Investigating the inoculum dynamics of Cladosporium on the surface of raspberry fruits and in the air.

Raspberry production is under threat from the emerging fungal pathogenic genus Cladosporium. We used amplicon-sequencing, coupled with qPCR, to investigate how fruit age, fruit location within a polytunnel, polytunnel location and sampling date affected the fruit epiphytic microbiome. Fruit age was the most important factor impacting the fungal microbiome, followed by sampling date and polytunnel location. In contrast, polytunnel location and fruit age were important factors impacting the bacterial microbiome composition, followed by the sampling date. The within-tunnel location had a small significant effect on the fungal microbiome and no effect on the bacterial microbiome. As fruit ripened, fungal diversity increased and the bacterial diversity decreased. Cladosporium was the most abundant fungus of the fruit epiphytic microbiome, accounting for nearly 44% of all fungal sequences. Rotorod air samplers were used to study how the concentration of airborne Cladosporium inoculum (quantified by qPCR) varied between location (inside and outside the polytunnel) and time (daytime vs. nighttime). Quantified Cladosporium DNA was significantly higher during the day than the night and inside the polytunnel than the outside. This study demonstrated the dynamic nature of epiphytic raspberry fruit microbiomes and airborne Cladosporium inoculum within polytunnels, which will impact disease risks on raspberry fruit.

Effect on microbial communities in apple orchard soil when exposed short-term to climate change abiotic factors and different orchard management practices.

AIM: We assessed the effect of exposing apple orchard soil to different temperatures and CO2 levels on the resident microbiome of soils from a conventionally managed and an organically managed apple orchard. The key difference between these two orchards was that synthetic fertilizers and pesticides are routinely used in the former one. METHODS AND RESULTS: To investigate the effect of CO2 and temperature, soil samples from each site at two depths were exposed to either elevated temperature (29°C) at either 5000 or 10 000 ppm for five weeks or control conditions (25°C + 400 ppm). Both bacterial and fungal communities were profiled with amplicon-sequencing. The differences between the two orchards were the most significant factor affecting the bacterial and fungal communities, contributing to 53.7-14.0% of the variance in Bray-Curtis ß diversity, respectively. Elevated CO2 concentration and increased temperature affected organic orchard microbial diversity more than the conventionally managed orchard. A number of candidate beneficial and pathogenic microorganisms had differential abundances when temperature and CO2 were elevated, but their effect on the plant is unclear. CONCLUSIONS: This study has highlighted that microbial communities in bulk soils are most significantly influenced by crop management practices compared to the climate conditions used in the study. The studied climate conditions had a more limited effect on microbial community diversity in conventionally managed soil samples than in organically managed soils.

De novo genome assembly and functional annotation for Fusarium langsethiae.

BACKGROUND: Fusarium langsethiae is a T-2 and HT-2 mycotoxins producing species firstly characterised in 2004. It is commonly isolated from oats in Northern Europe. T-2 and HT-2 mycotoxins exhibit immunological and haemotological effects in animal health mainly through inhibition of protein, RNA and DNA synthesis. The development of a high-quality and comprehensively annotated assembly for this species is therefore essential in providing the molecular understanding and the mechanism of T-2 and HT-2 biosynthesis in F. langsethiae to help develop effective control strategies. RESULTS: The F. langsethiae assembly was produced using PacBio long reads, which were then assembled independently using Canu, SMARTdenovo and Flye. A total of 19,336 coding genes were identified using RNA-Seq informed ab-initio gene prediction. Finally, predicting genes were annotated using the basic local alignment search tool (BLAST) against the NCBI non-redundant (NR) genome database and protein hits were annotated using InterProScan. Genes with blast hits were functionally annotated with Gene Ontology. CONCLUSIONS: We developed a high-quality genome assembly of a total length of 59 Mb and N50 of 3.51 Mb. Raw sequence reads and assembled genome is publicly available and can be downloaded from: GenBank under the accession JAFFKB000000000. All commands used to generate this assembly are accessible via GitHub: https://github.com/FadyMohareb/fusarium_langsethiae .

Lactobacillus plantarum strain HT-W104-B1: potential bacterium isolated from Malaysian fermented foods for control of the dermatophyte Trichophyton rubrum.

