Welcome to IMC 2018 International Mycological Congress
Mycotoxins and food security: deciphering the impacts of climate change scenarios
- N. Magan
- C. Verheecke-Vaesson
- E. Garcia-Cela
- A. Medina
There is interest in the impacts that climate change (CC) factors will have on the infection of staple food commodities by fungal diseases, pre-harvest and post-harvest. This applies to contamination of staple commodities with spoilage and mycotoxigenic moulds. These will have an impact on the food security agenda. CC is due to the interaction between three key environmental factors of elevated CO2 (400 vs 800/1200 ppm), temperature increases (+2-4oC) and drought stress. There is now some evidence that CC impacts on plant physiology including growth and yield of staple crops. We have been particularly interested in the impact that changes in CC scenarios may have on the growth/mycotoxin production by key spoilage fungi in staple food commodities. Thus, we have examined the effect of CC environmental factors on growth and mycotoxin production by Fusarium graminearum and F.langsethiae (type B and type A trichothecenes respectively), Aspergillus flavus (aflatoxins) and A.westerdijkiae and A.carbonarius (ochratoxin A). We have examined the impact that CC factors may have on growth as well as gene clusters involved in mycotoxin production. For example, by using RNAseq and information on aflatoxin B1 production we have been able to examine the impact that such CC environmental actors may have on functioning of the biosynthesis of aflatoxins and other key secondary metabolites. Studies on mycotoxigenic Aspergillus species colonising coffee and pistachio nuts, suggest differential effects on mycotoxin contamination in vitro and in situ when exposed to CC conditions. In addition, acclimatisation to CC conditions needs to be considered. This could also have implications for the legislative limits for mycotoxins in certain food commodities. These results will be discussed in the context of the food security agenda and the implications that CC scenarios may have on the resilience of staple food crops.
Biocontrol to reduce the impact of toxigenic fungi and the entry of mycotoxins into the food chain
- S. Chulze
The occurrence of plant pathogens and toxigenic fungi and subsequently mycotoxin contamination in different crops around the world have significant implications for food and feed safety, food security and international trade. The main mycotoxins detected as natural contaminants in oily seeds and cereals include aflatoxins, trichothecenes, fumonisins , ochratoxin A and zearalenone. Species of Fusarium and those within Aspergillus sections Flavi and Nigri are producers of these toxic secondary metabolites. Fusarium head blight (FHB) is a devastating disease that causes extensive yield and quality losses to wheat and other small cereal grains worldwide. Different strategies including crop rotation, tillage practices, fungicide application and planting less susceptible cultivars are used in order to reduce the impact of mycotoxins in these cereal-based food and feed chains. The development of fungicide resistance together with the rising of public concern of risks associated with pesticides use has led to the search for environmentally friendly alternatives. Biocontrol offers an alternative approach that can be used in the framework of an integrated pest management (IPM) strategy to reduce the accumulation of mycotoxins in food and feed chains. Aspergillus section Flavi can infect peanuts and maize pre-harvest stage, especially during drought stress episodes resulting in aflatoxin contamination. Biocontrol based on competitive exclusion by using atoxigenic Aspergillus flavus strains is one of the most promising strategies for minimising aflatoxin contamination in both these commodities. Populations of native atoxigenic Aspergillus flavus strains were evaluated based on phenotypic, physiological and genetic characteristics. Selected atoxigenic strains of A. flavus with no capacity for aflatoxin or cyclopiazonic acid production were evaluated in field trials. Reductions of aflatoxin contamination were between 78 - 90% in treated plots in comparison with control plots. Two potential biocontrol agents, Bacillus velezensis RC 218 and Streptomyces albidoflavus RC 87B, have also been evaluated for reduction of Fusarium head blight severity and deoxynivalenol (DON) in bread and durum wheats. Both these strains effectively reduced FHB incidence (up to 30%), severity (up to 25%) and DON accumulation (up to 51%) in durum wheat under field conditions.
Molecular detection of Penicillium nordicum in cured meat products: food quality and safety implications.
