Morphological changes and growth of filamentous fungi in the presence of high concentrations of PAHs

In this study, we evaluated the effect of low and high molecular weight polycyclic aromatic hydrocarbons (PAHs), i.e., Phenanthrene, Pyrene and Benzo[a]pyrene, on the radial growth and morphology of the PAH-degrading fungal strains Aspergillus nomius H7 and Trichoderma asperellum H15. The presence of PAHs in solid medium produced significant detrimental effects on the radial growth of A. nomius H7 at 4,000 and 6,000 mg L⁻¹ and changes in mycelium pigmentation, abundance and sporulation ability at 1,000–6,000 mg L⁻¹. In contrast, the radial growth of T. asperellum H15 was not affected at any of the doses tested, although sporulation was observed only up to 4,000 mg L⁻¹ and as with the H7 strain, some visible changes in sporulation patterns and mycelium pigmentation were observed. Our results suggest that fungal strains exposed to high doses of PAHs significantly vary in their growth rates and sporulation characteristics in response to the physiological and defense mechanisms that affect both pigment production and conidiation processes. This finding is relevant for obtaining a better understanding of fungal adaptation in PAH-polluted environments and for developing and implementing adequate strategies for the remediation of contaminated soils..(AU)

Compatibility of conventional agrochemicals used in rice crops with the entomopathogenic fungus Metarhizium anisopliae

The toxicological impact of chemical pesticides on fungal entomopathogens and their use in tank-mixing can be directly measured through in vitro compatibility tests. This study reports the in vitro toxicity of eight insecticides, four fungicides and five herbicides in the conidial germination, vegetative growth and conidiation of Metarhizium anisopliae (strain CG 168). A conidial suspension containing the pesticide at recommended field dosage was subjected to constant agitation in a rotary shaker for 3h to simulate a tank mixing. Then, aliquots of each suspension were used to determine conidial germination, vegetative growth and conidiation on potato dextrose agar (PDA). The fungicides difenoconazole (69 mL ha-1), propiconazole (75 mL ha-1), trifloxystrobin (313 g ha-1) and azoxystrobin (56 mL ha-1) were the most harmful products to all biological stages of M. anisopliae and they should not be applied together with this fungus in tank mixing. The insecticides exhibited the least degree of toxicity to this fungal pathogen, whereas the herbicides had the greatest impact on mycelial growth. The agrochemicals compatible with M. anisopliae were the insecticides methyl parathion (240 mL ha-1), thiamethoxam (31 g ha-1), and lambda-cyhalothrin (6.3 mL ha-1) and the herbicides glyphosate (1560 mL ha-1), bentazon (720 mL ha-1), and imazapic+ imazapyr (84 g ha-1). The compatible pesticides could be simultaneously used with this bio-control agent for integrated pest management in rice production systems.

Compatibility of conventional agrochemicals used in rice crops with the entomopathogenic fungus Metarhizium anisopliae

The toxicological impact of chemical pesticides on fungal entomopathogens and their use in tank-mixing can be directly measured through in vitro compatibility tests. This study reports the in vitro toxicity of eight insecticides, four fungicides and five herbicides in the conidial germination, vegetative growth and conidiation of Metarhizium anisopliae (strain CG 168). A conidial suspension containing the pesticide at recommended field dosage was subjected to constant agitation in a rotary shaker for 3h to simulate a tank mixing. Then, aliquots of each suspension were used to determine conidial germination, vegetative growth and conidiation on potato dextrose agar (PDA). The fungicides difenoconazole (69 mL ha-1), propiconazole (75 mL ha-1), trifloxystrobin (313 g ha-1) and azoxystrobin (56 mL ha-1) were the most harmful products to all biological stages of M. anisopliae and they should not be applied together with this fungus in tank mixing. The insecticides exhibited the least degree of toxicity to this fungal pathogen, whereas the herbicides had the greatest impact on mycelial growth. The agrochemicals compatible with M. anisopliae were the insecticides methyl parathion (240 mL ha-1), thiamethoxam (31 g ha-1), and lambda-cyhalothrin (6.3 mL ha-1) and the herbicides glyphosate (1560 mL ha-1), bentazon (720 mL ha-1), and imazapic+ imazapyr (84 g ha-1). The compatible pesticides could be simultaneously used with this bio-control agent for integrated pest management in rice production systems.