The objective was to screen and evaluate the anti-fungal activity of lactic acid bacteria (LABs) isolated from Malaysian fermented foods against two Trichophyton species. A total of 66 LAB strains were screened using dual culture assays. This showed that four LAB strains were very effective in inhibiting growth of T. rubrum but not T. interdigitale. More detailed studies with Lactobacillus plantarum strain HT-W104-B1 showed that the supernatant was mainly responsible for inhibiting the growth of T. rubrum. The minimum inhibitory concentration (MIC), inhibitory concentration, the 50% growth inhibition (IC50) and minimum fungicide concentration (MFC) were 20 mg/mL, 14 mg/mL and 30 mg/mL, respectively. A total of six metabolites were found in the supernatant, with the two major metabolites being L-lactic acid (19.1 mg/g cell dry weight (CDW)) and acetic acid (2.2 mg/g CDW). A comparative study on keratin agar media showed that the natural mixture in the supernatants predominantly contained L-lactic and acetic acid, and this significantly controlled the growth of T. rubrum. The pure two individual compounds were less effective. Potential exists for application of the natural mixture of compounds for the treatment of skin infection by T. rubrum.

Inhibitory effects of climate change on the growth and extracellular enzyme activities of a widespread Antarctic soil fungus.

Temperatures approaching or exceeding 20°C have been measured during summer in polar regions at the surfaces of barren fellfield soils under cloudless skies around solar noon. However, despite the upper temperature limit for the growth of cold-adapted microbes-which are abundant in polar soils and have pivotal roles in nutrient cycling-typically being close to this temperature, previous studies have not addressed the consequences of climate change for the metabolism of these organisms in the natural environment. Here in a 5-year field experiment on Alexander Island in the southern maritime Antarctic, we show that the abundance of Pseudogymnoascus roseus, the most widespread decomposer fungus in maritime Antarctic fellfield soils, is reduced by 1-2 orders of magnitude when irrigated and nutrient-amended soils are warmed to >20°C during summer. Laboratory experiments under conditions mimicking those during midsummer in the natural environment indicated that the hyphal extension rates of P. roseus isolates and the activities of five extracellular enzymes are reduced by 54%-96% at high water availability after exposure to temperatures cycling daily from 2 to 21°C and 2 to 24°C, relative to temperatures cycling from 2 to 18°C. Given that the temperatures of surface soils at the study site already reach 19°C during midsummer, the observations reported here suggest that, at predicted rates of warming arising from moderate greenhouse gas emissions, inhibitory effects of climate change on the metabolism of P. roseus could manifest themselves within the next few decades. Furthermore, with peak temperatures at the surfaces of fellfield soils at other maritime Antarctic locations and in High Arctic and alpine regions already exceeding 20°C during summer, the observations suggest that climate warming has the potential to inhibit the growth of other cold-adapted microbes, with negative effects on soils as the Earth's climate continues to warm.

Phytopathogenic organisms and mycotoxigenic fungi: Why do we control one and neglect the other? A biological control perspective in Malaysia.

In this review, we present the current information on development and applications of biological control against phytopathogenic organisms as well as mycotoxigenic fungi in Malaysia as part of the integrated pest management (IPM) programs in a collective effort to achieve food security. Although the biological control of phytopathogenic organisms of economically important crops is well established and widely practiced in Malaysia with considerable success, the same cannot be said for mycotoxigenic fungi. This is surprising because the year round hot and humid Malaysian tropical climate is very conducive for the colonization of mycotoxigenic fungi and the potential contamination with mycotoxins. This suggests that less focus has been made on the control of mycotoxigenic species in the genera Aspergillus, Fusarium, and Penicillium in Malaysia, despite the food security and health implications of exposure to the mycotoxins produced by these species. At present, there is limited research in Malaysia related to biological control of the key mycotoxins, especially aflatoxins, Fusarium-related mycotoxins, and ochratoxin A, in key food and feed chains. The expected threats of climate change, its impacts on both plant physiology and the proliferation of mycotoxigenic fungi, and the contamination of food and feed commodities with mycotoxins, including the discovery of masked mycotoxins, will pose significant new global challenges that will impact on mycotoxin management strategies in food and feed crops worldwide. Future research, especially in Malaysia, should urgently focus on these challenges to develop IPM strategies that include biological control for minimizing mycotoxins in economically important food and feed chains for the benefit of ensuring food safety and food security under climate change scenarios.

