Lactic acid bacteria and Bacillus spp. as fungal biological control agents.

Fungal pathogens are one of the most important agents affecting crop production and food safety, and agrochemical application is one of the main approaches to reduce phytopathogenic fungi contamination in agricultural products. However, excessive and inadequate use can cause environmental damage, human and animal hazard, and increased phytopathogen resistance to fungicides. Biological control using lactic acid bacteria (LAB) and Bacillus spp. is an environmentally friendly strategy for phytopathogenic fungi management. Several molecules produced by these bacteria indeed affect fungal growth and viability in different plant crops. In this article, the activity spectra are reviewed along with the antifungal effect and antifungal compounds produced by LAB (e.g. organic acids, peptides, cyclic dipeptides, fatty acids, and volatile compounds) and Bacillus spp. (e.g. peptides, enzymes, and volatile compounds).

Potential of yeasts as biocontrol agents against Fusarium graminearum in vitro and on corn.

AIMS: The antifungal effect of the yeast species Kluyveromyces marxianus, Meyerozyma caribbica, and Wickerhamomyces anomalus was evaluated against two Fusarium graminearum strains (FRS 26 and FSP 27) in vitro and on corn seeds. METHODS AND RESULTS: The antifungal effect of the yeasts against F. graminearum was evaluated using scanning electron microscopy and extracellular chitinase and glucanase production to further elucidate the biocontrol mode of action. In addition, the germination percentage and vigor test were investigated after applying yeast on corn seeds. All the yeast strains inhibited fungal growth in vitro (57.4%-100.0%) and on corn seeds (18.9%-87.2%). In co-culture with antagonistic yeasts, F. graminearum showed collapsed hyphae and turgidity loss, which could be related to the ability of yeasts to produce chitinases and glucanases. The three yeasts did not affect the seed corn germination, and W. anomalus and M. caribbica increased corn seed growth parameters (germination percentage, shoot and root length, and shoot dry weight). CONCLUSION: Meyerozyma caribbica and W. anomalus showed satisfactory F. graminearum growth inhibition rates and did not affect seed growth parameters. Further studies are required to evaluate the application of these yeasts to the crop in the field.

Yeasts as sustainable biocontrol agents against ochratoxigenic Aspergillus species and in vitro optimization of ochratoxin A detoxification.

AIMS: The aims of this study were to evaluate the potential of Hanseniaspora opuntiae, Meyerozyma caribbica, and Kluyveromyces marxianus for in vitro biocontrol of Aspergillus ochraceus, A. westerdijkiae, and A. carbonarius growth, the ochratoxin A (OTA) effect on yeast growth, and yeast in vitro OTA detoxification ability using an experimental design to predict the combined effects of inoculum size, incubation time, and OTA concentration. METHODS AND RESULTS: Predictive models were developed using an incomplete Box-Behnken experimental design to predict the combined effects of inoculum size, incubation time, and OTA concentration on OTA detoxification by the yeasts. The yeasts were able to inhibit fungal growth from 13% to 86%. Kluyveromyces marxianus was the most efficient in inhibiting the three Aspergillus species. Furthermore, high OTA levels (100 ng ml-1) did not affect yeast growth over 72 h incubation. The models showed that the maximum OTA detoxification under optimum conditions was 86.8% (H. opuntiae), 79.3% (M. caribbica), and 73.7% (K. marxianus), with no significant difference (P > 0.05) between the values predicted and the results obtained experimentally. CONCLUSION: The yeasts showed potential for biocontrol of ochratoxigenic fungi and OTA detoxification, and the models developed are important tools for predicting the best conditions for the application of these yeasts as detoxification agents.

Compulsory notification of paracoccidioidomycosis: A 14-year retrospective study of the disease in the state of Paraná, Brazil.

