The CRZ1 transcription factor in plant fungi: regulation mechanism and impact on pathogenesis.

Calcium (Ca2+) is a universal signaling molecule that is tightly regulated, and a fleeting elevation in cytosolic concentration triggers a signal cascade within the cell, which is crucial for several processes such as growth, tolerance to stress conditions, and virulence in fungi. The link between calcium and calcium-dependent gene regulation in cells relies on the transcription factor Calcineurin-Responsive Zinc finger 1 (CRZ1). The direct regulation of approximately 300 genes in different stress pathways makes it a hot topic in host-pathogen interactions. Notably, CRZ1 can modulate several pathways and orchestrate cellular responses to different types of environmental insults such as osmotic stress, oxidative stress, and membrane disruptors. It is our belief that CRZ1 provides the means for tightly modulating and synchronizing several pathways allowing pathogenic fungi to install into the apoplast and eventually penetrate plant cells (i.e., ROS, antimicrobials, and quick pH variation). This review discusses the structure, function, regulation of CRZ1 in fungal physiology and its role in plant pathogen virulence.

Aflatoxins are natural scavengers of reactive oxygen species.

The role of aflatoxins (AFs) in the biology of producing strains, Aspergillus sect. Flavi, is still a matter of debate. Over recent years, research has pointed to how environmental factors altering the redox balance in the fungal cell can switch on the synthesis of AF. Notably, it has been known for decades that oxidants promote AF synthesis. More recent evidence has indicated that AF synthesis is controlled at the transcriptional level: reactive species that accumulate in fungal cells in the stationary growth phase modulate the expression of aflR, the main regulator of AF synthesis-through the oxidative stress related transcription factor AP-1. Thus, AFs are largely synthesized and secreted when (i) the fungus has exploited most nutritional resources; (ii) the hyphal density is high; and (iii) reactive species are abundant in the environment. In this study, we show that AFs efficiently scavenge peroxides and extend the lifespan of E. coli grown under oxidative stress conditions. We hypothesize a novel role for AF as an antioxidant and suggest its biological purpose is to extend the lifespan of AFs-producing strains of Aspergillus sect. Flavi under highly oxidizing conditions such as when substrate resources are depleted, or within a host.

Preclinical Antileukemia Activity of Tramesan: A Newly Identified Bioactive Fungal Metabolite.

Despite improvements that occurred in the last decades in the acute myeloid leukemia (AML) treatment, clinical results are still unsatisfactory. More effective therapies are required, and innovative approaches are ongoing, including the discovery of novel antileukemia natural compounds. Several studies have described the activity of extracts from mushrooms which produce compounds that exhibited immunological and antitumor activities. The latter has been demonstrated to be promoted in vitro by mushroom polysaccharides via induction of apoptosis. However, the antileukemia activity of these compounds on primary cells is still not reported. In the present study, we examined the in vitro effects of Tramesan (TR), a bioactive compound extracted from Trametes versicolor, on leukemic cell lines and primary cells. Our results demonstrated that TR induced a marked growth inhibition of leukemic cell lines and primary cells from AML patients. The antiproliferative effects of TR were associated in primary AML cells with a significant increase of apoptosis. No significant cytotoxic effects were observed in normal peripheral blood mononuclear cells (MNC) from healthy donors. Our data demonstrated a cytotoxic activity of TR on leukemia cells prompting further translational applications. Ongoing studies are elucidating the molecular mechanisms underlying its antileukemic activity.

Buckwheat achenes antioxidant profile modulates Aspergillus flavus growth and aflatoxin production.