Glycerol enhances fungal germination at the water-activity limit for life.

For the most-extreme fungal xerophiles, metabolic activity and cell division typically halts between 0.700 and 0.640 water activity (approximately 70.0-64.0% relative humidity). Here, we investigate whether glycerol can enhance xerophile germination under acute water-activity regimes, using an experimental system which represents the biophysical limit of Earth's biosphere. Spores from a variety of species, including Aspergillus penicillioides, Eurotium halophilicum, Xerochrysium xerophilum (formerly Chrysosporium xerophilum) and Xeromyces bisporus, were produced by cultures growing on media supplemented with glycerol (and contained up to 189 mg glycerol g dry spores-1 ). The ability of these spores to germinate, and the kinetics of germination, were then determined on a range of media designed to recreate stresses experienced in microbial habitats or anthropogenic systems (with water-activities from 0.765 to 0.575). For A. penicillioides, Eurotium amstelodami, E. halophilicum, X. xerophilum and X. bisporus, germination occurred at lower water-activities than previously recorded (0.640, 0.685, 0.651, 0.664 and 0.637 respectively). In addition, the kinetics of germination at low water-activities were substantially faster than those reported previously. Extrapolations indicated theoretical water-activity minima below these values; as low as 0.570 for A. penicillioides and X. bisporus. Glycerol is present at high concentrations (up to molar levels) in many types of microbial habitat. We discuss the likely role of glycerol in expanding the water-activity limit for microbial cell function in relation to temporal constraints and location of the microbial cell or habitat. The findings reported here have also critical implications for understanding the extremes of Earth's biosphere; for understanding the potency of disease-causing microorganisms; and in biotechnologies that operate at the limits of microbial function.

Development of a HOG-based real-time PCR method to detect stress response changes in mycotoxigenic moulds.

There is a need to understand the mechanism of adaptation of toxigenic fungal species which are able to colonise highly specialised foods such as cured meats where there is a high osmotic stress due to the presence up to 20-22% NaCl during the ripening process. A new tool able to detect changes in stress related genes would be useful to understand the ecological reasons for the ability of these species to grow in specialised niches. In this work a real-time PCR (qPCR) using SYBR Green was developed. Primers were designed from the Hog1 gene involved in osmo-adaptation in fungi. For this, conserved regions resulting from the alignment of 26 published partial sequences of such gene were used. Specificity of primers HogF2/R2 was demonstrated when amplified, producing a unique 131-bp PCR product with a Tm value of 84 °C. The qPCR method showed an efficiency of 98%, R(2) value > 0.99 and a detection limit of 0.7 log Hog1 gene copies. The qPCR method to measure changes in the Hog1 gene expression in relation to growth in ionic and non-ionic stressed environments (using 10-40% NaCl and sorbitol concentrations) was found to be suitable for two mycotoxigenic species (Penicillium nordicum, P. expansum). This assay will be a valuable tool for generating relevant Hog1 expression data from different mould species in relation to different stresses in food habitats. It will also be a good tool for a better understanding of the ability of xerophilic and xerotolerant species to colonise extreme environments.

Impacts of environmental stress on growth, secondary metabolite biosynthetic gene clusters and metabolite production of xerotolerant/xerophilic fungi.

This paper examines the impact that single and interacting environmental stress factors have on tolerance mechanisms, molecular ecology and the relationship with secondary metabolite production by a group of mycotoxigenic species of economic importance. Growth of these fungi (Aspergillus flavus, A.ochraceus, A.carbonarius, Penicillium nordicum and P. verrucosum) is influenced by water and temperature interactions and type of solute used to induce water stress. Such abiotic stresses are overcome by the synthesis of increased amounts of low molecular weight sugar alcohols, especially glycerol and erythritol, to enable them to remain active under abiotic stress. This is accompanied by increased expression of sugar transporter genes, e.g., in A. flavus, which provides the nutritional means of tolerating such stress. The optimum conditions of water activity (a w) × temperature stress for growth are often different from those for secondary metabolite production. The genes for toxin production are clustered together and their relative expression is influenced by abiotic interacting stress factors. For example., A. flavus synthesises aflatoxins under water stress in non-ionic solutes. In contrast, P. nordicum specifically occupies a high salt (0.87 a w = 22% NaCl) niche such as cured meats, and produces ochratoxin A (OTA). There is differential and temporal expression of the genes in the secondary metabolite clusters in response to a w × temperature stress. We have used a microarray and integrated data on growth, relative expression of key genes in the biosynthetic pathways for secondary metabolite production and toxin production using a mixed growth model. This was used to correlate these factors and predict the toxin levels produced under different abiotic stress conditions. This system approach to integrate these different data sets and model the relationships could be a powerful tool for predicting the relative toxin production under extreme stress conditions, including climate change scenarios. This approach will facilitate a better functional understanding of the influence that environmental stress has on these mycotoxigenic fungi and enable better prevention strategies to be developed based on this system-based approach.

