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.

Evaluation of Strains of Metarhizium anisopliae and Beauveria bassiana against Spodoptera litura on the Basis of Their Virulence, Germination Rate, Conidia Production, Radial Growth and Enzyme Activity

Ten strains of the entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana were evaluated to find the most effective strain for optimization studies. The first criterion tested for strain selection was the mortality (> 50%) of Spodoptera litura larvae after inoculation of the fungus for 4 days. Results on several bioassays revealed that B. bassiana BNBCRC showed the most virulence on mortality S. litura larvae (80% mortality). B. bassiana BNBCRC also showed the highest germination rate (72.22%). However, its conidia yield (7.2 x 10(8) conidia/mL) was lower than those of B. bassiana B 14841 (8.3 x 10(8) conidia/mL) and M. anisopliae M6 (8.2 x 10(8) conidia/mL). The highest accumulative radial growth was obtained from the strain B14841 (37.10 mm/day) while the strain BNBCRC showed moderate radial growth (24.40 mm/day). M. anisopliae M6 possessed the highest protease activity (145.00 mU/mL) while M. anisopliae M8 possessed the highest chitinase activity (20.00 mU/mL) during 96~144 hr cultivation. Amongst these criteria, selection based on virulence and germination rate lead to the selection of B. bassiana BNBCRC. B. bassiana B14841 would be selected if based on growth rate while M. anisopliae M6 and M8 possessed the highest enzyme activities.

Monitoring the effect of pyrene on the germination and radial growth of the wild and mutant strains of Rhizopus arrhizus UCP402

The physiological mutant of Rhizopus arrhizus was obtained in the pyrene resistance gradient test. Comparative studies were carried out about the behavior of the germination process and the radial growth of the mutant and wild strains of R. arrhizus UCP 402. Sabouraud Sucrose and Yeast Malt Broth cultures containing pyrene (10 mg/L) induced the germination process of the sporangiospores of the wild and mutant strains of R. arrhizus. The radial growth of the strains was inversely proportional to the pyrene concentration in the culture medium. The results showed an adaptation of R. arrhizus UCP 402x (mutant) in the pyrene (50mg/L) and suggested a higher ability of application in the removal of pyrene from the contaminated areas.

O mutante fisiológico de Rhizopus arrhizus foi obtido pelo teste do gradiente de resistência ao pireno. Estudos comparativos conduzidos sobre o comportamento do processo de germinação e o crescimento radial foram realizados entre as amostras selvagem e mutante de R. arrhizus UCP 402. Os meios Sabouraud Sacarose e Caldo de Levedura e Malte contendo pireno (10 mg/L) induziram ao processo de germinação de esporangiosporos das amostras selvagem e mutante de R. arrhizus. O crescimento radial das amostras foi inversamente proporcional à concentração de pireno no meio de cultura. Os resultados demonstraram uma excelente adaptação da amostra mutante de R. arrhizus UCP 402x na concentração de pireno (50 mg/L), sugerindo uma alta habilidade e possibilidade de aplicação na remoção de pireno em áreas contaminadas.

Selection of culture media and in vitro assessment of temperature-dependent development of Nomuraea rileyi / Seleção do meio de cultura e avaliação in vitro do desenvolvimento de Nomuraea rileyi dependente da temperatura

In vitro development - radial growth and sporulation - of Nomuraea rileyi isolates from Anticarsia gemmatalis Hübner and Plusiinae larvae was studied on four culture media: Sabouraud, maltose, agar and yeast - SMAY; SMAY plus rice extract - SMAYR; complete medium for N. rileyi - CMNr and maltose agar yeast with potato extract - MAYP. Their development on a selected medium (MAYP) was analysed at five temperatures. Two mathematical models were fitted to in vitro and in vivo temperature dependent radial growth rates and thermal requirements were estimated. The medium with potato and yeast extract induced the highest growth rate in most cases, while SMAY induced the lowest rates for the tested isolates. Estimated optimum temperatures for mycelium in vitro growth ranged from 22°C to 26°C. No differences between the proportion of sporulation of colonies maintained at 20°C and 26°C were detected. Few or no colonies sporulated at 12, 16 and 30°C. The relative production of conidia per fungal biomass was very variable, ranging from 0.5 to 16 conidia per centigram of mycelium. Therefore, this was not a useful criterion for selecting a culture medium. Based on present findings, a medium based on potato extract, or enriched slices could be used for N. rileyi experimental and eventually mass production. Because of the similarities found between in vitro and in vivo thermal requirements, thermal traits of the mycosis could be simply estimated on the basis of the environmental temperature.

