Cocultivation of phytopathogenic Fusarium and Alternaria strains affects fungal growth and mycotoxin production

Toxicity of Metabolites Produced by the "Alternaria"

Plant growth-promoting rhizobacteria (PGPR) with antagonistic activity toward plant pathogenic fungi are valuable candidates for the development of novel plant protection products based on biocontrol activity. The very first step in the formulation of such products is to screen the potential effectiveness of the selected microorganism(s). In this study, non-pathogenic rhizobacteria were isolated from the rhizosphere of tomato plants and evaluated for their biocontrol activity against three species of mycotoxin-producing Alternaria. The assessment of their biocontrol potential involved investigating both fungal biomass and Alternaria toxin reduction. A ranking system developed allowed for the identification of the 12 best-performing strains among the initial 85 isolates. Several rhizobacteria showed a significant reduction in fungal biomass (up to 76%) and/or mycotoxin production (up to 99.7%). Moreover, the same isolates also demonstrated plant growth-promoting (PGP) traits such as siderophore or IAA production, inorganic phosphate solubilization, and nitrogen fixation, confirming the multifaceted properties of PGPRs. Bacillus species, particularly B. amyloliquefaciens and two strains of B. subtilis, showed the highest efficacy in reducing fungal biomass and were also effective in lowering mycotoxin production. Isolates such as Enterobacter ludwigii, Enterobacter asburiae, Serratia nematodiphila, Pantoea agglomerans, and Kosakonia cowanii showed moderate efficacy. Results suggest that by leveraging the diverse capabilities of different microbial strains, a consortium-based approach would provide a broader spectrum of effectiveness, thereby signaling a more encouraging resolution for sustainable agriculture and addressing the multifaceted nature of crop-related biotic challenges.

A QuEChERS (quick, easy, cheap, effective, rugged, and safe)-based multi-mycotoxin method was developed, analyzing 24 (17 free and 7 modified) Alternaria and Fusarium toxins in cereals via ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). A modified QuEChERS approach was optimized for sample preparation. Quantification was conducted using a combination of stable isotope dilution analysis (SIDA) for nine toxins and matrix-matched calibration for ten toxins. Quantification via a structurally similar internal standard was conducted for four analytes. Alternariol-9-sulfate (AOH-9-S) was measured qualitatively. Limits of detection (LODs) were between 0.004 µg/kg for enniatin A1 (ENN A1) and 3.16 µg/kg for nivalenol (NIV), while the limits of quantification were between 0.013 and 11.8 µg/kg, respectively. The method was successfully applied to analyze 136 cereals and cereal-based foods, including 28 cereal-based infant food products. The analyzed samples were frequently contaminated with Alternaria toxins, proving their ubiquitous occurrence. Interestingly, in many of those samples, some modified Alternaria toxins occurred, mainly alternariol-3-sulfate (AOH-3-S) and alternariol monomethyl ether-3-sulfate (AME-3-S), thus highlighting the importance of including modified mycotoxins in the routine analysis as they may significantly add to the total exposure of their parent toxins. Over 95% of the analyzed samples were contaminated with at least one toxin. Despite the general contamination, no maximum or indicative levels were exceeded.Graphical Supplementary InformationThe online version contains supplementary material available at 10.1007/s00216-024-05486-4.

The presence of mycotoxins and other toxic metabolites in hops (Humulus lupulus L.) was assessed for the first time. In total, 62 hop samples were sampled in craft breweries, and analyzed by a multi-toxin LS-MS/MS method. The study collected samples from craft breweries in all of the Croatian counties and statistically compared the results. Based on previous reports on Alternaria spp. and Fusarium spp. contamination of hops, the study confirmed the contamination of hops with these toxins. Alternaria toxins, particularly tenuazonic acid, were found in all tested samples, while Fusarium toxins, including deoxynivalenol, were present in 98% of samples. However, no Aspergillus or Penicillium metabolites were detected, indicating proper storage conditions. In addition to the Alternaria and Fusarium toxins, abscisic acid, a drought stress indicator in hops, was also detected, as well as several unspecific metabolites. The findings suggest the need for monitoring, risk assessment, and potential regulation of Alternaria and Fusarium toxins in hops to ensure the safety of hop usage in the brewing and pharmaceutical industries. Also, four local wild varieties were tested, with similar results to the commercial varieties for toxin contamination, but the statistically significant regional differences in toxin occurrence highlight the importance and need for targeted monitoring.

