Valeria Scala

How Peroxisomes Affect Aflatoxin Biosynthesis in Aspergillus Flavus

In filamentous fungi, peroxisomes are crucial for the primary metabolism and play a pivotal role in the formation of some secondary metabolites. Further, peroxisomes are important site for fatty acids β-oxidation, the formation of reactive oxygen species and for their scavenging through a complex of antioxidant activities. Oxidative stress is involved in different metabolic events in all organisms and it occurs during oxidative processes within the cell, including peroxisomal β-oxidation of fatty acids. In Aspergillus flavus, an unbalance towards an hyper-oxidant status into the cell is a prerequisite for the onset of aflatoxin biosynthesis. In our preliminary results, the use of bezafibrate, inducer of both peroxisomal β-oxidation and peroxisome proliferation in mammals, significantly enhanced the expression of pex11 and foxA and stimulated aflatoxin synthesis in A. flavus. This suggests the existence of a correlation among peroxisome proliferation, fatty acids β-oxidation and aflatoxin biosynthesis. To investigate this correlation, A. flavus was transformed with a vector containing P33, a gene from Cymbidium ringspot virus able to induce peroxisome proliferation, under the control of the promoter of the Cu,Zn-sod gene of A. flavus. This transcriptional control closely relates the onset of the antioxidant response to ROS increase, with the proliferation of peroxisomes in A. flavus. The AfP33 transformant strain show an up-regulation of lipid metabolism and an higher content of both intracellular ROS and some oxylipins. The combined presence of a higher amount of substrates (fatty acids-derived), an hyper-oxidant cell environment and of hormone-like signals (oxylipins) enhances the synthesis of aflatoxins in the AfP33 strain. The results obtained demonstrated a close link between peroxisome metabolism and aflatoxin synthesis.

Lipids in Aspergillus flavus-maize interaction

In some filamentous fungi, the pathways related to the oxidative stress and oxylipins production are involved both in the process of host-recognition and in the pathogenic phase. In fact, recent studies have shown that the production of oxylipins in filamentous fungi, yeasts and chromists is also related to the development of the organism itself and to mechanisms of communication with the host at the cellular level. The oxylipins, also produced by the host during defense reactions, are able to induce sporulation and to regulate the biosynthesis of mycotoxins in several pathogenic fungi. In A. flavus, the oxylipins play a crucial role as signals for regulating the biosynthesis of aflatoxins, the conidiogenesis and the formation of sclerotia. To investigate the involvement of an oxylipins based cross-talk into Z. mays and A. flavus interaction, we analyzed the oxylipins profile of the wild type strain and of three mutants of A. flavus that are deleted at the Aflox1 gene level also during maize kernel invasion. A lipidomic approach has been addressed through the use of LC-ToF-MS, followed by a statistical analysis of the principal components (PCA). The results showed the existence of a difference between the oxylipins profile generated by the WT and the mutants onto challenged maize. In relation to this, aflatoxin synthesis which is largely hampered in vitro, is intriguingly restored. These results highlight the important role of maize oxylipin in driving secondary metabolism in A. flavus.

Genotypic and Phenotypic Versatility of Aspergillus flavus during Maize Exploitation

Aspergillus flavus is a cosmopolitan fungus able to respond to external stimuli and to shift both its trophic behaviour and the production of secondary metabolites, including that of the carcinogen aflatoxin (AF). To better understand the adaptability of this fungus, we examined genetic and phenotypic responses within the fungus when grown under four conditions that mimic different ecological niches ranging from saprophytic growth to parasitism. Global transcription changes were observed in both primary and secondary metabolism in response to these conditions, particularly in secondary metabolism where transcription of nearly half of the predicted secondary metabolite clusters changed in response to the trophic states of the fungus. The greatest transcriptional change was found between saprophytic and parasitic growth, which resulted in expression changes in over 800 genes in A. flavus. The fungus also responded to growth conditions, putatively by adaptive changes in conidia, resulting in differences in their ability to utilize carbon sources. We also examined tolerance of A. flavus to oxidative stress and found that growth and secondary metabolism were altered in a superoxide dismutase (sod) mutant and an alkyl-hydroperoxide reductase (ahp) mutant of A. flavus. Data presented in this study show a multifaceted response of A. flavus to its environment and suggest that oxidative stress and secondary metabolism are important in the ecology of this fungus, notably in its interaction with host plant and in relation to changes in its lifestyle (i.e. saprobic to pathogenic).