Buckwheat (Fagopyrum spp.) is a "pseudo-cereal" of great interest in the production of healthy foods since its flour, derived from achenes, is enriched with bioactive compounds and, due to the absence of gluten, may be used in composition of celiac diets. Amongst buckwheat species, F. tataricum achenes possess a larger amount of the antioxidant flavenol rutin than the common buckwheat F. esculentum. Ongoing climate change may favor plant susceptibility to the attack by pathogenic, often mycotoxigenic, fungi with consequent increase of mycotoxins in previously unexploited feeds and foodstuffs. In particular, Aspergillus flavus, under suitable environmental conditions such as those currently occurring in Italy, may produce aflatoxin B1 (AFB1), the most carcinogenic compound of fungal origin which is classified by IARC as Category 1. In this study, the viable achenes of two buckwheat species, F. tataricum (var. Golden) and F. esculentum (var. Aelita) were inoculated with an AFB1-producing A. flavus NRRL 3357 to analyze their relative performances against fungal invasion and toxin contamination. Notably, we sought the existence of a correlation between the amount of tocols/flavonols in the achenes of buckwheat, infected and non-infected with A. flavus, and to analyze the ability of the pathogen to grow and produce toxin during achene infection. Results suggest that achenes of F. tataricum, the best producer of antioxidant compounds in this study, are less susceptible to A. flavus infection and consequently, but not proportionally, to mycotoxin contamination compared with F. esculentum. Moreover, rutin-derived quercetin appears to be more efficient in inhibiting aflatoxin biosynthesis than the parent compound.

Grape variety related trans-resveratrol induction affects Aspergillus carbonarius growth and ochratoxin A biosynthesis.

The paper reports the results of a study performed to investigate the influence of the grape variety on the growth of Aspergillus carbonarius on grape berries and the correlation between the amount of ochratoxin A (OTA) and the content of trans-resveratrol produced after fungal contamination. Variations in the amount of OTA produced by the fungus are observed depending on both grape variety and on the induction of trans-resveratrol determined during the infection. The obtained data suggest that if an increase in trans-resveratrol production in grape berries occurs early after the fungal infection, the berry exploits this compound to control OTA synthesis. If the increase in trans-resveratrol concentration is delayed after fungal infection (40 h), a control of OTA accumulation can not be achieved. The possibility of exerting significant control of OTA biosynthesis by this phytoalexin seems to rely in the promptness of its production, as occurs also in other fungus plant interactions and, in turn, seems to be dependent also on grape cultivar. In this fungus-plant system, trans-resveratrol appears to represent a defence-related compound toward A. carbonarius and OTA contamination.

Study on the role of patulin on pathogenicity and virulence of Penicillium expansum.

Although the antibacterial activity and toxicity to humans and animals of the mycotoxin patulin are well known, its role in the postharvest decay of apples by Penicillium expansum has never been investigated. In the present study the gene disruption technique was used to alter the sequence of 6-methyl-salicylic acid synthase, an enzyme involved in the first committed step of patulin biosynthesis. Thirty-nine mutants were obtained, however only two of them (M5 and M21) passed the sub-cultural and molecular confirmation tests. They proved to produce 33-41% less patulin than their wild-type (WT) strain, although no difference in the growth and morphology of the colony was observed. Moreover, the mutants showed a significantly reduced pathogenicity and virulence on artificially inoculated apples. In particular, a 33-34% and 47-54% reduction of disease incidence and severity were recorded for M5 and M21, respectively. As confirmation, when the biomass of the mutants was quantified in vivo by Real-time PCR, a significant difference was recorded as compared to the WT and even between mutants. Moreover, when patulin production potential of mutants was restored by exogenous application of the mycotoxin, their ability to cause the disease was not significantly different from that of WT. Finally, mutants showed an increased susceptibility to the application of the antioxidant quercetin, their pathogenicity and virulence being significantly reduced at only 1/100 of the concentration needed for the WT. Based on these findings, patulin seems to have a role in the development of blue mold decay on apples.

Early detection of toxigenic fungi on maize by hyperspectral imaging analysis.