Oats are highly susceptible to infection by Fusarium species, especially F. langsethiae, F. poae and F. sporotrichioides which contaminate the grain with mycotoxins. Climate change is expected to affect fungal colonisation and associated mycotoxin production. The objective of this study was to examine the effect of acclimatisation to elevated CO2 on the growth and mycotoxin production capacity of these fungal species. Strains of F. langsethiae (FL; seven strains), F. poae (FP; two strains) and F. sporotrichioides (FS; one strain) were acclimatised by sub-culturing for 10 generations at either 400 or 1000 ppm CO2 under diurnal temperature conditions. At each sub-culturing, the effect of acclimatisation to elevated CO2 on (a) lag phase prior to growth, (b) growth rate on oat-based media was assessed. Additionally, the production of type A trichothecenes and related toxic secondary metabolites of sub-cultures after 1, 7 and 10 generations were assessed using LC-MS/MS qTRAP. The results showed that Fusarium strains had an increased lag time and growth rate in response to the combined effect of sub-culturing and elevated CO2 levels. T-2 + HT-2 production was affected by elevated CO2 in strain FL4 (7.1-fold increase) and a decrease in strain FL1 (2.0-fold decrease) at the first sub-culturing and FS (1.3-fold decrease) after 7 sub-cultures compared to ambient conditions. The effect of sub-culturing on T-2 + HT-2 production varied depending on the fungal strain. For strain FL4, significantly less T-2 + HT-2 toxins were produced after 10 generations (4.4-fold decrease) as compared to that under elevated CO2 conditions after one sub-culture, and no change was observed under ambient conditions. The FS strain showed significant stimulation of T-2 + HT-2 toxin production after 10 sub-cultured generations (1.1-fold increase) compared to the initial sub-culture of this strain under elevated CO2 conditions. The production of other toxic secondary metabolites was generally not impacted by elevated CO2 conditions or by sub-culture for 10 generations, with the exceptions of FL1 and FP1. FL1 produced significantly more neosolaniol after 10 generations, when compared to those after 1 and 7, regardless of the CO2 conditions. For FP1, elevated CO2 significantly triggered beauvericin production after an initial sub-culture when compared to ambient conditions at the same sub-culture stage (29-fold). FP1 acclimatisation to elevated CO2 led to a decrease of beauvericin production after 10 generations when compared to 1 (6-fold). In contrast, sub-culturing for 10 generations compared to 1 under ambient CO2 conditions resulted in an increase in this toxin (12-fold).

The objective of this study was to evaluate the preharvest application of γ-aminobutyric acid (GABA), melatonin (MT), and oxalic acid (OA), at different concentrations and application frequencies, on the physicochemical and microbiological quality of dried figs (cv. Calabacita) at commercial harvest and after 3 and 6 months of refrigerated storage. A further aim was to determine their impact on fungal populations and mycotoxin production. The results showed that untreated dried figs had a higher frequency of Aspergillus welwitschiae, A. tubingensis, and Aspergillus section Flavi, whereas elicitor-treated figs exhibited a lower incidence of toxigenic fungi. A. welwitschiae was the main ochratoxin A (OTA)-associated species detected, although the proportion of OTA-positive figs was lower in elicitor-treated samples than in the control. Aflatoxins (AFs) were detected only sporadically in 2 mM OA treatments, consistent with the limited activity of A. flavus at low storage temperatures. Conversely, Penicillium spp. were widespread but were associated with citrinin (CIT) production only under 2 mM OA treatments. Among the Alternaria toxins, alternariol (AOH) was detected solely in dried figs treated with 1 mM OA. Notably, all investigated mycotoxins were below the limit of detection (<LOD) in dried figs treated with 0.5 mM MT. Moderate elicitor concentrations (e.g., 0.5 mM MT and 50 mM GABA) and multiple preharvest applications generally provided the best balance between fungal suppression and fruit quality, significantly reducing Aspergillus spp. occurrence without promoting the growth of undesirable species. Overall, elicitor treatments decreased the incidence of toxigenic fungi, most likely through direct antifungal effects in senescent dried fruit rather than by inducing host defences. The combined use of preharvest elicitors with appropriate drying and storage conditions is a promising strategy to control fungal contamination and mycotoxin accumulation in dried figs while maintaining quality from preharvest storage. Further research is needed to optimise elicitor concentrations and application timing.

