiosis patients was carried out from January 2010 to July 2016. Clinical information and blood samples were collected and evaluated by the BG and Pi-Ab assays. Linear mixed-effect models were used to compare BG and Pi-Ab levels. The in vitro susceptibility test was performed with all P. insidiosum isolates from culture-positive cases. A total of 50 patients were enrolled: 45 survived and 5 died during follow-up. The survivors had a significantly shorter time to medical care (P < 0.0001) and a significantly shorter waiting time to the first surgery (P < 0.0001). There were no differences in BG levels among the groups at diagnosis (P = 0.33); however, BG levels among survivors were significantly lower than those of the deceased group at 0.5 months (P < 0.0001) and became undetectable after 3 months. Survivors were able to maintain an enzyme-linked immunosorbent assay (ELISA) value (EV) of Pi-Ab above 8, whereas the EV among deceased patients was less than 4. In vitro susceptibility results revealed no synergistic effects between itraconazole and terbinafine. This study showed that BG and Pi-Ab are potentially valuable markers to monitor the disease after treatment initiation. An unchanged BG level at 2 weeks after surgery should prompt an evaluation for residual disease.

Antibiotic-producing Pseudomonas fluorescens mediates rhizome rot disease resistance and promotes plant growth in turmeric plants.

Rhizome rot of turmeric caused by Pythium aphanidermatum is a major threat to turmeric-cultivating areas of India. This study intends to evaluate the performance of fluorescent pseudomonads against Rhizome rot disease and understand the resistance mechanism in Turmeric plants. Fluorescent pseudomonads were screened against Pythium aphanidermatum using dual culture. Selected strains were evaluated for the performance of growth promoting attributes and the presence of antibiotic genes through PCR analysis. Strain FP7 recorded the maximum percent inhibition of P. aphanidermatum under in vitro conditions. Strains FP7 and TPF54 both increased plant growth in turmeric plants in vitro. Strain FP7 alone contained all the evaluated antibiotic biosynthetic genes. Talc and liquid-based formulations were prepared with effective strain and tested for its biocontrol activities under both glasshouse and field conditions. Enzymatic activities of the induced defense enzymes such as PO, PPO, PAL, CAT and SOD were estimated and subjected to spectrophotometric analysis. A combination of rhizome dip and soil drench of FP7 liquid formulation treatment remarkably recorded the minimum disease incidence, higher defense enzymes, maximum plant growth and yield under glasshouse and field conditions. Application of strain FP7 increased the defense molecules, plant growth and yield in turmeric plants thereby reducing the incidence of rhizome rot disease. Moreover, this study has a potential to be adopted for sustainable and eco-friendly turmeric production.

Leap forward in the treatment of Pythium insidiosum keratitis.

BACKGROUND: Pythium insidiosum is a parafungus that causes keratitis resembling fungal keratitis. This study compares outcome in a large cohort of patients with P insidiosum keratitis treated with antifungal drugs, to a pilot group treated with antibacterial antibiotics. METHODS: Between January 2014 and December 2016, 114 patients with culture positive P insidiosum keratitis were included in the study. A subset of culture isolates was tested in vitro for response to nine antibacterial antibiotics by disc diffusion and E test. Patients were treated with topical natamycin in 2014, 2015 and up until mid 2016. Thereafter, the patients received a combination of topical linezolid and topical and oral azithromycin. Therapeutic penetrating keratoplasty (TPK) was done for patients not responding to medical therapy. RESULTS: In vitro disc diffusion assay showed linezolid to be most effective. The rate of TPK was significantly higher in 2015 compared with 2016 (43/45, 95.6% vs 22/32, 68.8%; p=0.002). Eighteen patients were treated with antibacterial and 14 were treated with antifungal antibiotic in 2016. One patient was lost to follow-up in each group. The rate of TPK was higher and proportion of healed ulcers was lower (p=0.21, Fisher's exact test) in the group on antifungal therapy (TPK-11/13, 84.6%; Healed-2/13, 15.3%) compared with the group on antibacterial therapy (TPK-11/17, 64.7%; Healed-6/17, 35.2%). CONCLUSIONS: We report favourable but not statistically significant response of P insidiosum keratitis to antibacterial agents in a pilot series of patients. Further evaluation of this strategy in larger number of patients is recommended.

Oils extracted from Eupatorium adenophorum leaves show potential to control Phythium myriotylum in commercially-grown ginger.