- G. Perrone
- M. Ferrara
- D. Magistà
- V. Lippolis
- F. Epifani
- S. Cervellieri
- A. Susca
Fermented and cured meat products are unique and often represented as an element of culinary heritage and gastronomic identity. Together with meat enzymes and bacteria, molds are very important in the ripening of some dry fermented meat products. They contribute to the development of the typical sausage flavor, prevent lipid oxidations and counteract undesirable microorganisms. Various genera of fungi could colonize salami but Penicillium species are predominant, and above all P. nalgiovense, P. chrysogenum and a new recently described species P. salamii. On the other hand, depending on its peculiar composition, the surface could be colonized by undesirable molds, like P. nordicum an important and consistent producer of the potent nephrotoxic ochratoxin A (OTA). Addressing the safety of seasoning of meat products we developed different molecular approaches to detect the presence of P. nordicum and monitor OTA contamination risk. A sensitive and easy to use Loop-mediated isothermal ampliﬁcation (LAMP) assay for P. nordicum detection on salami surface was set up targeting otapksPN gene, a key gene in the biosynthesis of OTA in P. nordicum. Positive reactions were detected directly in-tube by color transition of hydroxynaphthol blue from violet to sky blue. The assay was proved to be specific for P. nordicum and able to detect down to 100 fg of target DNA. In addition, gene expression of otapksPN gene in P. nordicum and OTA production were monitored throughout the seasoning process up to 30 days in a small-scale experiment. The expression of otapksPN gene was already detected after 4 days of seasoning and increased signiﬁcantly after 7 days, reaching the maximum expression level after 10 days. Consistent with gene expression data, OTA was detected from the 4th day and its content increased signiﬁcantly from the 7th day, reaching the maximum level after 10 days. Finally, the LAMP assay was tested to detect the persistence of P. nordicum during the seasoning process of sausages after co-inoculation of the fungus with P. nalgiovense at different contamination rates. After 14 days of seasoning, LAMP assay was able to detect the presence of P. nordicum down to 2.5% of P. nordicum contamination. The analysis of toxin content at the end of seasoning, revealed that OTA was accumulated both in mycelium and dry-cured meat when P. nordicum contamination rate ranged from 25% to 100% of inoculum, while OTA was not detected in dry-cured meat at 2.5% and 0.25%. These results evidenced that contamination of dry-cured meat products by P. nordicum could represent a serious concern for salami production and therefore molecular tools, such as LAMP and gene expression assay, should be considered for new HACCP plans in order to prevent and control OTA risk in dry-cured meat production.
Prevention of mold spoilage and mycotoxin production: using the right methodology.
- E. Rico-Munoz
Fungal spoilage of foods and beverages imposes significant annual global revenue losses. Mold spoilage can also be a food safety issue due to the production of mycotoxins by these molds. To prevent mold spoilage and mycotoxin production, several hurdles can be used: (1) reducing the water activity, (2) thermal processing, (3) addition of preservatives, (4) reduction of oxygen in the packaging using vacuum, oxygen scavengers or modified atmosphere packaging (MAP), and (5) refrigerated storage. These hurdles individually target a different group of spoilage fungi; the use of two or more hurdles will reduce the number of molds that can spoil the product. This is called the associated mycobiota that typically comprises only a few mold species. It is essential that the associated mycobiota be adequately isolated and accurately identified. While classic mycological detection methods can detect a broad range of fungi using well validated protocols, they are time consuming, require skilled personnel, and some methods have low sensitivity. Molecular methods for the detection of fungi from spoiled foods are faster than conventional methods but require good DNA isolation techniques, expensive equipment and may detect non-viable fungi that are unlikely to spoil a specific product. One of their advantages, especially in PCR-based methods, is the specific detection of small amounts of target organisms by amplifying their DNA in a considerably short time frame. Identification based on phenotypic characters can be time consuming and well-trained staff is needed. It is therefore more prone to erroneous identifications than a sequence-based identification. However, the results of a sequence-based identification heavily depend on the quality of the database. In order to prevent misidentifications, it is strongly recommended to use sequence-based techniques in conjunction with morphological techniques. Strain typing – distinguishing between different strains of the same species – is used to get insight in the genetic diversity of spoilage agents (is the contamination caused by the same strain?) or can be used to trace the source of the contamination. Although there is no complete or easy method for the detection of fungi in foods it is important to be aware of the limitations of each methodology. This