Fungal growth and the production of mycotoxins are influenced by several factors. Environmental conditions such as temperature, water activity, and humidity affect mycotoxin production and fungal growth. Other factors such as pH, fungal strain, and substrate also play roles. Common mycotoxins include aflatoxins, fumonisins, trichothecenes, sterigmatocystin (STC), citrinin, ergot alkaloids, ochratoxins, zearalenones (ZEAs), patulin, deoxynivalenol (DON), Alternaria toxins, tremorgenic mycotoxins, fusarins, cyclochlorotine, sporidesmin, 3-nitropropionic acid, etc. These toxins cause many health conditions in animals and humans, including death. A comprehensive approach starting from the field before planting, continuing throughout the entire food chain is required to control mycotoxin contamination. Good practices, such as proper field practices before and after planting, good harvest practices and postharvest handling, and proper drying and storage measures, help reduce mycotoxin contamination. Several physical, biological, and chemical techniques have been applied to help reduce/eliminate mycotoxin contamination. Food processing also play slight role in mycotoxins removal.

Chapter 4 - Factors Affecting Mycotoxin Production in Fruits

Decaying macrophytes are an important source of carbon and nutrients in fungal and bacterial communities of northern prairie wetlands. Dead macrophytes do not collapse into the water column immediately after death, and decomposition by fungi and bacteria begins while the plants are standing. The seasonal variations in fungal biomass and production on Scirpus lacustris stems, both above and below water, were measured to assess which environmental factors were dominant in affecting these variations in a typical prairie wetland. Fungal biomass and production were measured from early May to November, just prior to freeze-up. Fungal decomposition began and was greatest in the spring despite low water temperatures. The fungal production, as measured by the incorporation of [1-(14)C]acetate into ergosterol, ranged from 1.8 to 376 microg of C g of ash-free dry mass (AFDM)(-1) day(-1), and the biomass, as estimated by using ergosterol, ranged from nondetectable to 5.8 mg of C g of AFDM(-1). There was no significant difference in biomass or production between aerial and submerged portions of Scirpus stems. The water temperature was correlated with fungal production (r = 0.7, P < 0.005) for aerial stem pieces but not for submerged pieces. However, in laboratory experiments water temperature had a measurable effect on both biomass and production in submerged stem pieces. Changes in fungal biomass and productivity on freshly cut green Scirpus stems decaying in the water either exposed to natural solar radiation or protected from UV radiation were monitored over the summer. There was no significant difference in either fungal biomass (P = 0.76) or production (P = 0.96) between the two light treatments. The fungal biomass and rates of production were within the lower range of the values reported elsewhere, probably as a result of the colder climate and perhaps the lower lability of Scirpus stems compared to the labilities of the leaves and different macrophytes examined in other studies performed at lower latitudes.

The phenolic extracts obtained from rice bran cultivated with Rhizopus oryzae were evaluated about their abitily to inhibit the production of trichotecenes and emerging mycotoxins by Fusarium strains on sweet corn cobs (Zea mays L.). The rice bran was cultured with R. oryzae for 48 h at 30 °C. The phenolic compounds were extracted, clarified, lyophilized and ressuspend as water solution with different concentrations. The phenolic acid extracts were applied on sweet corn inoculated by F. cerealis, F. graminearum and F. poae incubated by 15 days at 25 °C and afterward the fermented corn was autoclaved and frozen. The mycotoxins were quantified by LC-MS/MS. The mycotoxin production by all Fusarium strains evaluated was reduced in relation to the control. F. poae was the most sensitive, because its production of the toxins T-2 and HT-2 and BEA was inhibited by phenolic extract 0,5% (p/v), while extract 1% (p/v) inhibited 96% of the DON production. DON, 3ADON and 15ADON production by F. graminearum and F. cerealis was also reduced. Therefore, the extract is a promising antimicotoxinogenic against Fusarium toxins production on sweet corn cobs and should be a good alternative to reuse wastes from agroindustry.