BACKGROUND: Paracoccidioidomycosis, caused by fungi of the Paracoccidioides genus, is one of the most important endemic mycoses in Brazil. The disease is not of mandatory reporting in the country; however, some Brazilian states, such as Paraná, have included it on their local lists of public health-reportable diseases. OBJECTIVE: Describe the epidemiology of the positive paracoccidioidomycosis cases in the state of Paraná based on analysis of reporting forms and mortality data. SUBJECTS AND METHODS: Data of positive cases of state residents 2007-2020 were obtained from public health databases and frequency, incidence, geographic distribution, mortality and trends were analysed. Mortality of the disease was also compared to other mycoses such as cryptococcosis, aspergillosis, histoplasmosis, candidiasis and sporotrichosis. RESULTS: 670 patients were positive for the disease. The cumulative and average annual incidence was, respectively, 6.4 and 0.46 cases/100,000 inhabitants. The new cases of paracoccidioidomycosis were reported mainly by specialised health units, including tertiary centres, and 285 days was the mean from the beginning of the symptoms until the diagnosis. The western region showed the highest incidence and mortality by the disease over the other state mesoregions. During the period, a decreasing trend was observed in the confirmed cases and stability in the mortality rate with an average annual mortality of 1.17 per million/inhabitants in the state; however, paracoccidioidomycosis had the highest mortality when compared to other mycoses. CONCLUSIONS: Paracoccidioidomycosis is an important endemic mycosis in Paraná and this study provides an epidemiological baseline for future modifications of paracoccidioidomycosis surveillance.

First report of Paracoccidioides brasiliensis infection in fish.

The thermodimorphic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis (PCM), a deep mycosis endemic in Latin American countries that affects mainly male rural workers. Infection by P. brasiliensis has also been reported in several species of terrestrial animals; however, the capacity of the fungus to infect aquatic organisms is poorly known. The aim of this study was to detect P. brasiliensis in a fish species, Nile tilapia (Oreochromis niloticus), the most farmed and widely distributed fish in endemic areas for human PCM in Brazil. As a first step, the humoral immune response against the fungus was evaluated in an experimental group of three fish immunized with inactivated P. brasiliensis yeast cells. For the seroepidemiological study, serum samples of Nile tilapia raised in cages (n = 109) and in ponds (n = 105), collected from a fish slaughterhouse, were analyzed for P. brasiliensis antibodies by ELISA using gp43 as antigen. All the inoculated fish produced antibodies against the fungus. The seropositivity observed in fish raised in cages and ponds was 17.4 and 5.7%, respectively. Due to the higher seropositivity observed in caged fish, 100 tissue samples (encephalon, liver, and kidney), from another group of tilapia raised in cages, were analyzed by polymerase chain reaction (PCR; Pb-ITSR and Pb-ITSE). Three tissue samples (liver n = 1, kidney n = 1, and enchepahlon n = 1) from three different fish resulted positive to PCR. This is the first report to show serological and molecular evidence of P. brasiliensis infection in a fish species.

Paracoccidioides restrepiensis B339 (PS3) and P. lutzii LDR2 yeast cells and soluble components display in vitro hemolytic and hemagglutinating activities on human erythrocytes.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by thermodimorfic fungi of Paracoccidioides species complex. Several pathogenic fungi produce hemagglutinins and hemolysins, which are virulence factors involved in adhesion of pathogens to host tissues or cells and in destruction of erythrocytes. The present research investigated hemolytic and hemagglutinating activities of yeast cells and soluble components from P. restrepiensis (PS3; former P. brasiliensis B339) and P. lutzii (LDR2). Different concentrations of live and heat-killed yeast cells and soluble components from cell free antigen preparation (CFA) (native or heated - 56 and 100 °C, 30 min) were mixed with 1% human erythrocyte suspension. Yeast cells from both species caused hemolysis, but P. lutzii LDR2 was more hemolytic than P. restrepiensis B339, while the opposite phenomena occurred with soluble components in most conditions. Live or heat-killed yeast cells of both fungi agglutinated erythrocytes, but only heated soluble components from P. restrepiensis B339 showed hemagglutinating activity. In conclusion, yeast cells of P. restrepiensis B339 and P. lutzii LDR2 produce hemolysins and hemagglutinins, which most likely are more restricted to yeast cells in P. lutzii LDR2 and are more released in soluble form byP. restrepiensis B339, requiring further study.

Detection of Fusarium verticillioides by PCR-ELISA based on FUM21 gene.