LDS1-produced oxylipins are negative regulators of growth, conidiation and fumonisin synthesis in the fungal maize pathogen Fusarium verticillioides

Oxylipins are fatty acid-derived signaling compounds produced by all eukaryotes so far investigated; in mycotoxigenic fungi, they modulate toxin production and interactions with the host plants. Among the many enzymes responsible for oxylipin generation, Linoleate Diol Synthase 1 (LDS1) produces mainly 8-hydroperoxyoctadecenoic acid and subsequently different di-hydroxyoctadecenoic acids. In this study, we inactivated a copy of the putative LDS1 ortholog (acc. N. FVEG_09294.3) of Fusarium verticillioides, with the aim to investigate its influence on the oxylipin profile of the fungus, on its development, secondary metabolism and virulence. LC-MS/MS oxylipin profiling carried out on the selected mutant strain revealed significant quali-quantitative differences for several oxylipins when compared to the WT strain. The Fvlds1-deleted mutant grew better, produced more conidia, synthesized more fumonisins and infected maize cobs faster than the WT strain. We hypothesize that oxylipins may act as regulators of gene expression in the toxigenic plant pathogen F. verticillioides, in turn causing notable changes in its phenotype. These changes could relate to the ability of oxylipins to re-shape the transcriptional profile of F. verticillioides by inducing chromatin modifications and exerting a direct control on the transcription of secondary metabolism in fungi.

Quantitative profiling of oxylipins through comprehensive LC-MS/MS analysis of Fusarium verticillioides and maize kernels

Fusarium verticillioides is one of the most important fungal pathogens causing ear and stalk rot in maize, even if frequently asymptomatic, producing a harmful series of compounds named fumonisins. Plant and fungal oxylipins play a crucial role in determining the outcome of the interaction between the pathogen and its host. Moreover, oxylipins result as signals able to modulate the secondary metabolism in fungi. In keeping with this, a novel, quantitative LC-MS/MS method was designed to quantify up to 17 different oxylipins produced by F. verticillioides and maize kernels. By applying this method, we were able to quantify oxylipin production in vitro – F. verticillioides grown into Czapek–Dox/yeast extract medium amended with 0.2% w/v of cracked maize – and in vivo, i.e. during its growth on detached mature maize ears. This study pinpoints the role of oxylipins in a plant pathogen such as F. verticillioides and sets up a novel tool aimed at understanding the role oxylipins play in mycotoxigenic pathogens during their interactions with respective hosts.

Fusarium verticillioides and maize interaction in vitro: relationship between oxylipin cross-talk and fumonisin synthesis

Fusarium verticillioides is one of the most important fungal pathogens causing ear and stalk rot in maize. Even if frequently asymptomatic, it can produce a harmful series of compounds named fumonisins. Plant and fungal oxylipins play a crucial role in determining the outcome of the interaction between the pathogen and its host. Moreover, oxylipins are factors able to modulate the secondary metabolism in fungi. To uncover the existence of the relationship between oxylipin production and fumonisin synthesis in F. verticillioides, we analysed some molecular and physiological parameters, such as the expression of genes whose products are related to oxylipin synthesis (i.e. lipoxygenase, diol synthases and fatty acid oxidase), the oxylipin profile of both cracked maize and the pathogen by using a lipidomic approach (i.e. combining LC-TOF and LC-MS/MS approaches with a robust statistical analysis) and the synthesis of fumonisin B1. The results suggested a close relationship between the modification of the path...

Maize lipids play a pivotal role in the fumonisin accumulation

The role of lipids in maize – Fusarium verticillioides interaction and fumonisin production in natural field conditions were investigated. In 2010, ten maize hybrids were grown in fields located in 3 districts in Northern Italy and sampled at 4 growing stages, from early dough to full ripe. Chemical composition, fungal incidence and free and hidden fumonisin contamination were determined in all grain samples. All the hybrids considered within this study showed a strong fungal incidence, with Fusarium section Liseola as prevalent, already at the early dough maturity and along the ripening period. Fumonisins accumulated over the growing season and reached the maximum level at the full ripe stage; hidden fumonisins were found significant in all the considered samples (~57% of the free form at harvest). Hybrid H9 showed more than 50% of kernels infected by Aspergillus flavus and no hidden fumonisins were detected. This finding stresses the relevance of monitoring both free and total fumonisins for a comprehen...