Concomitant osmotic and chaotropicity-induced stresses in Aspergillus wentii: compatible solutes determine the biotic window.

Whereas osmotic stress response induced by solutes has been well-characterized in fungi, less is known about the other activities of environmentally ubiquitous substances. The latest methodologies to define, identify and quantify chaotropicity, i.e. substance-induced destabilization of macromolecular systems, now enable new insights into microbial stress biology (Cray et al. in Curr Opin Biotechnol 33:228-259, 2015a, doi: 10.1016/j.copbio.2015.02.010 ; Ball and Hallsworth in Phys Chem Chem Phys 17:8297-8305, 2015, doi: 10.1039/C4CP04564E ; Cray et al. in Environ Microbiol 15:287-296, 2013a, doi: 10.1111/1462-2920.12018 ). We used Aspergillus wentii, a paradigm for extreme solute-tolerant fungal xerophiles, alongside yeast cell and enzyme models (Saccharomyces cerevisiae and glucose-6-phosphate dehydrogenase) and an agar-gelation assay, to determine growth-rate inhibition, intracellular compatible solutes, cell turgor, inhibition of enzyme activity, substrate water activity, and stressor chaotropicity for 12 chemically diverse solutes. These stressors were found to be: (i) osmotically active (and typically macromolecule-stabilizing kosmotropes), including NaCl and sorbitol; (ii) weakly to moderately chaotropic and non-osmotic, these were ethanol, urea, ethylene glycol; (iii) highly chaotropic and osmotically active, i.e. NH4NO3, MgCl2, guanidine hydrochloride, and CaCl2; or (iv) inhibitory due primarily to low water activity, i.e. glycerol. At ≤0.974 water activity, Aspergillus cultured on osmotically active stressors accumulated low-M r polyols to ≥100 mg g dry weight(-1). Lower-M r polyols (i.e. glycerol, erythritol and arabitol) were shown to be more effective for osmotic adjustment; for higher-M r polyols such as mannitol, and the disaccharide trehalose, water-activity values for saturated solutions are too high to be effective; i.e. 0.978 and 0.970 (25 ºC). The highly chaotropic, osmotically active substances exhibited a stressful level of chaotropicity at physiologically relevant concentrations (20.0-85.7 kJ kg(-1)). We hypothesized that the kosmotropicity of compatible solutes can neutralize chaotropicity, and tested this via in-vitro agar-gelation assays for the model chaotropes urea, NH4NO3, phenol and MgCl2. Of the kosmotropic compatible solutes, the most-effective protectants were trimethylamine oxide and betaine; but proline, dimethyl sulfoxide, sorbitol, and trehalose were also effective, depending on the chaotrope. Glycerol, by contrast (a chaotropic compatible solute used as a negative control) was relatively ineffective. The kosmotropic activity of compatible solutes is discussed as one mechanism by which these substances can mitigate the activities of chaotropic stressors in vivo. Collectively, these data demonstrate that some substances concomitantly induce chaotropicity-mediated and osmotic stresses, and that compatible solutes ultimately define the biotic window for fungal growth and metabolism. The findings have implications for the validity of ecophysiological classifications such as 'halophile' and 'polyextremophile'; potential contamination of life-support systems used for space exploration; and control of mycotoxigenic fungi in the food-supply chain.

Effect of interaction between Aspergillus carbonarius and non-ochratoxigenic grape-associated fungal isolates on growth and ochratoxin A production at different water activities and temperatures.

