Effective chemical management for prevention of aflatoxins in maize

The presence of aflatoxins in maize grain has been an increasing problem in the Mediterranean area, possibly due to climate change such as increased temperatures and extended drought periods. It is therefore important to prevent the growth of aflatoxigenic Aspergillus species in the field. There are no fungicides registered for control A. flavus in maize, so this study investigated the efficacy of azoxystrobin, boscalid, cyprodinil, fludioxonil and cyprodinil + fludioxonil to reduce A. flavus growth, sporulation and aflatoxin production in in vitro, and in maize field studies. Based on in vitro inhibition of mycelial growth, the most effective fungicides were cyprodinil (EC50 < 0.05 μg mL-1) and fludioxonil (EC50 <0.11 μg mL-1), while the least effective was boscalid (EC50 4.35-4.50 μg mL-1). Azoxystrobin almost completely inhibited the conidium germination at > 0.5 μg mL-1. Further evaluation of the fungicides on maize seeds infected with A. flavus demonstrated that all the fungicides reduced conidium production by 76 to 94%, and reduced aflatoxin contamination. In a 2-year field study, application of cyprodinil + fludioxonil reduced A. flavus ear rot severity by 40%, and was the most effective formulation for reducing aflatoxin contamination, by 83%. The other four single ingredient fungicides also decreased aflatoxin production on maize kernels (fludioxonil by 80%, cyprodinil by 75%, boscalid by 74% and azoxystrobin by 67%). Field data from this study provide farmers with a new effective chemical approach to control A. flavus and aflatoxin production in maize within an integrated strategy for management of aflatoxins in maize

Analysis of volatile emissions from grape berries infected with Aspergillus carbonarius using hyphenated and portable mass spectrometry

Mycotoxins represent a serious risk for human and animal health. Οchratoxin A (OTA) is a carcinogenic mycotoxin produced by A. carbonarius that constitutes a severe problem for viticulture. In this study, we investigate the development of novel detection and on-line monitoring approaches for the detection of OTA in the field (i.e. out of the chemical laboratory) using advanced molecular sensing. Both stand-alone and hyphenated mass spectrometry (MS) based systems (e.g. Time-of-Flight ToF–MS and gas chromatography GC combined with MS) and compact portable membrane inlet MS (MIMS) have been employed for the first time to detect and monitor volatile emissions of grape berries infected by the fungus Aspergillus carbonarius. In vacuo (electron impact—EI) and ambient ionisation (electrospray ionisation—ESI) techniques were also examined. On-line measurements of the volatile emissions of grape berries, infected by various strains of A. carbonarius with different toxicity levels, were performed resulting in different olfactory chemical profiles with a common core of characteristic mass fragments, which could be eventually used for on-site detection and monitoring allowing consequent improvement in food security.ISSN:2045-232

The Transcriptional Repressor TupA in Aspergillus niger Is Involved in Controlling Gene Expression Related to Cell Wall Biosynthesis, Development, and Nitrogen Source Availability.

The Tup1-Cyc8 (Ssn6) complex is a well characterized and conserved general transcriptional repressor complex in eukaryotic cells. Here, we report the identification of the Tup1 (TupA) homolog in the filamentous fungus Aspergillus niger in a genetic screen for mutants with a constitutive expression of the agsA gene. The agsA gene encodes a putative alpha-glucan synthase, which is induced in response to cell wall stress in A. niger. Apart from the constitutive expression of agsA, the selected mutant was also found to produce an unknown pigment at high temperatures. Complementation analysis with a genomic library showed that the tupA gene could complement the phenotypes of the mutant. Screening of a collection of 240 mutants with constitutive expression of agsA identified sixteen additional pigment-secreting mutants, which were all mutated in the tupA gene. The phenotypes of the tupA mutants were very similar to the phenotypes of a tupA deletion strain. Further analysis of the tupA-17 mutant and the DeltatupA mutant revealed that TupA is also required for normal growth and morphogenesis. The production of the pigment at 37 degrees C is nitrogen source-dependent and repressed by ammonium. Genome-wide expression analysis of the tupA mutant during exponential growth revealed derepression of a large group of diverse genes, including genes related to development and cell wall biosynthesis, and also protease-encoding genes that are normally repressed by ammonium. Comparison of the transcriptome of up-regulated genes in the tupA mutant showed limited overlap with the transcriptome of caspofungin-induced cell wall stress-related genes, suggesting that TupA is not a general suppressor of cell wall stress-induced genes. We propose that TupA is an important repressor of genes related to development and nitrogen metabolism

Genome Sequencing and Comparative Transcriptomics of the Model Entomopathogenic Fungi Metarhizium anisopliae and M. acridum