Aflatoxin Control in Maize by Trametes versicolor

Aspergillus flavus is a well-known ubiquitous fungus able to contaminate both in pre- and postharvest period different feed and food commodities. During their growth, these fungi can synthesise aflatoxins, secondary metabolites highly hazardous for animal and human health. The requirement of products with low impact on the environment and on human health, able to control aflatoxin production, has increased. In this work the effect of the basidiomycete Trametes versicolor on the aflatoxin production by A. flavus both in vitro and in maize, was investigated. The goal was to propose an environmental loyal tool for a significant control of aflatoxin production, in order to obtain feedstuffs and feed with a high standard of quality and safety to enhance the wellbeing of dairy cows. The presence of T. versicolor, grown on sugar beet pulp, inhibited the production of aflatoxin B1 in maize by A. flavus. Furthermore, treatment of contaminated maize with culture filtrates of T. versicolor containing ligninolytic enzymes, showed a significant reduction of the content of aflatoxin B1.

ROS and 9-oxylipins are correlated with deoxynivalenol accumulation in the germinating caryopses of Triticum aestivum after Fusarium graminearum infection

Wheat germinating caryopses may represent a starting point for the Fusarium Head Blight disease; however, only few studies concern the defence repertoire of wheat caryopses against fungal challenge. The germinating caryopses of two wheat commercial varieties (Blasco and Sagittario), differentially susceptible to FHB in the field, were inoculated with F. graminearum and the redox status in the interaction milieu, oxylipin production, the expression profile of some host-defence related genes, and programmed cell death in the aleuronic layer, were analysed. In Sagittario, the redox balance was profoundly modified and 9-oxylipins accumulated during fungal contamination. In this variety, F. graminearum produced a high quantity of deoxynivalenol whilst programmed cell death, also through metacaspases activation, was enhanced in the aleuronic layer of its caryopses. In Blasco, the expression of tolerance factors such as Pathogenesis-Related-protein1, glucosyl-transferase and glutathione transferase genes was up-regulated consequent to infection. Results show that unscavenged ROS and 9-oxylipins may be related to deoxynivalenol accumulation in the germinating caryopses of wheat after F. graminearum infection.

Climate, Soil Management, and Cultivar Affect Fusarium Head Blight Incidence and Deoxynivalenol Accumulation in Durum Wheat of Southern Italy

Fusarium head blight (FHB) is a multifaceted disease caused by some species of Fusarium spp. A huge production of mycotoxins, mostly trichothecenes, often accompanied this disease. Amongst these toxic compounds, deoxynivalenol (DON) and its derivatives represent a major issue for human as well as for animal health and farming. Common and durum wheat are amongst the hosts of trichothecene-producing Fusaria. Differences in susceptibility to fungal infection and toxin accumulation occur in wheat cultivars. Recently, increasing incidence and severity of Fusarium infection and a higher DON accumulation in durum wheat were observed in Italy, especially in Northern regions. In this study, we analyzed wheat yield, technological parameters, the incidence of Fusarium infection and DON content in kernel samples of durum wheat coming from three locations of Southern Italy with different climatic conditions and grown during two seasons, with two methods of cultivation. Four different durum wheat cultivars prevalently cultivated in Southern Italian areas were chosen for this study. Our analysis showed the effects of environment and cultivar types on wheat productivity and key technological parameters for the quality level of the end-product, namely pasta. Notably, although a low rate of mycotoxin contamination in all study sites was assessed, an inverse relation emerged between fungal infection/DON production and durum wheat yield. Further, our study pinpoints the importance of environment conditions on several quality traits of durum wheat grown under Mediterranean climate. The environmental conditions at local level (microscale) and soil management practices may drive FHB outbreak and mycotoxin contamination even in growing area suitable for cropping this wheat species.