Fusarium verticillioides is a primary corn pathogen and fumonisin producer which is associated with toxic effects in humans and animals. The traditional methods for detection of fungal contamination based on morphological characteristics are time-consuming and show low sensitivity and specificity. Therefore, the objective of this study was to develop a PCR-ELISA based on the FUM21 gene for F. verticillioides detection. The DNA of the F. verticillioides, Fusarium sp., Aspergillus sp. and Penicillium sp. isolates was analyzed by conventional PCR and PCR-ELISA to determine the specificity. The PCR-ELISA was specific to F. verticillioides isolates, showed a 2.5 pg detection limit and was 100-fold more sensitive than conventional PCR. In corn samples inoculated with F. verticillioides conidia, the detection limit of the PCR-ELISA was 1 × 104 conidia/g and was also 100-fold more sensitive than conventional PCR. Naturally contaminated corn samples were analyzed by PCR-ELISA based on the FUM21 gene and PCR-ELISA absorbance values correlated positively (p < 0.05) with Fusarium sp. counts (CFU/g). These results suggest that the PCR-ELISA developed in this study can be useful for F. verticillioides detection in corn samples.

Eighty Years of Mycopathologia: A Retrospective Analysis of Progress Made in Understanding Human and Animal Fungal Pathogens.

Mycopathologia was founded in 1938 to 'diffuse the understanding of fungal diseases in man and animals among mycologists.' This was an important mission considering that pathogenic fungi for humans and animals represent a tiny minority of the estimated 1.5-5 million fungal inhabitants on Earth. These pathogens have diverged from the usual saprotrophic lifestyles of most fungi to colonize and infect humans and animals. Medical and veterinary mycology is the subdiscipline of microbiology that dwells into the mysteries of parasitic, fungal lifestyles. Among the oldest continuing scientific publications on the subject, Mycopathologia had its share of 'classic papers' since the first issue was published in 1938. An analysis of the eight decades of notable contributions reveals many facets of host-pathogen interactions among 183 volumes comprising about 6885 articles. We have analyzed the impact and relevance of this body of work using a combination of citation tools (Google Scholar and Scopus) since no single citation metric gives an inclusive perspective. Among the highly cited Mycopathologia publications, those on experimental mycology accounted for the major part of the articles (36%), followed by diagnostic mycology (16%), ecology and epidemiology (15%), clinical mycology (14%), taxonomy and classification (10%), and veterinary mycology (9%). The first classic publication, collecting nearly 200 citations, appeared in 1957, while two articles published in 2010 received nearly 150 citations each, which is notable for a journal covering a highly specialized field of study. An empirical analysis of the publication trends suggests continuing interests in novel diagnostics, fungal pathogenesis, review of clinical diseases especially with relevance to the laboratory scientists, taxonomy and classification of fungal pathogens, fungal infections and carriage in pets and wildlife, and changing ecology and epidemiology of fungal diseases around the globe. We anticipate that emerging and re-emerging fungal pathogens will continue to cause significant health burden in the coming decades. It remains vital that scientists and physicians continue to collaborate by learning each other's language for the study of fungal diseases, and Mycopathologia will strive to be their partner in this increasingly important endeavor to its 100th anniversary in 2038 and beyond.

Serological Evidence of Infection by Paracoccidioides brasiliensis in Dogs with Leishmaniasis.

Paracoccidioidomycosis is a systemic mycosis prevalent in Latin American countries, caused by the dimorphic fungi Paracoccidioides brasiliensis and P. lutzii. The habitat of these fungi in nature remains undefined, although it is believed that infection occurs by inhalation of infective propagules present in soil. Sentinel animals, such as dogs, can be valuable epidemiological markers of paracoccidioidomycosis. Taking into account that paracoccidioidomycosis and visceral leishmaniasis may occur in the same area, the objective of this study was to evaluate the occurrence of P. brasiliensis infection in dogs positive for Leishmania sp. Serum samples of dogs positive (n = 199) and negative (n = 101) for Leishmania sp. were analyzed by the immunodiffusion test using P. brasiliensis exoantigen, and 22 samples (7.3%) were positive. The serum samples positive in the immunodiffusion test were also analyzed by Western blotting using the P. brasiliensis gp43 recombinant protein, and 86% of the samples were positive. A high positive correlation (r = 0.96) between positivity for Leishmania sp. and P. brasiliensis was observed. These data suggest an association between leishmaniasis and paracoccidioidomycosis in dogs.

