Ecology, climate change and control strategies for aspergillus flavus colonisation and aflatoxin contamination of pistachio nuts.

Pistachio nuts (Pistacia vera L.) have become one of the most important products in the economy of many countries including the USA, Iran, Syria, Greece, Turkey, China, EU and the Middle East. Pistachio nuts are very commonly colonized by spoilage mycobiota especially aflatoxigenic species because they are very hygroscopic and can adsorb water. Aspergillus flavus can contaminate pistachio nuts with aflatoxins (AFs), especially aflatoxin B1 (AFB1) classified as a class 1a carcinogen. The objectives of this project were (a) to examined the mycobiota and the aflatoxin producing strains of Aspergillus section Flavi species in pistachio nuts originating from different countries and sourced in the Kingdom of Saudi Arabia (KSA), (b) investigated the effect of the interactions between temperature and water activity (aw) on the ecology and molecular ecology growth and AFB₁ production by Aspergillus flavus strains in vitro on pistachio nut-based media and in stored raw pistachio nuts (c) evaluate the effect of Climate Change (CC) interacting factors on growth and AFB1 production by strains of A.flavusand on relative genes expression of the aflD and aflR genes involved in the biosynthetic pathway for AFB₁ production, (d) examine whether acclimatisation to 1000 ppm CO₂ of A.flavus strains AB3 and AB10 for 5 generations affected growth and AFB₁ production; and (e) to examine the use of gaseous O₃ for the control of germination, A.flavus populations and AFB₁ contamination of stored pistachio nuts for up to 4 weeks. Pistachio samples were colonized by a range of Aspergillus and Penicillium species. In some samples, typically phyllosphere fungi such as yeasts, Mucor, Rhizopus, Alternaria, Epicoccum and Phoma species were isolated. 10 different species of A.flavus were isolated and molecularly identified. The relative toxigenic nature of strains was evaluated using selective media and HPLC and confirmed using molecular tools. Four strains were used in ecological studies and two (AB3, AB10) in other studies. The ecological studies showed that optimum growth of AB3 and AB10 strainswas at 0.98 or 0.95 aw and 30-35°C. The effect of the same factors on aflR gene expression of the two strains showed optimum condition at 30-35°C and 0.98 aw; with optimum conditions for AFB₁ production at 35°C and 0.98 aw for strain AB3. There was little difference between the effect of using a non-ionic (glycerol) or ionic (NaCl) to modify water stress in in vitro studies. The effect of interacting CC factors on growth of A.flavus colonisation was not significant. However, AFB₁ production was stimulated. With regards to aflD gene expression, at 35°C, the relative expression was higher in current CO₂ conditions (400 ppm) for both strains except that for strain AB3 the gene expression was higher at 1000 ppm CO2 at 0.95 aw. However, at 37°C, the expression was generally higher in the 1000 ppm CO₂ than with existing atmospheric CO₂ levels. The aflR gene expression was higher at 1000 ppm CO₂ at 37°C for both strains. AFB₁ production was higher at 35°C at the two CO₂ levels for both strains. At the same temperature, AFB1 production was significantly increased at 1000 ppm CO₂ and 0.98 aw. At 37°C, AFB₁ production was either decreased in strain AB3 or similar as in strain AB10 when exposed to 1000 ppm CO₂. This suggests that CC factors may have a differential effect depending on the interacting conditions of temperature (35 or 37°C) as in some cases for AFB₁. Acclimatisation influenced growth of one strain while there was no significant effect on another strain when colonising pistachio nuts. For AFB₁, the production was significantly stimulated after ten days colonisation after acclimatisation for one strain, while there was no significant increase for the other strain. This suggests that there may be intra-strain differences in effects of acclimatisation and this could influence mycotoxin contamination of such commodities as mixed population of contaminant fungi often occurs. Exposure of conidia to gaseous O₃ initially had lower germination percentages when compared to the controls at different aw levels. Complete inhibition of germinations was observed after 12 h treatment of 200 ppm O₃ at 0.98 aw. However, spore viability appeared to recover and the germination was increased after 24 h and reached 100% after 48 h. Growth rates of mycelial colonies were decreased with increasing of O₃ dose and colony extension was significantly inhibited by O₃ treatment at 0.98 aw. Variable effects on AFB1 production during exposure to O₃ treatment after in vitro exposure of colonies of A.flavus incubated for ten days at 30°C. The populations of A.flavus were significantly decreased by O₃ exposure; however, there was little difference between 50-200 ppm treatment levels. A reduction in AFB₁ was only observed in the 50 ppm O₃ × 0.98 aw treatment in stored pistachio nuts. The relationship between exposure concentration × time of exposure and prevailing aw level to determine the efficacy in terms of toxin control needs to be better understand.PhD in Environment and Agrifoo