Genomic comparisons of biocontrol Aspergillus flavus strains revealed rearrangements that disrupt secondary metabolite gene clusters
- K. Pennerman
- G. Yin
- J. Bennett
- S. Hua
Aspergillus flavus is a prevalent saprophytic and pythopathogenic fungus that causes loss of billions of dollars globally due to damage to and mycotoxin contamination of pre- and postharvest crops, and negative health effects in humans and domesticated animals. The A. flavus mycotoxins of greatest concern are the aflatoxins. Use of non-aflatoxigenic strains of A. flavus to compete against aflatoxin-producing strains has emerged as one of the best management practices for reducing aflatoxins contamination. We recently sequenced the genome and transcriptome of a new potential A. flavus biocontrol agent isolated from almond. This strain, WRRL 1519, does not produce aflatoxins or cyclopiazonic acid. The genome of WRRL 1519 was similar to other strains in size (38.0 Mb), GC content (47.2%) and number of putative proteins (12,121). Compared to aflatoxigenic A. flavus strains, strain WRRL 1519 had low shared identity or deletions for many genes and proteins required for aflatoxins and cyclopiazonic acid (CPA) syntheses. Over half of the aflatoxin synthesis gene cluster was missing, while the CPA gene cluster could not be identified. The new strain also appeared to maintain functional sequences of genes known to be involved in infectivity, particularly a pectinase gene that is thought to be required for aggressive growth in plant hosts. These results indicated that strain WRRL 1519 would be a good candidate for reducing aflatoxins and CPA accumulation by out-competing toxigenic strains in infected host crops, and warrants further experimental study. We additionally compared the genomic arrangements of predicted protein-coding genes of WRRL 1519 and other naturally-occurring biocontrol strains NRRL 21882 (Afla-Guard), NRRL 18543 (AF36) and NRRL 30797 (K49) to those of the aflatoxigenic strain NRRL 3357. While the aflatoxin synthesis gene clusters were disrupted by deletions and point mutations, our work revealed that chromosomal transpositions also appeared to disrupt several secondary metabolite gene clusters in strain WRRL 1519. The loss of secondary metabolites may affect growth rate, toxicity and effectiveness of biocontrol. Continued computational analyses and experimental work on the A. flavus genomes will identify defense, metabolic and infectivity genes of atoxigenic A. flavus strains that promote biocontrol-related management of toxin contamination.
Ergot alkaloid synthetic capacity of Penicillium camemberti
- S. Fabian
- M. Maust
- D. Panaccione
Penicillium camemberti plays a major role in the ripening process of brie and camembert type cheeses. Investigation of the recently sequenced P. camemberti genome revealed the presence of a cluster of five genes previously shown to be required for ergot alkaloid synthesis in other fungi. Clustered with the five ergot alkaloid synthesis genes (eas genes) were two additional genes that had the apparent capacity to encode enzymes involved in secondary metabolism. We analyzed samples of brie and camembert cheeses as well as cultures of P. camemberti grown under different conditions by HPLC with fluorescence detection and LC-MS and did not detect any known ergot alkaloids, indicating the P. camemberti eas genes were either not expressed or encoded non-functional enzymes. We used a heterologous expression strategy to investigate the theoretical biosynthetic capacity of P. camemberti. Based on studies with the related ergot alkaloid-producing fungus Neosartorya fumigata (Aspergillus fumigatus), the five known eas genes found clustered in the P. camemberti genome should give the fungus the capacity to produce the ergot alkaloid chanoclavine-I aldehyde. We used a chanoclavine-I aldehyde-accumulating mutant of N. fumigata as a recipient strain in which to express the two uncharacterized P. camemberti eas cluster genes (named easH and easQ) to create a functioning facsimile of the P. camemberti cluster. Expression of easH and easQ in the chanoclavine-I-accumulating N. fumigata strain resulted in the accumulation of a pair of compounds of m/z 269.1289 in positive mode LC-MS. Since this m/z is consistent with the molecular ion of the isomeric pair of [rugulovasine A/B + H]+, we analyzed a culture of the rugulovasine producer Penicillium biforme (a recent ancestor of P. camemberti) and found the same isomeric pair of analytes with the same retention times. Fragmentation of the analytes yielded fragments typical of those resulting from fragmentation of rugulovasine A/B. The deduced activities of the products of easH and easQ indicate the capacity to catalyze theoretical reactions that provide a reasonable pathway from the precursor chanoclavine-I aldehyde to the products rugulovasine A/B. The chanoclavine-I aldehyde-accumulating mutant of N. fumigata transformed with the P. camemberti easQ gene alone yielded an abundant analyte of m/z 271.1, consistent with the addition of oxygen to chanoclavine-I aldehyde. Transformation of the N. fumigata strain with P. camemberti easH alone did not yield a novel product, indicating that EasH acts after EasQ in the pathway. The data indicate that P. camemberti has the genes to produce the ergot alkaloids rugulovasine A/B but that during domestication isolates that failed to produce alkaloids were selected.