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After providing a picture of the global transcriptomic changes of grapevine responses to “stolbur” phytoplasma, the recovery status and molecular responses to the phytoplasma and virus co-presence were analyzed. NimbleGen ® Vitis vinifera genome arrays were used. Lower transcript abundance of the genes involved in photosynthesis, trehalose, phospholipids was observed in response to the presence of “stolbur” phytoplasma. The expression of the genes involved in tetrapyrrole increased. The recovered plants showed that the transcripts involved in ATP synthesis and amino acid metabolism, secondary metabolism and biotic stress-related pathways increased. Recovery was associated with tetrapyrrole pathway repression. Co-infection with viruses induced the genes involved in the hormone categories (cytokinin, gibberellin, salicylic acid and jasmonates).

In Aspergillus nidulans, Aspergillus flavus, and Aspergillus parasiticus, lipoperoxidative signalling is crucial for the regulation of mycotoxin biosynthesis, conidiogenesis, and sclerotia formation. Resveratrol, which is a lipoxygenase (LOX) and cyclooxygenase inhibitor, downmodulates the biosynthesis of ochratoxin A (OTA) in Aspergillus ochraceus. In the genome of A. ochraceus, a lox-like sequence (AoloxA ; National Center for Biotechnology Information (NCBI) accession number: DQ087531) for a lipoxygenase-like enzyme has been found, which presents high homology (100 identities, 100 positives %, score 555) with a lox gene of Aspergillus fumigatus (NCBI accession number: XM741370). To study how inhibition of oxylipins formation may affect the A. ochraceus metabolism, we have used a DeltaAoloxA strain. This mutant displays a different colony morphology, a delayed conidia formation, and a high sclerotia production. When compared to the wild type, the DeltaAoloxA strain showed a lower basal activity of LOX and diminished levels of 13-hydroperoxylinoleic acid (HPODE) and other oxylipins derived from linoleic acid. The limited oxylipins formation corresponded to a remarkable inhibition of OTA biosynthesis in the DeltaAoloxA strain. Also, wheat seeds (Triticum durum cv Ciccio) inoculated with the DeltaAoloxA mutant did not accumulate 9-HPODE, which is a crucial element in the host defence system. Similarly, the expression of the pathogenesis-related protein 1 (PR1) gene in wheat seeds was not enhanced. The results obtained contribute to the current knowledge on the role of lipid peroxidation governed by the AoloxA gene in the morphogenesis, OTA biosynthesis, and in host-pathogen interaction between wheat seeds and A. ochraceus.

Pleurotus eryngii and P. ferulae, two species belonging to the P. eryngii complex, synthesize laccases, ligninolytic enzymes that play a role in the host-pathogen interaction in the first step of infection. Ecological studies have shown that although both fungi have been recognized as saprophytes, P. eryngii weakly pathogenic when colonizing the roots and stems of Eryngium campestre, whereas P. ferulae is mostly pathogenic to Ferula communis. The paper describes the genomic organization of four putative laccase genes (lac1, lac2, lac3, and lac5-like gene; gene names were assigned on the basis of sequence homologies) of P. eryngii and P. ferulae. The mRNA expression and enzymatic activity of the laccases were analysed under culture conditions where a source of lignin (wheat bran) or lyophilized roots of E. campestre or F. communis were present. These experiments indicated that the four lac-like genes were differentially regulated in the two mushrooms. Specifically, the addition of the lyophilized roots of the respective host plant to the culture media induced an advance in the mRNA expression of the four lac-like genes and a seven-fold higher total laccase activity in P. ferulae than in P. eryngii. The results obtained are discussed in relation to the possible role of laccases in the interaction of P. eryngii and P. ferulae with their respective host.