Paracoccidioides brasiliensis-associated dermatitis and lymphadenitis in a dog.

Paracoccidioidomycosis (PCM) is an endemic disease of humans from Latin America that is caused by Paracoccidioides brasiliensis and P. lutzii, with most cases of PCM in domestic animals being associated with P. brasiliensis. This study presents the clinical, cytological, mycological, serological, and molecular findings associated with P. brasiliensis in a dog from Southern Brazil. Fine needle biopsies were collected from the skin and several lymph nodes of a 5-year-old female Labrador dog that had enlargement of most superficial lymph nodes. Cytology of the skin and lymph nodes revealed pyogranulomatous dermatitis and lymphadenitis associated with fine-necked, budding fungal structures consistent with the Paracoccidioides genus of organisms; mycological culture derived from the lymph node aspirate demonstrated similar budding structures. Serological assays using exoantigens obtained from the fungal culture demonstrated that the fungal organisms derived from the lymph node were antigenically similar to P. brasiliensis by immunodiffusion and Western blot. A PCR assay, using the fungal culture as input, amplified a partial segment of the internal transcribed spacer 1 and 2 regions of P. brasiliensis; direct sequencing and phylogenetic analyses confirmed the PCR product as P. brasiliensis. The combined cytological, mycological, serological, and molecular findings confirmed a diagnosis of fungal dermatitis and lymphadenitis due to P. brasiliensis in this dog. This case represents the third description of clinical PCM in dogs and the first confirmation of mycotic dermatitis associated with P. brasiliensis in this species. The participation of dogs in the possible dissemination of PCM is reviewed, and it is proposed that dogs are probable accidental hosts in the epidemiological cycle associated with P. brasiliensis.

Paracoccidioides brasiliensis Infection in Small Wild Mammals.

Paracoccidioidomycosis (PCM) is a systemic mycosis prevalent in Brazil and other Latin American countries. The etiological agents of PCM are the thermo-dimorphic fungi Paracoccidioides brasiliensis and P. lutzii. Taking into account that the natural habitat of Paracoccidioides spp. is still undefined, domestic and wild animals could be useful as indicators of Paracoccidioides spp. presence in endemic areas. The objective of this study was to evaluate the infection of small wild mammals by P. brasiliensis in an endemic area for human PCM. Samples from 38 wild mammals from different species such as Akodon sp., Thaptomys nigrita, Euryoryzomys russatus, Oligoryzomys nigripes, Monodelphis sp., Sooretamys angouya, Abrawayaomys angouya, Abrawayaomys ruschii and Akodontinae sp. were evaluated by ELISA, immunodiffusion, histopathology, nested PCR and culture. The overall positivity to gp43 observed in the ELISA was 23.7%. Samples from heart and liver of one O. nigripes were PCR positive, and the animal was also seropositive to gp43 in ELISA. This study showed that wild animals living in endemic areas for PCM are infected with P. brasiliensis and can be valuable epidemiological markers of the fungus presence in the environment. This is the first evidence of PCM infection in Akodon sp., E. russatus, T. nigrita and O. nigripes.

Immunoblotting of soluble antigens in Paracoccidioides brasiliensis culture.

This study investigated the major soluble antigens produced by Paracoccidioides brasiliensis (Pb339) cultured in solid Sabouraud (pH 5.6 and 8.5), Sabouraud plus brain heart infusion and liquid tomato juice-enriched complex medium media at intervals of 3 days over 30 days by immunoblotting and concluded that, to optimize the source of each antigen, both time and growth conditions should be considered.

Paracoccidioides brasiliensis infection in domestic rabbits (Oryctolagus cuniculus).