Fungi can grow on many food commodities. Some fungal species, such as Aspergillus flavus, Aspergillus parasiticus, Aspergillus niger and Fusarium spp., can produce, under suitable conditions, mycotoxins, secondary metabolites which are toxic for humans and animals. Toxigenic fungi are a real issue, especially for the cereal industry. The aim of this work is to carry out a non destructive, hyperspectral imaging-based method to detect toxigenic fungi on maize kernels, and to discriminate between healthy and diseased kernels. A desktop spectral scanner equipped with an imaging based spectrometer ImSpector- Specim V10, working in the visible-near infrared spectral range (400-1000 nm) was used. The results show that the hyperspectral imaging is able to rapidly discriminate commercial maize kernels infected with toxigenic fungi from uninfected controls when traditional methods are not yet effective: i.e. from 48 h after inoculation with A. niger or A. flavus.

Early detection of Aspergillus carbonarius and A. niger on table grapes: a tool for quality improvement.

Aspergillus carbonarius and A. niger aggregate are the main fungal contaminants of table grapes. Besides their ability to cause black rot, they can produce ochratoxin A (OTA), a mycotoxin that has attracted increasing attention worldwide. The objective of this work was to set up a simple and rapid molecular method for the early detection of both fungi in table grapes before fungal development becomes evident. Polymerase chain reaction (PCR)-based assays were developed by designing species-specific primers based on the polyketide synthases (PKS(S)) sequences of A. carbonarius and A. niger that have recently been demonstrated to be involved in OTA biosynthesis. Three table grape varieties (Red globe, Crimson seedless, and Italia) were inoculated with A. carbonarius and A. niger aggregate strains producing OTA. The extracted DNA from control (non-inoculated) and inoculated grapes was amplified by PCR using ACPKS2F-ACPKS2R for A. carbonarius and ANPKS5-ANPKS6 for A. niger aggregate. Both primers allowed a clear detection, even in symptomless samples. PCR-based methods are considered to be a good alternative to traditional diagnostic means for the early detection of fungi in complex matrix for their high specificity and sensitivity. The results obtained could be useful for the definition of a 'quality label' for tested grapes to improve the safety measures taken to guarantee the production of fresh table grapes.

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.

DNA-fingerprinting (AFLP and RFLP) for genotypic identification in species of the Pleurotus eryngii complex.

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.

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.

beta-Glucan synthase induction in mushrooms grown on olive mill wastewaters.

beta-1-3-Glucan synthase activity and its induction by olive mill wastewaters (OMW) was studied in ten fungal strains (Auricularia auricula-judae, Lentinula edodes, Pleurotus eryngii, Stropharia aeruginosa, Agrocybe aegerita, P. pulmonarius, Armillaria mellea, P. ferulae, P. ostreatus, P. nebrodensis). A microtiter-based enzymatic assay on beta-1-3-glucan synthase activity was carried out on all mycelia growth both on the control medium and on OMW. Among the fungi assayed, L. edodes beta-1-3-glucan synthase was highly enhanced in OMW. The main components of OMW, i.e. phenols and lipids, were added separately to the control medium, to highlight the mechanism of L. edodes beta-1-3-glucan synthase induction. A Southern blot analysis and PCR with degenerated primers were carried out to detect the presence of fks1-like genes in these Basidiomycetes. The sequences obtained from the ten Basidiomycota were remarkably similar to fks1 from Filobasidiella neoformans. Spectrofluorimetric and RT-PCR analyses of beta-1-3-glucan synthase were performed on the mycelia of L. edodes. In this fungus, a strong stimulation of beta-1-3-glucan synthase mRNA and protein was recorded in the presence of OMW and phenols.

Genetic diversity and population structure of the Italian fungi belonging to the taxa Pleurotus eryngii (DC.:Fr.) Quèl and P. ferulae (DC.:Fr.) Quèl.