O desenvolvimento in vitro de isolados de Nomuraea rileyi, obtidos de larvas de Anticarsia gemmatalis Hübner e de Plusiinae, foi estudados em quatro meios de cultura: Sabouraud, maltose, agar e levedura - SMAY; SMAY com extrato de arroz - SMAYR; meio completo para N. rileyi - CMNr; e maltose, agar, levedura com extrato de batata - MAYP. Seu crescimento radial em MAYP foi analisado sob cinco temperaturas. Foi estudada a esporulação em relação à temperatura, e a produção de conídios nos quatro meios testados. Dois modelos matemáticos foram aplicados para descrever as taxas de desenvolvimento radial dependentes da temperatura in vitro e in vivo. MAYP permitiu a maior taxa de crescimento, porém SMAY provocou as taxas mais baixas para os isolados testados. As temperaturas ótimas estimadas para o crescimento do micélio in vitro variam de 22°C a 26°C. A esporulação não variou entre 20°C e 26°C. As temperaturas de 12, 16 e 30°C observou-se pouca ou nenhuma esporulação. A produção relativa dos conídios por biomassa de fungo foi muito variável, de 0,5 a 16 conídios por centigrama de micélio, não sendo considerada um critério adequado para escolher o meio de cultura. Com base nos presentes resultados, um meio à base de extrato de batata ou fatias de batata enriquecidas poderiam ser usados para a produção experimental e, eventualmente, massal de N. rileyi. Dadas as similaridades entre exigências térmicas in vitro e in vivo aqui descritas, as características térmicas da micose poderiam ser simplesmente estimadas com base na temperatura ambiente.

Effect of ERG6 Gene Extra Copies on Aspergillus fumigatus / 微生物学通报

To investigate the role of ERC,6 gene in the growth rate and antifungal susceptibility,Aspergillus fumigatus strain with extra copies of ERG6 gene was constructed.Open reading frame (ORF) of putative ERG6 gene was searched in A.fumigatus genome.A PCR fragment, ERG6 ORF sandwiched by its flanking sequences (about 1 kb respectively),was amplified and was then subcloned into vector pRG-AMA1- NotI to produce a recombinant plasmid pERG6,which was further transformed into uracil auxotroph A.fumigatus strain AF293.1 to produce the transformant AF-pERG6.In the same time,empty plasmid pRG-AMA1-NotI was also transformed into A.fumigatus strain AF293.1 to produce the transformant AF-empty as a control.Radial growth of the transformants was tested on minimal medium (MM) and YAG medi- um.Antifungal susceptibilities of these resahing transformants,AF-pERG6 and AF-empty,to the common antifungal agents were performed by using both disk diffusion and broth microdillution methods.A.fumigatus genome contains a ERG6 gene,of which the ORF size is 1 256 bp.Comparing to Candida albicans and Sacchromyces cerivisiae sterol methyltransferase,A.fumigatus Erg6p had 57% and 50% identity, and had 70% and 63% similarity in amino acid sequences,respectively.Radial growth of transformant AF-pERG6 was slower than that of transformant AF-empty.The antifungal susceptibilties of transformant AF-pERG6 to the antifungal drugs itraconazole,voriconazole,terbin- aline,amphotericin B,caspofungin and grisefolvin were same to that of transformant AF-empty.In A.fumigatus,extra copies of ERG6 gene have no effect on antifungal susceptibilities to itraconazole,voriconazole,terbinafine,amphotericin B,caspofungin and grisefolvin.Radial growth of A.fumigatus harboring extra copies of ERG6 gene becomes slower compared to the control.