Im Rahmen der vorliegenden Arbeit wurde unter Klimakammer- und Freilandbedingungen der Einfluß von Witterung, Standort, Sorte, Vorfrucht und Blattapplikation mit verschiedenen Fungizidklassen auf die Mykotoxinbildung von Fusarium spp. für den Klimaraum Niedersachsen überprüft. Erstmalig wurde dabei die spezifische Mykotoxinbildung, d.h. die Menge Mykotoxin die pro Einheit F. graminearum DNA gebildet wird im Feld und im Gewächshaus ermittelt. Vorherige Untersuchungen stellten für die spezifische Mykotoxinbildung große Schwankungsbreiten in verschiedenen Versuchsjahren fest, die bisher nur unzureichend mit Witterungseinflüssen erklärt wurden. Im Verlauf dieser Arbeit wurden verschiedene Umweltfaktoren in Versuchen unter kontrollierten Umweltbedingungen identifiziert, die diese komplexen Interaktionen zwischen Mykotoxinmenge und Pilzbiomasse teilweise erklärten. Haupteinflussfaktor auf die Mykotoxinbildung im FAEN-Feldversuch war erwartungsgemäß der Jahres- bzw. Witterungseffekt mit einem Anteil von ca. 20% an der Gesamtvariation. Dies beruhte auf der notwendigen Koinzidenz von Weizenpflanzen, die sich im Entwicklungssta-dium der Vollblüte befanden mit einer relativ lang anhaltenden, feucht warmen Witterung mit Niederschlagsereignissen. Mit 12% Anteil an der Gesamtvariation des DON-Gehaltes stellte die Vorfrucht den zweitbedeutendsten Versuchsfaktor dar. Neben der Förderung des Ährenbefalls durch F. graminearum führten die Vorfruchtvarianten Mais in allen drei Versuchsjahren zu den höchsten Mykotoxinkontaminationen im Ernteprodukt. Für die Vorfruchtvarianten Winterweizen und Zuckerrübe waren die Effekte nicht eindeutig, sie waren von der Befallsstärke des jeweiligen Jahres abhängig. Unter schwachen bis mittleren Befallsbedingungen galt für die drei Versuchsjahre die Abstufung: Mais &gt; Winterweizen &gt; Zuckerrübe; hingegen bei Starkbefall: Mais &gt; Winterweizen = Zuckerrübe. Ursache dieser Verschiebungen sind wahrscheinlich luftbürtige Infektionen durch Askosporenwolken, die in Starkbefallsjahren flächenbezogene Vorfruchteffekte überlagern. Die Mykotoxingehalte der Sorte Centrum unterschieden sich bei Ährenbefall signifikant von der Sorte Ritmo. Der ermittelte Anteil der Sortenresistenz an der Gesamtvarianz aller drei Versuchsjahre war mit einem Anteil von 8% im Vergleich zu den Erfahrungswerten aus der Literatur von bis zu 40% überraschend gering. Wurden diese Analysen nur auf Ebene der einzelnen Versuchsjahre durchgeführt, stieg der Anteil der Sortenresistenz an der Jahresvarianz jedoch deutlich an. Der Einfluß der Sortenresistenz war als einziger der Versuchsfaktoren im Feldversuch unabhängig vom spezifischen Befallsniveau des Versuchsjahrs. Im Gegensatz dazu war der Einfluß von Standort und Vorfrucht auf den Mykotoxingehalt nur in Jahren mit hohem Befallsniveau wie 2007 und 2009 signifikant. Bis zu 70% der Varianz des Deoxynivalenolgehaltes im jeweiligen Versuchsjahr ließ sich mittels der vier Versuchsfaktoren Vorfrucht, Sorte, Standort und Fungizidapplikation erklären. Der Anteil der Fungizidapplikationen an der Gesamtvariation des DON-Gehaltes war nur gering, jedoch war dieser Effekt über die drei Versuchsjahre signifikant. Eine Blattapplikation von Fungiziden wie Strobilurinen, die die Pflanzenphysiologie beeinflussen, führte zwar nicht zu einer signifikanten Erhöhung der Mykotoxinkonzentration im Korn, jedoch konnte in jedem Versuch dieser Arbeit eine solche Tendenz festgestellt werden. Unter Freilandbedingun-gen wiesen mit Strobilurinen behandelte Weizenpflanzen eine später einsetzende Seneszenz auf. Möglicherweise stand auf solchen Pflanzen dem Pathogen bei günstigeren Wassergehal-ten ein längerer Zeitraum für die Mykotoxinproduktion zur Verfügung, was eine der Ursachen für die erhöhten DON-Mengen in den Strobilurinvarianten sein könnte. Andererseits könnte juvenileres Gewebe auch eine partielle Resistenz gegen nekrotrophe Pathogene besitzen, so dass der erhöhte Stress des Pathogens bei der Gewebebesiedlung zu einer Erhöhung der Mykotoxinbildung beitrug. Die spezifische Mykotoxinbildung war im Feld hochgradig abhängig von den Jahres- bzw. Witterungseffekten. Es bestätigte sich, dass hohe Mykotoxingehalte im Erntegut, aufgrund günstiger Infektions- und feuchter Abreifebedingungen, i.d.R. mit geringeren spezifischen Mykotoxinbildungen assoziiert sind. Das Pilzwachstum reagierte sensitiver auf veränderte Umweltbedingungen als die Mykotoxinproduktion, was in Klimakammerexperimenten bestätigt wurde. Günstige Umweltbedingungen mit hohen Tagesdurchschnittstemperaturen und Luftfeuchten führten in planta zu einem verhältnismäßig stärkeren vegetativen Wachstum des Pathogens und somit der DNA-Menge von F. graminearum im Vergleich zu den Mykotoxinmengen. Resistentere Sorten wiesen in dieser Arbeit in allen Versuchen unter Freilandbedingungen eine signifikant geringere spezifische Mykotoxinbildung auf als anfälligere Sorten, was im Gegensatz zu bisherigen Ergebnissen aus der Literatur steht. In Feldversuchen sanken mit zunehmender Sortenresistenz die Mengen der Pilzbiomasse und der DON-Gehalte linear ab. Die Mykotoxinmengen verringerten sich dabei deutlich schneller, was zu der geringeren spezifischen Mykotoxinbildung in den resistenten Sorten führte. Unter kontrollierten Umweltbedingungen kehrte sich diese Tendenz jedoch um und führte zu höheren spezifischen Mykotoxinbildungen in den resistenteren Sorten. Als Konsequenz dieser distinkten Wechselwirkungen wurden zwei Modelle einerseits für Versuche unter kontrollierten Umweltbedingungen z.B. im Gewächshaus und andererseits für Feldversuche entwickelt, die den Verläufen der spezifischen Mykotoxinbildung Rechnung tragen. Darüber hinaus zeigten die Pathogenitätstests der aus Zuckerrüben isolierten Fusarium-Arten, dass auch diese in der Lage waren Weizenähren zu besiedeln und erhebliche Mengen an Mykotoxinen in Körnern zu bilden. Klassische FHB-Bildner wie F. graminearum und F. culmorum besiedelten die inokulierten Ähren systemisch und führten zur Entwicklung der fusarientypischen weißlichen Schmachtkörner. Die Pathogense von F. tricinctum, F. equiseti, F. redolens und F. oxysporum blieb auf einzelne infizierte Ährchen beschränkt und verursachte dort atypische Verbräunungen an den Deck- und Hüllspelzen. Dort kam es auch zu der Ausbildung des sogenannten Black Point Symptoms an ansonsten normal entwickelten Körnern. Im Vergleich zu symptomlosen Körnern wies diese Kornfraktion einen deutlich erhöhten Gehalt an Mykotoxinen auf. br/&gt; Mit den Ergebnissen dieser Arbeit wird es in Zukunft möglich sein unter verschiedenen Witterungsszenarien den Einfluss agronomischer Faktoren wie der Vorfrucht, dem Standort und der Sortenresistenz auf die Pathogenentwicklung von F. graminearum und die Mykotoxingehalte im Getreidekorn exakter zu bewerten.