Interacting Abiotic Factors Affect Growth and Aflatoxin B1 Production Profiles of Aspergillus flavus Strains on Pistachio-Based Matrices and Pistachio Nuts

© 2021 Baazeem, Garcia-Cela, Medina and Magan. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Pistachio nuts are an economically important commodity produced by many countries. They can be colonized by mycotoxigenic fungi, especially Aspergillus flavus, resulting in contamination with aflatoxins (AFs), especially aflatoxin B1 (AFB1), a Class 1a carcinogen. The objectives were to examine the effect of interactions between the two key abiotic factors, temperature and water activity (aw) on (a) in vitro growth and AFB1 production by four strains of A. flavus isolated from pistachio nuts, on a milled pistachio nut medium modified ionically (NaCl) and non-ionically (glycerol) in the range 20–35°C and 0.995–0.85 aw, (b) colonization of layers of raw pistachio nuts stored at different interacting temperature x aw conditions and on relative AFB1 production and (c) develop models to produce contour maps of the optimal and marginal boundary conditions for growth and AFB1 production by up to 4 strains of this species. On pistachio nut-based media, optimum growth of four strains of A. flavus was at 0.98–0.95 aw and 30–35°C. Optimum AFB1 production was at 30–35°C and 0.98 aw. No significant differences in growth was found on ionic and non-ionically modified media. Colonization of layers of raw pistachio nuts was slower and contamination with AFB1 significantly less than in in vitro studies. Contour maps based on the pooled data for up to four strains (in vitro, in situ) showed the optimum and marginal conditions for growth and AFB1 production. These data can be used to identify those conditions which represent a high, intermediate or low risk of colonization and AFB1 contamination in the pistachio nut processing chain. These results are discussed in the context of the development of appropriate intervention strategies to minimize AFB1 contamination of this economically important commodity.Peer reviewedFinal Published versio

Climate change, food security and mycotoxins: do we know enough?

Climate change (CC) scenarios are predicted to have significant effects on the security of staple commodities. A key component of this impact is the infection of such crops by mycotoxigenic moulds and contamination with mycotoxins. The impacts of CC on mycotoxigenic fungi requires examination of the impacts of the three-way interactions between elevated CO2 (350–400 vs 650–1200 ppm), temperature increases (+2–5 °C) and drought stress on growth/mycotoxin production by key spoilage fungi in cereals and nuts. This review examines the available evidence on the impacts of interacting CC factors on growth and mycotoxin production by key mycotoxigenic fungi including Alternaria, Aspergillus, Fusarium and Penicillium species. Aspergillus flavus responsible for producing aflatoxin B1 (AFB1) is a class 1A carcinogen and its growth appears to be unaffected by CC factors. However, there is a significant stimulation of AFB1 production both in vitro and in vivo in maize. In contrast, studies on Aspergillus section Circumdati and Nigri species responsible for ochratoxin A contamination of a range of commodities and F. verticillioides and fumonisins suggest that some species are more resilient than others, especially in terms of mycotoxin production. Acclimatisation of mycotoxigenic fungal pathogens to CC factors may result in increased disease and perhaps mycotoxin contamination of staple cereals. Predictive modelling approaches to help identify regions where maximum impact may occur in terms of infection by mycotoxigenic fungi and toxin contamination of staple crops is hindered by the lack of reliable inputs on effects of the interacting CC factors. The present available knowledge is discussed in the context of the resilience of staple food chains and the impact that interacting CC factors may have on the availability of food in the future