Cgl-SLT2 is required for appressorium formation, sporulation and pathogenicity in Colletotrichum gloeosporioide

The mitogen-activated protein (MAP) kinase pathways has been implicated in the pathogenicity of various pathogenic fungi and plays important roles in regulating pathogenicity-related morphogenesis. This work describes the isolation and characterization of MAP kinase gene, Cgl-SLT2, from Colletotrichum gloeosporioides. A DNA sequence, including 1,633 bp of Cgl-SLT2 open-reading frame and its promoter and terminator regions, was isolated via DNA walking and cloned. To analyze gene function, a gene disruption cassette containing hygromycin-resistant gene was constructed, and Cgl-SLT2 was inactivated via gene deletion. Analysis on Cgl-slt2 mutant revealed a defect in vegetative growth and sporulation as compared to the wild-type strain. When grown under nutrient-limiting conditions, hyperbranched hyphal morphology was observed in the mutant. Conidia induction for germination on rubber wax-coated hard surfaces revealed no differences in the percentage of conidial germination between the wild-type and Cgl-slt2 mutant. However, the percentage of appressorium formation in the mutant was greatly reduced. Bipolar germination in the mutant was higher than in the wild-type at 8-h post-induction. A pathogenicity assay revealed that the mutant was unable to infect either wounded or unwounded mangoes. These results suggest that the Cgl-SLT2 MAP kinase is required for C. gloeosporioides conidiation, polarized growth, appressorium formation and pathogenicity.

Growth inhibition and morphological alterations of Trichophyton rubrum induced by essential oil from Cymbopogon winterianus Jowitt ex Bor

Trichophyton rubrum is one of the most common fungi causer of dermatophytosis, mycosis that affect humans and animals around the world. Researches aiming new products with antifungal activity become necessary to overcome difficulties on treatment of these infections. Accordingly, this study aimed to investigate the antifungal activity of essential oil from Cymbopogon winterianus against the dermatophyte T. rubrum. The antifungal screening was performed by solid medium diffusion method with 16 T. rubrum strains, minimum inhibitory concentration (MIC) and minimum fungicide concentration (MFC) were determined using the microdilution method. The effects on mycelial dry weight and morphology were also observed. Screening showed essential oil in natura inhibited all the tested strains, with inhibition zones between 24-28 mm diameter. MIC50 and MIC90 values of the essential oil were 312 µg/mL for nearly all the essayed strains (93.75 %) while the MFC50 and MFC90 values were about eight times higher than MIC for all tested strains. All tested essential oil concentrations managed to inhibit strongly the mycelium development. Main morphological changes on the fungal strains observed under light microscopy, which were provided by the essential oil include loss of conidiation, alterations concerning form and pigmentation of hyphae. In the oil presence, colonies showed folds, cream color and slightly darker than the control, pigment production was absent on the reverse and with evident folds. It is concluded that C. winterianus essential oil showed activity against T. rubrum. Therefore, it could be known as potential antifungal compound especially for protection against dermatophytosis.

Chemical composition and antifungal activity of Hyptis suaveolens (L.) poit leaves essential oil against Aspergillus species

This study aimed to identify the constituents of the essential oil from Hyptis suaveolens (L.) leaves using a Gas Chromatograph -Mass Spectrometer and assess its inhibitory effect on some potentially pathogenic Aspergilli (A. flavus, A. parasiticus, A. ochraceus, A. fumigatus and A. niger). Eucaliptol (47.64 %) was the most abundant component in the oil, followed for gama-ellemene (8.15 %), beta-pynene (6.55 %), (+)3-carene (5.16 %), trans-beta-cariophyllene (4.69 %) and germacrene (4.86 %). The essential oil revealed an interesting anti-Aspergillus property characterized by a Minimum Inhibitory Concentration and Minimum Fungicidal Concentration of 40 and 80 µL/mL, respectively. The oil at 80 and 40 µL/mL strongly inhibited the mycelial growth of A. fumigatus and A. parasiticus along 14 days. In addition, at 10 and 20 µL/mL the oil was able to cause morphological changes in A. flavus as decreased conidiation, leakage of cytoplasm, loss of pigmentation and disrupted cell structure suggesting fungal wall degeneration. These findings showed the interesting anti-Aspergillus property of H. suaveolens leaves essential oil supporting its possible rational use as alternative source of new antifungal compounds to be applied in the aspergillosis treatment.