The influence of nutrients on fungal growth, productivity, and sporulation during leaf breakdown in streams

Adverse human health effects from the consumption of mycotoxins have occurred for many centuries. Although mycotoxin contamination of agricultural products still occurs in the developed world, the application of modern agricultural practices and the presence of a legislatively regulated food processing and marketing system have greatly reduced mycotoxin exposure in these populations. At the mycotoxin contamination levels generally found in food products traded in these market economies, adverse human health effects have largely been overcome. However, in the developing world, where climatic and crop storage conditions are frequently conducive to fungal growth and mycotoxin production, much of the population relies on subsistence farming or on unregulated local markets. The extent to which mycotoxins affect human health is difficult to investigate in countries whose health systems lack capacity and in which resources are limited. Aflatoxin B1, the toxin on which major resources have been expended, has long been linked to liver cancer, yet its other effects, such as immune suppression and growth faltering previously observed in veterinary studies, are only now being investigated and characterized in human populations. The extent to which factors such as immune suppression contribute to the overall burden of infectious disease is difficult to quantify, but is undoubtedly significant. Thus, food safety remains an important opportunity for addressing current health problems in developing countries.

Two species of Lactobacilli (Lactobacillus rhamnosus and Lactobacillus paracasie subsp. paracasie) were tested for their ability to inhibit growth and mycotoxins production by three species of Fusarium, (F. graminearum, F. culmorum and F. proliferation) which are the main producers of mycotoxins deoxynivalenol, zearalenone and fumonisin B1, respectively. L. paracasie subsp. paracasie was found to be effective in reducing the amount of toxins produced, although fungal growth was not affected. The inhibition levels of Deoxynivalenol, Zearalenone and Fumonisin B1 production reached to 56.8, 73.0 and 76.5%, respectively. Meanwhile, L. rhamnosus showed the highest inhibitory activity against both fungal growth and mycotoxins production. It completely suppressed mycelium growth of all the studied Fusarium species and consequently, no toxin was produced in the presence of this bacterium. The obtained results confirm that, selected species of Lactic acid bacteria may be successfully used as a biological control agent of food contamination with molds and mycotoxins. This bio-preservation action has interesting technological possibilities for a variety of fermented food and dairy products.

How will climate change affect mycotoxins in food?