Lentinula edodes enhances the biocontrol activity of Cryptococcus laurentii against Penicillium expansum contamination and patulin production in apple fruits.

Penicillium expansum is a post-harvest pathogen of apples which can produce the hazardous mycotoxin patulin. The yeast Cryptococcus laurentii (LS28) is a biocontrol agent able to colonize highly oxidative environments such as wounds in apples. In this study culture filtrates of the basidiomycete Lentinula edodes (LF23) were used to enhance the biocontrol activity of LS28. In vitro L. edodes culture filtrates improved the growth of C. laurentii and the activity of its catalase, superoxide dismutase and glutathione peroxidase, which play a key role in oxidant scavenging. In addition, LF23 also delayed P. expansum conidia germination. The biocontrol effect of LS28 used together with LF23 in wounded apples improved the inhibition of P. expansum growth and patulin production in comparison with LS28 alone, under both experimental and semi-commercial conditions. The biocontrol effect was confirmed by a semi-quantitative PCR analysis set up for monitoring the growth of P. expansum.

Apyap1 affects aflatoxin biosynthesis during Aspergillus parasiticus growth in maize seeds.

It is demonstrated that, in fungal cells grown in synthetic media, the Apyap1 gene is implicated in the modulation of aflatoxin biosynthesis following the perturbation of redox balance. This study suggests that an association between oxidative stress and aflatoxin biosynthesis also occurs in maize seeds. We used DeltaApyap1, a strain in which the gene Apyap1 was disrupted, to verify whether this oxidative stress-related transcription factor, by affecting cell redox balance, can have a role in the modulation of aflatoxin synthesis. The amount of hydroperoxides (ROOH) produced by wild type (WT) and DeltaApyap1, both grown in potato dextrose broth, was assayed in the filtrate. In maize seeds (30 g), inoculated with WT and DeltaApyap1conidia and incubated at 30 degrees C for 15 days, lipoxygenase activity (LOX), lipoperoxides (LOOH) production, fungal growth and aflatoxin biosynthesis was analysed. It was observed that DeltaApyap1 released more hydroperoxides in the culture media and more aflatoxins in seeds, possibly through stronger stimulation of LOX, which, in turn led to greater LOOH production in the seeds. On the basis of the results, a hypothesis regarding strategies to control aflatoxin synthesis is formulated.

Oxidant/antioxidant balance inAspergillus parasiticus affects aflatoxin biosynthesis.

A close correlation between lipoperoxide formation in cells ofAspergillus parasiticus and aflatoxin biosynthesis has been established in rich and poor media in which oxidative stress was induced by addition of cumene hydroperoxide, a lipoperoxidation inducer. The presence of hydroperoxides of linoleic acid inA. parasiticus mycelia was analysed by liquid chromatography-mass spectrometry (LC-MS). This relation appears to be driven by activation of certain oxidative stress related transcription factors, such asyap1-like,skn7-like andhsf2-like. Activation of these factors then leads to the promotion of transcription of genes encoding antioxidant-related enzymes, such as superoxide dismutase, catalase and glutathione peroxidase.The incomplete seavenging of intracellular oxidation inA. parasiticus cells can lead to aflatoxin biosynthesis. The relationship between oxidative stress and aflatoxin biosynthesis is indicated by the high correlation among increased activity of lipoperoxidation and the antioxidant defence system with formation of aflatoxins.With regard to the relationship of oxidative stress and aflatoxin biosynthesis, the mechanism of action of butylated hydroxyl anisole (BHA), an antioxidant compound, in the control of aflatoxin biosynthesis was also investigated. Results indicate this compound can act,per se, by inhibiting lipoperoxidation and by inducing antioxidative defence responses of the fungal cell.

Antioxidant enzymes stimulation in Aspergillus parasiticus by Lentinula edodes inhibits aflatoxin production.

Biosynthesis of aflatoxins, toxic metabolites produced by Aspergillus parasiticus, is correlated to the fungal oxidative stress and cell ageing. In this paper, the mechanism underlying the aflatoxin-inhibiting effect of the Lentinula edodes culture filtrates was studied by analysing their anti-oxidant activity and beta-glucan content. Mushroom beta-glucans are pharmacologically active compounds stimulating anti-oxidant responses in animal cells. L. edodes lyophilised filtrates stimulate A. parasiticus anti-oxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase) and aflatoxin inhibition was better correlated with beta-glucan content than with anti-oxidant activity of the filtrates. RT-PCR analyses on treated mycelia showed a delay in the activation of aflR, and norA, genes of aflatoxin cluster and a synchronous activation of hsf2-like, a homologue of a yeast transcription factor involved in oxidative stress responses. The first evidence of hsf2-like in A. parasiticus and its activation during aflatoxin biosynthesis is reported. L. edodes filtrates could play a role as external stimulus affecting the anti-oxidant status in the fungal cell that, in turn, leads to aflatoxin inhibition. In the fungal cell, beta-glucans present in the filtrates could stimulate the activation of transcription factors related to anti-oxidant response and anti-oxidant enzyme activity with a contemporaneous delay of aflatoxin genes transcription, which led to a marked reduction of aflatoxin production. This research suggests new perspectives to set suitable strategies against aflatoxins and L. edodes could be considered a promising tool.