Effect of acclimatization in elevated CO2 on growth and aflatoxin B1 production by Aspergillus flavus strains on pistachio nuts

There is little knowledge of the effect of acclimatization of Aspergillus flavus strains to climate-related abiotic factors and the subsequent effects on growth and aflatoxin B1 (AFB1) production. In this study, two strains of A. flavus (AB3, AB10) were acclimatized for five generations in elevated CO2 (1000 ppm × 37 °C) on a milled pistachio-based medium. A comparison was made of the effects of non-acclimatized strains and those that were acclimatized when colonizing layers of pistachio nuts exposed to 35 or 37 °C, 400 or 1000 ppm CO2, and 0.93 or 0.98 water activity (aw), respectively. Acclimatization influenced the fitness in terms of the growth of one strain, while there was no significant effect on the other strain when colonizing pistachio nuts. AFB1, production was significantly stimulated after ten days colonization when comparing the non-acclimatized and the acclimatized AB3 strain. However, there was no significant increase when comparing these for strain AB10. This suggests that there may be inter-strain differences in the effects of acclimatization and this could have a differential influence on the mycotoxin contamination of such commodities

Impacts of climate change interacting abiotic factors on growth, aflD and aflR gene expression and aflatoxin B1 production by Aspergillus flavus strains in vitro and on pistachio nuts

Pistachio nuts are an important economic tree nut crop which is used directly or processed for many food-related activities. They can become colonized by mycotoxigenic spoilage fungi, especially Aspergillus flavus, mainly resulting in contamination with aflatoxins (AFs), especially aflatoxin B1 (AFB1). The prevailing climate in which these crops are grown changes as temperature and atmospheric CO2 levels increase, and episodes of extreme wet/dry cycles occur due to human industrial activity. The objectives of this study were to evaluate the effect of interacting Climate Change (CC)-related abiotic factors of temperature (35 vs. 37 °C), CO2 (400 vs. 1000 ppm), and water stress (0.98–0.93 water activity, aw) on (a) growth (b) aflD and aflR biosynthetic gene expression and (c) AFB1 production by two strains A. flavus (AB3, AB10) in vitro on milled pistachio-based media and when colonizing layers of shelled raw pistachio nuts. The A. flavus strains were resilient in terms of growth on pistachio-based media and the colonisation of pistachio nuts with no significant difference when exposed to the interacting three-way climate-related abiotic factors. However, in vitro studies showed that AFB1 production was significantly stimulated (p < 0.05), especially when exposed to 1000 ppm CO2 at 0.98–0.95 aw and 35 °C, and sometimes in the 37 °C treatment group at 0.98 aw. The relative expression of the structural aflD gene involved in AFB1 biosynthesis was decreased or only slightly increased, relative to the control conditions at elevated CO, regardless of the aw level examined. For the regulatory aflR gene expression, there was a significant (p < 0.05) increase in 1000 ppm CO2 and 37 °C for both strains, especially at 0.95 aw. The in situ colonization of pistachio nuts resulted in a significant (p < 0.05) stimulation of AFB1 production at 35 °C and 1000 ppm CO2 for both strains, especially at 0.98 aw. At 37 °C, AFB1 production was either decreased, in strain AB3, or remained similar, as in strain AB10, when exposed to 1000 ppm CO2. This suggests that CC factors may have a differential effect, depending on the interacting conditions of temperature, exposure to CO2 and the level of water stress on AFB1 production

Plants in vitro propagation with its applications in food, pharmaceuticals and cosmetic industries; current scenario and future approaches