The objective of this study was to evaluate the infection of domestic rabbits by Paracoccidioides brasiliensis. Initially two rabbits were experimentally infected with P. brasiliensis and the humoral immune response was evaluated by ELISA using gp43 as antigen. The two animals showed IgG response against gp43 although no signs of disease were observed. The seroepidemiological study was carried out in 170 rabbits (free range n = 81 and caged n = 89) living in an endemic area for human paracoccidioidomycosis and a positivity of 27% was observed in the ELISA using gp43 as antigen. The free-range rabbits showed a significantly higher positivity (34.6-51.7%) than the caged animals (11.1%). Sentinel rabbits exposed to natural infection with P. brasiliensis were followed up for 6 months and a seroconversion rate of 83.3% was observed. This is the first report of paracoccidioidomycosis in rabbits and suggests that this species can be useful sentinels for P. brasiliensis presence in the environment.

Detection of antibodies against Paracoccidioides brasiliensis in free-range domestic pigs (Sus scrofa).

Paracoccidioidomycosis, caused by the thermodimorphic fungus Paracoccidioides brasiliensis, is a human systemic mycosis prevalent in Latin America. Paracoccidioidomycosis affects mainly male rural workers, causing granulomatous lesions in several organs such as the lungs, liver and spleen. The participation of other animal species in the fungus epidemiology is not well understood. The objective of this study was to evaluate the infection of free-range domestic pigs by P. brasiliensis. Serum samples from 106 pigs were analyzed by ELISA and the immunodiffusion test, using P. brasiliensis gp43 and exoantigen as antigens, respectively. The overall positivity to gp43 in ELISA was 37.7 %, although no reactivity was observed in the immunodiffusion test and nor was P. brasiliensis detected in tissue samples (spleen, lung, liver and lymph nodes) from slaughtered animals submitted to culture, histopathological examination and PCR analysis. Five pigs seronegative to gp43 were exposed to natural infection by P. brasiliensis, and all animals seroconverted 3 months after exposure. The results suggest that free-range pigs are frequently infected with P. brasiliensis but are resistant to disease development. This is the first report of paracoccidioidomycosis in pigs.

Antifungal effect and some properties of cell-free supernatants of two Bacillus subtilis isolates against Fusarium verticillioides.

Fusarium verticillioides causes significant decrease in corn yield and quality, and produces fumonisins, which represent a serious risk to human and animal health. Bacillus species can be an effective and environmentally friendly alternative for F. verticillioides biological control. In this study, some properties of cell-free supernatants (CFSs) of two Bacillus spp. identified as Bacillus subtilis (NT1, NT2) as well as the antifungal effect against F. verticillioides 97L were evaluated. B. subtilis NT1 and NT2 were isolated from commercially available fermented whole soybeans (Natto). Antifungal activity was observed in both CFSs of B. subtilis isolates (50-59 mm) obtained by co-culture suggesting that antifungal compound production depends on interaction between bacteria and fungi. Cell-free supernatants from the two B. subtilis isolates inhibited mycelial growth (77%-94%) and conidial germination (22%-74%) of F. verticillioides 97L. In addition, CFSs caused significant morphological changes such as distorted and collapsed hyphae with wrinkled surfaces and the presence of a large amount of extracellular material compared to the control without CFSs. Both B. subtilis isolates (NT1 and NT2) produced extracellular proteases, biosurfactants and polar low molecular weight compounds that probably act synergistically and may contribute to the antifungal activity. Antifungal compounds showed heat and pH stability and resistance to proteolytic enzymes. Furthermore, antifungal compounds showed high polarity, high affinity to water and a molecular weight less than 10 kDa. These results indicated that the two B. subtilis (NT1 and NT2) have potential as biocontrol agents for F. verticillioides.

An atypical case of chronic paracoccidioidomycosis in a dog caused by a fungus from the Paracoccidioides brasiliensis complex.

Paracoccidioidomycosis (PCM) is a systemic mycosis endemic in Latin American countries and one of the most important fungal diseases regarding incidence and mortality in humans. PCM has also been described in some animal species such as dogs. In this study we describe a new case of PCM disease in a dog that differed from previous records in the literature which includes a progressive evolution of fungal dermatitis causing a deforming lesion in the nose, like those found in human patients, and humoral response against gp70 instead of gp43, the major diagnostic antigen for human PCM. The clinical isolate through the ITS and partial gp43 gene phylogenetic analysis was grouped in the Paracoccidioides brasiliensis complex. This case describes several features which may contribute to improving diagnosis and understanding of canine paracoccidioidomycosis.