Oils extracted from the leaves of Eupatorium adenophorum were tested in vitro and in vivo against the soilborne pathogen Pythium myriotylum which causes soft rot, a devastating disease of commercial ginger production in China. Twelve compounds accounting for 99.15% of the total oil composition were identified by GC-MS. The major components were 10Hß-9-oxo-agerophorone (37.03%), 10Hα-9-oxo-agerophorone (37.73%) and 9-oxo-10, 11-dehydro-agerophorone (23.41%). Antifungal activity was tested by the poisoned food technique against P. myriotylum, indicating minimum inhibitory concentrations of 100µg/ml after 7 days incubation. In addition, the oil extracts greatly inhibited the formation of both wet and dry mycelial biomass. The combination of E. adenophorum oil extracts and synthetic fungicides showed a strong synergistic effect, inhibiting the mycelial growth in in vitro assays. The synergistic effect of oil extracts with fungicides could allow fungicides to be used at reduced rates in the future which has environmental advantages. Oil extracts applied at 160 and 200µg/ml concentrations to ginger rhizomes before inoculation with P. myriotylum significantly reduced the infection rate in ginger. Examination by light and transmission electron microscopy revealed that oil extracts caused swelling of the hyphae, disruption of the cell wall, degradation of the cytoplasmic organelles and shortening of the cytoplasmic inclusion. These results suggested that the plasma membrane and endomembrane systems of P. myriotylum were severely damaged by the oil extracts of E. adenophorum which offer significant potential for use as a fungicide to control P. myriotylum.

Evolution of the Sterol Biosynthetic Pathway of Pythium insidiosum and Related Oomycetes Contributes to Antifungal Drug Resistance.

Pythiosis is a life-threatening infectious disease caused by the oomycete Pythium insidiosum Direct exposure to Py. insidiosum zoospores can initiate infections of the eye, limb, gastrointestinal tract, or skin/subcutaneous tissue. Treatments for pythiosis have mostly relied on surgery. Antifungal drugs are generally ineffective against Py. insidiosum However, one patient with an invasive Py. insidiosum infection recovered completely following treatment with terbinafine and itraconazole. Additionally, the drug target sterol biosynthetic enzymes have been identified in the oomycete Aphanomyces euteiches It remains an open question whether Py. insidiosum is susceptible to the antifungal drugs and harbors any of the known drug target enzymes. Here, we determined the in vitro susceptibilities of terbinafine and itraconazole against 30 isolates of Py. insidiosum We also analyzed endogenous sterols and searched for genes encoding the sterol biosynthetic enzymes in the genomes of Py. insidiosum and related oomycetes. The susceptibility assay showed that the growth of each of the Py. insidiosum isolates was inhibited by the antifungal agents, but only at difficult-to-achieve concentrations, which explains the clinical resistance of the drugs in the treatment of pythiosis patients. Genome searches of Py. insidiosum and related oomycetes demonstrated that these organisms contained an incomplete set of sterol biosynthetic enzymes. Gas chromatographic mass spectrometry did not detect any sterol end products in Py. insidiosum In conclusion, Py. insidiosum possesses an incomplete sterol biosynthetic pathway. Resistance to antifungal drugs targeting enzymes in the ergosterol biosynthetic pathway in Py. insidiosum was due to modifications or losses of some of the genes encoding the drug target enzymes.

In vitro susceptibility of the oomycete Pythium insidiosum to metallic compounds containing cadmium, lead, copper, manganese or zinc.

Pythium insidiosum is an aquatic oomycete that causes pythiosis, an important and severe disease of difficult treatment that affects humans, domestic and wild animals. This infection is often described in horses in Brazil and humans in Thailand. In clinical practice, we have observed many cases that do not respond to available therapies, indicating the need to explore alternative therapeutic approaches. In this sense, studies using metal compounds in conjunction with available antimicrobial agents have been demonstrated greater antimicrobial activity. Thus, in this research, we tested in vitro activities of metallic compounds containing cadmium, lead, copper, manganese, or zinc against 23 isolates of P. insidiosum. The assays were performed by broth microdilution based on CLSI M38-A2 document. The minimum inhibitory and fungicidal concentrations were established for all isolates. Copper acetate and cadmium acetate showed the highest inhibitory effects, with minimal inhibitory concentration ranging from 4-64 µg/ml and 16-256 µg/ml, respectively. The mean geometric for minimal fungicidal concentrations were, respectively, 26 µg/ml and 111.43 µg/ml for copper acetate and cadmium acetate. These results suggest that copper and cadmium can inhibit P. insidiosum growth, highlighting the greater inhibitory activity of copper acetate. In addition, our results propose that copper and/or cadmium compounds can be used in upcoming researches to formulate effective new complexed drugs against P. insidiosum in in vitro and in vivo experimental models.