The effect of water activity (0.90, 0.94, and 0.98 aw) and temperature (15, 20, and 25 °C) on the in vitro interactions between three ochratoxigenic strains of Aspergillus carbonarius (Ac-28, Ac-29, and Ac-33) and eleven non ochratoxigenic grape-associated fungal strains was assessed in this study. Fungal strains were allowed to grow in dual cultures on Synthetic Grape-juice Medium (SGM) for 15 days and fungal interactions were given a numerical score to obtain an Index of Dominance (ID) for each fungus. Results showed that in most cases A. carbonarius toxigenic strains were dominant against other fungal species. However, A. carbonarius presented mutual antagonism with A. section Nigri strains regardless of water activity (aw) and temperature. Moreover, interactions with Penicillium spinulosum and Cladosporium spp. at 15 °C, as well as Botrytis cinerea at 20 °C, showed that the antagonists were more competitive against A. carbonarius. In some cases, growth rates of A. carbonarius strains were either slightly stimulated or inhibited after interaction in dual cultures, depending on temperature, aw and competing species. Regarding OTA production, the presence of other species sometimes decreased the production or slightly enhanced it, depending on fungal competitor and environmental conditions. Overall, OTA production was higher at 15 °C/0.98 aw and 20 °C/0.98 aw for all target strains and at 20 °C/0.94 aw for Ac-33 strain only, but decreased at higher temperatures regardless of aw and interacting species.

Assessment of rhizospheric culturable bacteria of Phragmites australis and Juncus effusus from polluted sites.

This study aimed at the isolation and characterization of metal(loid)-tolerant bacteria from the rhizosphere of Phragmites australis and Juncus effusus plants growing in two long-term contaminated sites in Northern Portugal. Site 1 had higher contamination than Site 3. Bacteria were isolated using metal(loid)-supplemented (Cd, Zn, and As) media. Isolates were grouped by random amplified polymorphic DNA and identified by 16S rRNA gene sequencing. Strains were also examined for their metal(loid) tolerance. The counts of metal(loid)-tolerant bacteria were higher in Site 1 and ranged between log 7.17 CFU g(-1) soil in As-containing medium and log 7.57 CFU g(-1) soil in Zn-containing medium, while counts at Site 3 varied between log 5.33 CFU g(-1) soil in Cd-containing medium and log 6.97 CFU g(-1) soil in As-containing medium. The composition of bacterial populations varied between locations. In Site 1, the classes Actinobacteria (36%) and Bacilli (24%) were well represented, while in Site 3 strains were mainly affiliated to classes Actinobacteria (35%), γ-Proteobacteria (35%), and ß-Proteobacteria (12%). The order of metal(loid) toxicity for the isolated strains was Cd > As > Zn. Overall, 10 strains grew at 500 mg Cd L(-1) , 1000 mg Zn L(-1) , and 500 mg As L(-1) , being considered the most metal(loid)-tolerant bacteria. These strains belonged to genera Cupriavidus, Burkholderia, Novosphingobium, Sphingobacterium, Castellaniella, Mesorhizobium, Chryseobacterium, and Rhodococcus and were mainly retrieved from Site 1. The multiple metal(loid)-tolerant strains isolated in this study have potential to be used in bioremediation/phytoremediation.

Inter-row cropping and rootstock genotype selection in a UK cider orchard to combat apple replant disease.

Apple rootstock genotypes confer different levels of tolerance to apple replant disease (ARD) and vigour to a newly replanted apple tree. A hybrid management system of rotating the rootstock genotype planted between successive generations and inter-row planting in the alleyways of orchards may minimise the severity of ARD symptoms. High-throughput sequencing of the fungal ITS and bacterial 16S rDNA regions was used to investigate the diversity, and differential taxa present in soils displaying symptoms of ARD. Candidate pathogens and beneficial microorganisms were correlated with the above-ground establishment of each rootstock genotype in a UK cider orchard. Our results suggest that the same rootstock or rootstock with closely related parentage to the previous rootstock had more severe ARD symptoms. Planting in the alleyway appeared an effective strategy to minimise the severity of symptoms irrespective of rootstock genotype. The planting location effect had a higher contribution to the variation in the rhizosphere microbiome than that of the rootstock genotype. No predicted causal agents for ARD could be identified to a taxonomic level to predict their function but two species associated with mycorrhizae, Pteridiospora spinosispora and Paraglomus laccatum were identified as inversely correlated with ARD severity and could be candidate beneficial species for apple, warranting further investigation and research. Our findings suggest that planting in the alleyways and planting rootstocks genetically dissimilar to the previously planted rootstock can be beneficial for tree establishment. We have also identified species inversely associated with ARD severity, making candidates for future research to test the antagonistic effect of the species against ARD pathogens in apple roots. Supplementary Information: The online version contains supplementary material available at 10.1186/s42483-023-00184-y.