A study using allozymes and PCR fingerprinting was conducted to estimate the genetic diversity of Italian populations of two economically important cultivated fungal taxa, Pleurotus eryngii and P. ferulae. Very little is known about the genetic diversity distribution pattern of these taxa. Heterozygote deficiency was observed at few loci; in fact the inbreeding coefficients were not high, which demonstrates that mechanisms restrain the inbreeding act at the local level. Estimates of genetic differentiation indicated a pattern of greater variation within, rather than between, populations. These results were supported by AMOVA analysis, which attributed a low proportion of the total genetic variation to large geographical scale divergence, and indicated that most of the genetic diversity was because of differences within populations. This distribution pattern of genetic variation of P. eryngii and P. ferulae populations seems to be the result of high gene flow, by efficient basidiospore dispersal, and outcrossing mechanisms, which restrain inbreeding within populations.

Oxidative stress, growth factor production and budding in potato tubers during cold storage.

In order to verify the role played by oxidation in the budding of potato tubers (Solanum tuberosum L. cv. Kennebec), the physiological events occurring below bud at 4 degrees C have been studied for a period of 6 months. The low temperature storage induced an increase in the degree of unsaturation and a decrease in the ratio of saturated/unsaturated fatty acids of membrane polar lipids with a subsequent increase of lipid hydroperoxides (LOOH). Cold stress increased both enzymatic antioxidative activities (superoxide dismutase, SOD, E.C.1.15.1.1; catalase, CAT, E.C.1.11.1.6), and alpha-tocopherol levels thus protecting membrane's polyunsaturated lipids. Between 0 and 15 days of storage SOD/CAT ratio, alpha-tocopherol, LOOH levels and the degree of lipid unsaturation showed strong variations. After 30 to 120/150 days the antioxidative system seemed to reach a homeostasis different from that of time 0, accompanied by a constant increase of indole-3-acetic acid (IAA) after 60 days. The antioxidative system, after 150 days, lost its efficiency while LOOH levels were maintained higher than time 0 and IAA concentration was sufficient to allow sprouting.

Early physiological and cytological events induced by wounding in potato tuber.

The response of potato tuber (Solanum tuberosum L. cv. Kennebec) to mechanical wounding was investigated at different times. Changes in the levels of indole-3-acetic acid (IAA), polyunsaturated fatty acids (PUFAs) and lipid hydroperoxides (LOOHs) were monitored up to 120 min after wounding and related to the cytological events occurring up to 24 h. Twenty minutes after injury, an increase in IAA and LOOH levels and a decrease in the levels of PUFAs was observed. Wounding induced mitoses in differentiated (parenchyma) cells starting at 120 min, and promoted an increase of mitotic activity in the meristematic cells (procambium and bud dome), after 360 min. The inhibition of the increase in LOOHs and IAA by lipoxygenase (LOX) inhibitors, as well as the ability of in vitro peroxidated linoleic acid to enhance IAA production, suggest a close relationship among lipoperoxidation, IAA and mitotic activity in the response of potato tuber cells to injury, resulting in a specific growth response, i.e. bud growth and periderm formation.

Parma ham: Correlation between myotypological parameters of Biceps femoris muscle and qualitative characteristics of fresh and seasoned product.

The morphofunctional characterisation of the Biceps femoris muscle was studied in 128 pigs intended for use as Parma ham, as a means of evaluating the raw material in relation to its transformation into a seasoned product answering to the requirements of its trademark. Organoleptic tests were carried out on both fresh and seasoned product, and the seasoned product was chemically characterised. The following defects in fresh hams-muscle which was pale in colour and of unsatisfactory firmness, insufficent compactness, poor thickness of fat cover layer-were found to be strongly correlated to the increase in white fibres of the IIa type; no links were found, however, between the measurement of myofibre diameters and these defects. Principal component analysis, applied to the complete set of histoenzymatic and organoleptic variables considered, reveals the qualitative characteristics of seasoned ham as having no close correlation with the histoenzymatic parameters of the fresh product; the authors nevertheless hope that myotypological examination will become one of the parameters in the evaluation of meat quality.