Potential effects of fungicide and algaecide extracts of Annona glabra L. (Annonaceae) on the microalgae Raphidocelis subcapitata and on the oomycete Pythium

ABSTRACT Annona glabra L. is a semi-deciduous tree that contains several active substances, including secondary metabolites, with antifungal activity. Phytopathogenic strains of the genus Pythium cause billion dollar losses all over the world on natural and crop species. Searching for eco-friendly algaecides and fungicides, we analyzed the effects of acetone extracts of A. glabra leaves on the algae Rhaphidocelis subcapitata (Korshikov) and on the oomycete Pythium aphanidermatum (Edson). We evaluated ten extract concentrations for each organism - 0 to 400 mg L-1 for algae and 0-1000 µg disc1 for oomycete. The results showed no effect on algae up to 75 mg L-1, but a significant inhibitory effect at 125 mg L-1 and above, which reduced the growth rate and the final biomass of the algae. Extract concentrations above 200 mg L-1 were completely inhibitory. The half maximal inhibitory concentration for 72 and 96 h of exposure to our crude extracts are comparable to those obtained with commercial fungicides and herbicides used in aquatic ecosystems. The P. aphanidermatum inhibition concentrations have effects comparable to fungicides as Cycloheximide and Bifonazole. Some substances isolated from the extracts are described as antifungals, which could explain part of anti-oomycete activity. Our results highlight the importance of searching bioactive compounds from plants.

Pythium insidiosum: inhibitory effects of propolis and geopropolis on hyphal growth

Abstract Propolis and geopropolis are resinous products of bees showing antimicrobial effects. There is no data concerning their action against Pythium insidiosum - the causative agent of pythiosis, a pyogranulomatous disease of the subcutaneous tissue that affects mostly horses, dogs and humans. Fragments of 15 isolates of P. insidiodum were incubated with propolis and geopropolis extracts and evaluated for up to seven days to detect the minimal fungicidal concentration (MFC). Propolis inhibited three isolates at 1.0 mg mL-1 after 24 h and all other isolates at 3.4 mg mL-1. Geopropolis led to more variable results, exerting predominantly a fungistatic action than a fungicidal one. Propolis was more efficient than geopropolis in inhibiting P. insidiosum since lower concentrations led to no growth after 24 h. This effect may be due to propolis chemical composition, which has more active compounds than geopropolis. Propolis seemed to be a good candidate for in vivo studies, since treatment with conventional antifungal compounds is difficult in most of the cases, requiring extensive surgical debridement.

In Vitro Activity of Melaleuca alternifolia (Tea Tree) in Its Free Oil and Nanoemulsion Formulations Against Pythium insidiosum.

Pythium insidiosum is an important aquatic oomycete which can cause pythiosis in both animals and humans. This microorganism shows low susceptibility to antifungal drugs available. This study analyzed the in vitro antimicrobial activity of Melaleuca alternifolia in its free oil (FO) and nanoemulsion (NE) formulations against Brazilian P. insidiosum isolates. The antimicrobial activity evaluation was performed by the broth microdilution method according to CSLI M38-A2 document adapted to phytopharmaceuticals. Twenty-six P. insidiosum isolates were evaluated, and the minimum inhibitory concentration was determined at 100 % growth inhibition. Melaleuca alternifolia essential oil or FO was obtained commercially. The NE containing 1 % M. alternifolia essential oil was prepared by the spontaneous emulsification method. All P. insidiosum isolates evaluated showed minimum inhibitory concentrations (MIC) ranging from 531.5 to 2125 µg/mL for the FO formulation; MIC50 and MIC90 showed values between 1062.5 and 2125 µg/mL, respectively. When the NE formulation was evaluated, MIC values ranged from 132.7 to 2125 µg/mL and both MIC50 and MIC90 corresponded to 1062.5 µg/mL. FO and NE formulations of M. alternifolia showed antimicrobial activity against P. insidiosum. This study demonstrated that M. alternifolia oil can be an additional therapy in pythiosis treatment; however, further studies are needed to evaluate the applicability of the plant essential oils in the treatment of clinical pythiosis.