Mechanism of Sterigmatocystin Biosynthesis Regulation by pH in Aspergillus nidulans

External pH constitutes one of the most important environmental factors that control growth, metabolism and differentiation in microorganisms, including fungi. We have analyzed the effect of external pH on sterigmatocystin biosynthesis in Aspergillus nidulans. It was observed in repeated experiments that alkaline pH, in opposition to acid pH, increased sterigmatocystin production and the transcript levels of aflR, the master gene that regulates expression of the sterigmatocystin cluster in A. nidulans. It is known that pH effects in fungi operate mostly through the Pal/Pac signaling pathway, originally described in Aspergillus nidulans. Accordingly, we studied the role of this signaling pathway in ST biosynthesis. It was observed that aflR transcript levels were increased in the "alkalinity mimicking" mutant pacCc14 and were minimal in the "acidity mimicking" mutant palA1. No sterigmatocystin was produced by palA1 or pacC- mutants at neither acid or alkaline pH of incubation. Finally, fluG and flbA, genes known to regulate both conidiation and sterigmatocystin synthesis upstream in the regulatory cascade, were up-regulated at alkaline pH.

Effect of cinnamomum zeylanicum blume essential oil on the rowth and morphogenesis of some potentially pathogenic Aspergillus species

Cinnamomum zeylanicum Blume is known for a wide range of medicinal properties. This study aimed to assess the interference of C. zeylanicum essential oil on the growth and morphogenesis of some potentially pathogenic Aspergillus species. The essential oil presented strong antifungal effect causing the growth inhibition of the assayed strains and development of large growth inhibition zones. MIC50 and MIC90 values were 40 and 80 µL/mL, respectively. 80, 40 and 20 µL/mL of the oil strongly inhibited the radial mycelial growth of A. niger, A. flavus and A. fumigatus along 14 days. 80 and 40 µL/mL of the oil caused a 100% inhibition of the fungal spore germination. Main morphological changes observed under light microscopy provided by the essential oil in the fungal strains were decreased conidiation, leakage of cytoplasm, loss of pigmentation and disrupted cell structure indicating fungal wall degeneration. It is concluded that C. zeylanicum essential oil could be known as potential antifungal compound, particularly, to protect against the growth of Aspergillus species.

Cinnamomum zeylanicum Blume é uma planta conhecida por apresentar ampla variedade de propriedades medicinais. Portanto, este estudo teve por objetivo avaliar a interferência do óleo essencial C. zeylanicum sobre o crescimento e morfogênese de algumas espécies de Aspergillus potencialmente patogênicas. O óleo essencial testado apresentou potente efeito antifúngico demonstrado pela visualização de grandes zonas de inibição de crescimento de todas as linhagens testadas. Os valores de CIM50 e de CIM90 foram 40 e 80 µL/mL, respectivamente. Nas concentrações de 80, 40 e 20 µL/mL o óleo demonstrou um potente efeito fumigante, inibindo o crescimento micelial radial de A. niger, A. flavus e A. fumigatus ao longo de 14 dias de exposição. A 80 e 40 µL/mL o óleo essencial promoveu inibição de 100% da germinação de esporos, das três espécies de Aspergillus citadas anteriormente. Além disso, alterações morfológicas no crescimento fúngico foram observadas sob microscopia óptica após exposição ao óleo essencial, como diminuição da conidiação, perda citoplasmática, perda de pigmentação e rompimento da estrutura fúngica (hifa) indicando degeneração da parede celular. Diante do exposto, conclui-se que o óleo essencial de C. zeylanicum poderia ser empregado como potente composto antifúngico, particularmente, prevenindo crescimento de espécies de Aspergillus.