Acclimatisation of Fusarium langsethiae, F. poae and F. sporotrichioides to elevated CO2: Impact on fungal growth and mycotoxin production on oat-based media.

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).

Analysis of volatile fingerprints for monitoring anti-fungal efficacy against the primary and opportunistic pathogen Aspergillus fumigatus.

The aims of this study were to use qualitative volatile fingerprints obtained using a hybrid sensor array system to screen anti-fungals for controlling the important lung infecting fungus, Aspergillus fumigatus, especially in immunocompromised patients. SIFT-MS was also used to try and identify key volatiles produced by A. fumigatus. Initial studies were carried out to identify the ED(50) and ED(90) (effective dose) for inhibiting growth of A. fumigatus using three anti-fungal compounds, benomyl, tebuconazole and fluconazole. Subsequent studies involved inoculation of malt extract agar plates with spores of A. fumigatus (25 and 37°C) over periods of 24-72 h to examine the headspace volatile fingerprints generated from the sample treatments using the hybrid sensor array system to compare controls and ED(50)/ED(90) concentrations. The sensor responses showed discrimination between treatments after 48-h incubation when benomyl and tebuconazole were used against A. fumigatus at 37°C using Principal Components Analysis and Cluster Analysis. SIFT-MS analysis showed that methyl pentadiene, ethanol, isoprene and methanol were key biomarker volatiles produced by A. fumigatus in the presence of anti-fungal compounds. This may also be a good approach for the development of rapid screening of anti-microbial compounds and potentially useful for monitoring the possible build up of resistance to specific drug types. Volatile fingerprints produced by patient samples could also be used to evaluate whether lung infections are caused by bacteria or specific fungi to facilitate early diagnosis and enable the right drug treatment to be prescribed.

The effect of substrate, season, and agroecological zone on mycoflora and aflatoxin contamination of poultry feed from Khyber Pakhtunkhwa, Pakistan.

To study the effects of and interactions among feed types, seasons, and agroecological zones on the total fungal viable count and aflatoxins B1 (AFB1), B2 (AFB2), G1 (AFG1), and G2 (AFG2) production in poultry feed, an experiment was conducted using three-factorial design. A total of 216 samples of poultry feed ingredients, viz. maize, wheat, rice, cotton seed meal (CSM), and finished products, that is, starter and finisher broilers' rations, were collected from Peshawar, Swat, and D. I. Khan districts of Khyber Pakhtunkhwa, Pakistan, during the winter, spring, summer, and autumn seasons of the year 2007/2008. Analysis of variance showed that there was a complex interaction among all these factors and that this influenced the total fungal viable count and relative concentrations of the aflatoxins produced. Minimum total culturable fungi (6.43 × 10³ CFUs/g) were counted in CSM from D. I. Khan region in winter season while maximum (26.68 × 10³ CFUs/g) in starter ration from Peshawar region in summer. Maximum concentrations of AFB1 (191.65 ng/g), AFB2 (86.85 ng/g), and AFG2 (89.90 ng/g) were examined during the summer season whereas the concentration of AFG1 was maximum (167.82 ng/g) in autumn in finisher ration from Peshawar region. Minimum aflatoxins were produced in the winter season across all the three agroecological zones.

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.

Comparison of growth and aflatoxin B1 production profiles of Aspergillus flavus strains on conventional and isogenic GM-maize-based nutritional matrices.