Metarhizium spp. are being used as environmentally friendly alternatives to chemical insecticides, as model systems for studying insect-fungus interactions, and as a resource of genes for biotechnology. We present a comparative analysis of the genome sequences of the broad-spectrum insect pathogen Metarhizium anisopliae and the acridid-specific M. acridum. Whole-genome analyses indicate that the genome structures of these two species are highly syntenic and suggest that the genus Metarhizium evolved from plant endophytes or pathogens. Both M. anisopliae and M. acridum have a strikingly larger proportion of genes encoding secreted proteins than other fungi, while ∼30% of these have no functionally characterized homologs, suggesting hitherto unsuspected interactions between fungal pathogens and insects. The analysis of transposase genes provided evidence of repeat-induced point mutations occurring in M. acridum but not in M. anisopliae. With the help of pathogen-host interaction gene database, ∼16% of Metarhizium genes were identified that are similar to experimentally verified genes involved in pathogenicity in other fungi, particularly plant pathogens. However, relative to M. acridum, M. anisopliae has evolved with many expanded gene families of proteases, chitinases, cytochrome P450s, polyketide synthases, and nonribosomal peptide synthetases for cuticle-degradation, detoxification, and toxin biosynthesis that may facilitate its ability to adapt to heterogenous environments. Transcriptional analysis of both fungi during early infection processes provided further insights into the genes and pathways involved in infectivity and specificity. Of particular note, M. acridum transcribed distinct G-protein coupled receptors on cuticles from locusts (the natural hosts) and cockroaches, whereas M. anisopliae transcribed the same receptor on both hosts. This study will facilitate the identification of virulence genes and the development of improved biocontrol strains with customized properties

Biological control strategies of mycotoxigenic fungi and associated mycotoxins in Mediterranean basin crops

Fungi that belong to the genera Aspergillus, Fusarium, and Penicillium pose serious phytopathological and mycotoxicological risks at pre-harvest and post-harvest stages, as well as in processed food products because they can produce several mycotoxins. Mycotoxins pose a serious problem for animal and human health and have a significant economic impact worldwide. The Mediterranean basin is a large geographical region with a temperate climate supporting the cultivation of a wealth of field and greenhouse crops with a high risk of mycotoxin contamination. The most important mycotoxins that occur in the Mediterranean basin are aflatoxins (B1, B2, G1 and G2) in dried fruits and nuts, ochratoxin A in grapes and raisins as well as trichothecenes and fumonisins in cereals. A variety of chemical, biological and physical strategies have been developed to control the mycotoxigenic pathogens; to minimize mycotoxin production at pre- or post-harvest level; to contribute to decontamination and/or detoxification of mycotoxins from contaminated foods and feeds; or to inhibit mycotoxin absorption in the gastrointestinal tract. Biological control using microbial antagonists either alone or as part of an integrated control strategy to reduce pesticide inputs, has emerged as a promising approach for control of mycotoxins in crops, both pre- and post-harvest. Several organisms including atoxigenic Aspergilli, yeasts, bacteria and fungi have been tested for their ability to reduce both fungal infection and mycotoxin contamination. For instance, atoxigenic fungal strains are being used widely to prevent pre-harvest aflatoxin contamination of crops such as peanuts, pistachios, maize, and cottonseed in several parts of the world including the Mediterranean area. Recent advancements in the use of biocontrol strategies have led to registration of commercial products with increased practical applications for the benefit of growers in several countries

Endogenous Lipogenic Regulators of Spore Balance in Aspergillus nidulans

The ability of fungi to produce both meiospores and mitospores has provided adaptive advantages in survival and dispersal of these organisms. Here we provide evidence of an endogenous mechanism that balances meiospore and mitospore production in the model filamentous fungus Aspergillus nidulans. We have discovered a putative dioxygenase, PpoC, that functions in association with a previously characterized dioxygenase, PpoA, to integrate fatty acid derived oxylipin and spore production. In contrast to PpoA, deletion of ppoC significantly increased meiospore production and decreased mitospore development. Examination of the PpoA and PpoC mutants indicate that this ratio control is associated with two apparent feedback loops. The first loop shows ppoC and ppoA expression is dependent upon, and regulates the expression of, nsdD and brlA, genes encoding transcription factors required for meiospore or mitospore production, respectively. The second loop suggests Ppo oxylipin products antagonistically signal the generation of Ppo substrates. These data support a case for a fungal “oxylipin signature-profile” indicative of relative sexual and asexual spore differentiation

AFLA-PISTACHIO: Development of a Mechanistic Model to Predict the Aflatoxin Contamination of Pistachio Nuts

In recent years, very many incidences of contamination with aflatoxin B1 (AFB1) in pistachio nuts have been reported as a major global problem for the crop. In Europe, legislation is in force and 12 μg/kg of AFB1 is the maximum limit set for pistachios to be subjected to physical treatment before human consumption. The goal of the current study was to develop a mechanistic, weather-driven model to predict Aspergillus flavus growth and the AFB1 contamination of pistachios on a daily basis from nut setting until harvest. The planned steps were to: (i) build a phenology model to predict the pistachio growth stages, (ii) develop a prototype model named AFLA-pistachio (model transfer from AFLA-maize), (iii) collect the meteorological and AFB1 contamination data from pistachio orchards, (iv) run the model and elaborate a probability function to estimate the likelihood of overcoming the legal limit, and (v) manage a preliminary validation. The internal validation of AFLA-pistachio indicated that 75% of the predictions were correct. In the external validation with an independent three-year dataset, 95.6% of the samples were correctly predicted. According to the results, AFLA-pistachio seems to be a reliable tool to follow the dynamic of AFB1 contamination risk throughout the pistachio growing season