P-glycoproteins (P-gps) are efflux transporters found in cells of a broad range of both procaryotic and eukaryotic taxa, whose action is to relieve the cells of multiple, structurally dissimilar, toxic compounds. The possible role of P-gps in defence against the insecticides temephos and diflubenzuron was investigated in the mosquito Aedes caspius (Pallas), also known as Ochlerotatus (Aedes) caspius (Diptera: Culicidae), and the genomic DNA sequences encoding for P-gp transporters were isolated to provide molecular instruments for future research into the expression and characterization of genes codifying for P-gps in this mosquito species. Mosquito larvae were treated with insecticides alone and in conjunction with a sublethal dose of the P-gp inhibitor verapamil. The inhibition of P-gps reduced the LD(50) values of temephos and diflubenzuron by factors of 3.5 and 16.4, respectively, suggesting the potential involvement of P-gps in insecticide defence. Using a polymerase chain reaction (PCR)-based approach, a 481-bp sequence was isolated. The inferred nucleotide sequence shows high homology with the C-terminal sequence of known P-gps. The isolation and characterization of a putative P-gp sequence from Ae. caspius is the first step towards a better molecular understanding of the role played by multidrug transporters in the defence against insecticides in this species. This knowledge may open the way to a novel control strategy based on the inhibition of pest defences. The beneficial consequences of the inhibition of efflux pumps in improving insecticide performance are discussed.

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.

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.

Among the various factors correlated with toxin production in fungi, oxidative stress is a crucial one. In relation to this, an important role is played by oxidative stress-related receptors. These receptors can transduce the "oxidative message" to the nucleus and promote a transcriptional change targeted at restoring the correct redox balance in the cell. In Aspergillus parasiticus, the knockout of the ApyapA gene, a homologue of the yeast Yap-1, disables the fungus's capacity to restore the correct redox balance in the cell. As a consequence, the onset of secondary metabolism and aflatoxins synthesis is triggered. Some clues as to the involvement of oxidative stress in the regulation of ochratoxin A (OTA) synthesis in Aspergillus ochraceus have already been provided by the disruption of the oxylipin-producer AoloxA gene. In this paper, we add further evidence that oxidative stress is also involved in the regulation of OTA biosynthesis in A. ochraceus. In fact, the use of certain oxidants and, especially, the deletion of the yap1-homologue Aoyap1 further emphasize the role played by this stress in controlling metabolic and morphological changes in A. ochraceus. © 2012 Springer-Verlag.

Oxidative stress is recognized as a trigger of different metabolic events in all organisms. Various factors correlated with oxidation, such as the β-oxidation of fatty acids and their enzymatic or nonenzymatic by-products (e.g., precocious sexual inducer factors and lipoperoxides) have been shown to be involved in aflatoxin formation. In the present study, we found that increased levels of reactive oxygen species (ROS) were correlated with increased levels of aflatoxin biosynthesis in Aspergillus parasiticus . To better understand the role of ROS formation in toxin production, we generated a mutant (ΔAp yapA ) having the Ap yapA gene deleted, given that Ap yapA orthologs have been shown to be part of the antioxidant response in other fungi. Compared to the wild type, the mutant showed an increased susceptibility to extracellular oxidants, as well as precocious ROS formation and aflatoxin biosynthesis. Genetic complementation of the ΔAp yapA mutant restored the timing and quantity of toxin biosynthesis to the levels found in the wild type. The presence of putative AP1 (ApYapA orthologue) binding sites in the promoter region of the regulatory gene aflR further supports the finding that ApYapA plays a role in the regulation of aflatoxin biosynthesis. Overall, our results show that the lack of Ap yapA leads to an increase in oxidative stress, premature conidiogenesis, and aflatoxin biosynthesis.

Wild populations of edible species are important source of genetic variability for cultivated lines that can undergo a drastic loss of diversity resulting from man's selection. The development of tools aimed at the clear-cut and safe identification and assessment of genetic variability of the wild and cultivated strains is thus a fundamental goal of molecular genetic research. In this study, we used two polymerase chain reaction (PCR)-based fingerprinting methods-amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) of laccase and manganese peroxidase genes-to assess genetic differences among strains and independently evolving lineages belonging to the Pleurotus eryngii complex. Both laccase RFLP and AFLP have been proved to distinguish unambiguously the three taxa studied: Pleurotus ferulae, P. eryngii, and P. eryngii var. nebrodensis. AFLP also showed enough sensitivity to detect polymorphisms among the strains, proving to be an efficient DNA fingerprinting tool in studies of strain assignment. The divergent RFLP laccase and manganese peroxidase patterns are also discussed in relation to the role played by these genes in the interaction between these fungi and their host plants.