Maize grown in both North and South America are now predominantly genetically modified (GM) cultivars with some resistance to herbicide, pesticide, or both. There is little information on the relative colonisation and aflatoxin B1 (AFB1) production with maize meal-based nutritional matrices based on kernels of non-GM maize and isogenic GM-ones by strains of Aspergillus flavus. The objectives were to examine the effect of interacting conditions of temperature (25-35 °C) and water availability (0.99-0.90 water activity, aw) on (a) mycelial growth, (b) AFB1 production and (c) develop contour maps of optimum and marginal conditions of these parameters for four strains of A. flavus on three different non-GM and isogenic GM-maize based nutritional media. The growth of the four strains of A. flavus (three aflatoxigenic; one non-aflatoxigenic) was relatively similar in relation to the temperature × aw conditions examined on both non-GM and GM-based matrices. Optimum growth overall was at 30-35 °C and 0.99 aw for all four strains. Under water stress (0.90 aw) growth was optimum at 35 °C. Statistically: non-GM, GM cultivars, temperature and aw all significantly affected growth rates. For AFB1 production, all single and interacting factors were statistically significant except for non-GM × GM cultivar. In conclusion, colonisation of GM- and non-GM nutritional sources was similar for the different A. flavus strains examined. The contour maps will be very useful for understanding the ecological niches for both toxigenic and non-toxigenic strains in the context of the competitive exclusion of those producing aflatoxins.

Interacting Environmental Stress Factors Affect Metabolomics Profiles in Stored Naturally Contaminated Maize.

There is interest in understanding the relationship between naturally contaminated commodities and the potential for the production of different useful and toxic secondary metabolites (SMs). This study examined the impact of interacting abiotic stress parameters of water availability and temperature of stored naturally contaminated maize on the SM production profiles. Thus, the effect of steady-state storage water activity (aw; 0.80−0.95) and temperature (20−35 °C) conditions on SM production patterns in naturally contaminated maize was examined. The samples were analysed using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) to evaluate (a) the total number of known SMs, (b) their concentrations, and (c) changes under two-way interacting environmental stress conditions. A total of 151 metabolites were quantified. These included those produced by species of the Aspergillus, Fusarium and Penicillium genera and other unspecified ones by other fungi or bacteria. There were significant differences in the numbers of SMs produced under different sets of interacting environmental conditions. The highest total number of SMs (80+) were present in maize stored at 20−25 °C and 0.95 aw. In addition, there was a gradation of SM production with the least number of SMs (20−30) produced under the driest conditions of 0.80 aw at 20−30 °C. The only exception was at 35 °C, where different production patterns occurred. There were a total of 38 Aspergillus-related SMs, with most detected at >0.85 aw, regardless of the temperature in the 50−500 ng/g range. For Fusarium-related SMs, the pattern was different, with approx. 10−12 SMs detected under all aw × temperature conditions with >50% produced at 500 ng/g. A total of 40−45 Penicillium-related SMs (50−500 ng/g) were detected in the stored maize but predominantly at 20−25 °C and 0.95 aw. Fewer numbers of SMs were found under marginal interacting abiotic stress storage conditions in naturally contaminated maize. There were approx. eight other known fungal SM present, predominantly in low concentrations (<50 ng/g), regardless of interacting abiotic conditions. Other unspecified SMs present consisted of <20 in low concentrations. The effect of interacting abiotic stress factors for the production of different suites of SMs to take account of the different ecological niches of fungal genera may be beneficial for identifying biotechnologically useful SMs.

Comparison of multiple mycotoxins in harvested maize samples in three years (2018-2020) in four continents.

This study has examined the pattern of mycotoxin contamination of maize destined for animal feed in different global regions over a period of 3 years (2018-2020) with up to 1000+ samples analysed in each year. Overall, >75% of samples in each of the survey years were contaminated with multiple mycotoxins regardless of the global region (Europe, Africa, Asia, South Americas countries). Using LC-MS/MS, it was possible to quantify the relative contamination present in the samples in each year from the different regions of eight different mycotoxins including aflatoxin B1 (AFB1), ochratoxin A (OTA) deoxynivalenol (DON), fumonisin B1 (FB1) and B2, zearalenone (ZEA), T-2 and HT-2 toxins. The trends in mycotoxin contamination showed that there was a consistent contamination with DON in the 3 sampling years in all four regions. Interestingly, AFB1 contamination was prevalent in all regions in 2018, but more predominant in Europe and in 2019. In contrast, in 2020 it was found to be the major contaminant in Africa only. However, FB1 contamination of maize which was prevalent in Europe in 2018, became more prevalent in Asia and LATAM countries in 2019 and even in African maize in 2020. Comparisons of contamination with different mycotoxins in each of the years globally showed significant differences for AFB1, FB1, DON and ZEA between the different years. These results are discussed in relation to the trends of contamination of maize with mixtures of mycotoxins and the implication for their control in this key commodity used as an important ingredient in animal feed.