Plant tissue culture technique employed for the identification and isolation of bioactive phytocompounds has numerous industrial applications. It provides potential benefits for different industries which include food, pharmaceutical and cosmetics. Various agronomic crops i.e., cereals, fruits, vegetables, ornamental plants and forest trees are currently being used for in vitro propagation. Plant tissue culture coupled with biotechnological approaches leads towards sustainable agricultural development providing solutions to major food security issues. Plants are the rich source of phytochemicals with medicinal properties rendering them useful for the industrial production of pharmaceuticals and nutraceuticals. Furthermore, there are numerous plant compounds with application in the cosmetics industry. In addition to having moisturizing, anti‐ageing, anti‐wrinkle effects; plant-derived compounds also possess pharmacological properties such as antiviral, antimicrobial, antifungal, anticancer, antioxidant, anti-inflammatory, and anti-allergy characteristics. The in vitro propagation of industrially significant flora is gaining attention because of its several advantages over conventional plant propagation methods. One of the major advantages of this technique is the quick availability of food throughout the year, irrespective of the growing season, thus opening new opportunities to the producers and farmers. The sterile or endangered flora can also be conserved by plant micro propagation methods. Hence, plant tissue culture is an extremely efficient and cost-effective technique for biosynthetic studies and bio-production, biotransformation, or bioconversion of plant-derived compounds. However, there are certain limitations of in-vitro plant regeneration system including difficulties with continuous operation, product removal, and aseptic conditions. For sustainable industrial applications of in-vitro regenerated plants on a large scale, these constraints need to be addressed in future studies

Impacts of gaseous ozone (O3) on germination, mycelial growth, and aflatoxin B1 production in vitro and in situ contamination of stored pistachio nuts

Pistachio nuts can become colonized by mycotoxigenic fungi, especially Aspergillus flavus, resulting in contamination with aflatoxins (AFs). We examined the effect of gaseous O3 (50–200 ppm; 30 min; 6 L/min) on (a) in vitro germination, (b) mycelial growth, and (c) aflatoxin B1 (AFB1) production on a milled pistachio nut-based medium at different water activity (aw) levels and at 30 °C. This was complimented with in situ studies exposing raw pistachio nuts to 50–200 ppm of O3. Exposure of conidia to gaseous O3 initially resulted in lower germination percentages at different aw levels. However, 12 h after treatment, conidial viability recovered with 100% germination after 24–48 h. Growth rates of mycelial colonies were slightly decreased with the increase of the O3 dose, with significant inhibition only at 0.98 aw. The production of AFB1 after O3 treatment and storage for 10 days was stimulated in A. flavus colonies at 0.98 aw. Raw pistachio nuts inoculated with A. flavus conidia prior to O3 exposure showed a significant decrease in population after 20 days of storage. However, AFB1 contamination was stimulated in most O3 treatments. The relationship between exposure concentration, time and prevailing aw levels on toxin control needs to be better understood for these nuts

Comprehensive studies on amino acid based organometallic L-threoninum cobalt (II) sulfate (LTCS) single crystal and its antibacterial and antifungal properties

Highly transparent superior quality nonlinear single crystals of L-Threoninum Cobalt (II) Sulfate Heptahydrate were prepared by simple novel slow evaporation solution growth method. The space group, lattice parameters, crystal structure and crystalline nature of L-Threoninum Cobalt (II) Sulfate (LTCS) materials assessed through diffraction analysis (PXRD and SXRD). The optical behavior of LTCS crystals were checked via UV–Vis analysis and it confirms optical parameters often depend on photon energy with optical band gap Eg = 5.6 eV and LTCS material can be acceptable for optoelectronic devices. The hardness and work hardening coefficient of LTCS crystal were investigated using Vickers microhardness testing and the work hardening value 1.94 point out soft nature of the grown material. By use of Kurtz and Perry technique the nonlinear second harmonic generation (SHG) efficiency of prepared LTCS crystals was assessed. The LTCS material developed here is used to treat bacterial and fungal infections and is detected by an antimicrobial assay