Serological survey of paracoccidioidomycosis in cats.

The objective of the present study was to evaluate infection of cats by Paracoccidioides brasiliensis. Serum samples of 136 cats from rural (n = 86) and urban areas (n = 50) were analyzed by indirect ELISA and immunodiffusion test using P. brasiliensis gp43 and exoantigen as antigens, respectively, and an overall reactivity of 31.6 % was observed by ELISA although no reactivity was detected by immunodiffusion. The positivity observed in animals living in rural areas (48.8 %) with free access to soil was significantly higher (P < 0.0001) than among urban animals (2 %) with limited access to soil, although no significant difference was observed in relation to age or sex. The high rates of positivity observed in cats from rural areas suggest that not diagnosed cases of this mycosis may be occurring in cats living in endemic areas for human paracoccidioidomycosis. This is the first report showing serological evidence of P. brasiliensis infection in cats.

Limosilactobacillus reuteri as sustainable biological control agent against toxigenic Fusarium verticillioides.

Corn contamination with Fusarium verticillioides (Sacc.) Nirenberg is a worldwide problem that affects yield and grain quality resulting in severe economic losses and implications for food safety. Control of F. verticillioides is a challenge, but lactic acid bacteria (LAB) has high potential as a biological control agent. In this study, the antifungal effect of Limosilactobacillus reuteri (formerly Lactobacillus reuteri) LR-92 against F. verticillioides 97L was investigated. Cell-free supernatant (CFS) from L. reuteri showed concentration-dependent fungicidal and fungistatic activity against F. verticillioides 97L. The antifungal compounds from CFS showed heat stability and pH dependence, and antifungal activity was not affected by treatment with proteolytic enzymes. High-performance liquid chromatography analysis indicated that L. reuteri LR-92 produces lactic and acetic acids. After liquid-liquid extraction, electrospray ionization mass spectrometry analysis of the active ethyl acetate fraction containing antifungal compounds revealed the production of 3-phenyllactic acid, cyclo-(L-Pro-L-Leu), cyclo-(L-Pro-L-Phe), and cyclo-(L-Phe-trans-4-OH-L-Pro). L. reuteri LR-92 has potential as a biocontrol agent for F. verticillioides and contributes to food safety.

In vitro Arthrographis kalrae biofilm formation: Scanning electron microscopy and cytotoxic analysis.

To our knowledge, this study represents the first demonstration of Arthrographis kalrae biofilm formation in vitro by scanning electron microscopy and the distinct cytotoxic activity between planktonic and biofilm extracts on RAW 264.7 cell line. Higher activity was observed with biofilm. It could impact host immune response, that require furthers study.

Fusarium verticillioides: evaluation of fumonisin production and effect of fungicides on in vitro inhibition of mycelial growth.

The objectives of this study were to evaluate the fumonisin production by 16 F. verticillioides strains on corn cultures and the effect of quintozene and fludioxonil + metalaxyl-M fungicides on "in vitro" mycelial growth on agar. In addition, the effect of fludioxonil + metalaxyl-M on fumonisin production in defined liquid culture medium was analyzed. Fumonisin B(1) levels on corn cultures ranged from 2.41 to 3996.36 µg/g and the F. verticillioides 103F strain produced the highest level (3996.36 ± 390.49 µg/g, P < 0.05). F. verticillioides strains were inoculated in potato dextrose agar with the addition of quintozene (75 to 9,375 µg/ml) and fludioxonil + metalaxyl-M (1.5 + 0.6 to 187.5 + 75 µg/ml) in order to evaluate the effect of these fungicides on "in vitro" mycelial growth. The F. verticillioides strains showed great variability concerning ED(50) values, which were below the recommended application dose for quintozene, but above that for fludioxonil + metalaxyl-M. Moreover, fungicide addition to the culture medium increased mean FB(1) levels compared to the control, suggesting the importance of focusing on the effect of fungicides on mycotoxin production as well as on the phytopathogen control.