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Dry rot is an emerging issue for garlic production worldwide and Fusarium proliferatum is its major causal agent. Since the disease is seed-transmitted, sowing healthy cloves is crucial. In this study, some disinfection strategies were tested on garlic seeds, including steam, dry heat, chemical disinfectants and gaseous ozone (O3). Steam reduced the Colony Forming Units·g−1 (CFUs·g−1) by up to 92% in garlic seeds, but, at the same time, it affected their germination (−36%). Similarly, hydrogen peroxide (H2O2) and peracetic acid (C2H4O3) reduced the CFUs·g−1 by up to 83%; however, these methods also severely impaired germination (−40%). Dry heat did not negatively impact germination, but fungal contamination was not significantly reduced. The most promising strategy was gaseous O3 treatment; it decreased CFUs·g−1 by up to 96%, without causing any reduction of germination. The treatments applied were partially effective because the fungus is predominantly located in the outer layer of the seed, although it is also found in the inner portions. Some of these treatments can contribute to garlic protection from seed-borne pathogens and possibly reduce the occurrence of garlic dry rot.

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 five‐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 2–21 °C and 2–24 °C, relative to temperatures cycling from 2–18 °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.

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 five‐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 2–21 °C and 2–24 °C, relative to temperatures cycling from 2–18 °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.

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.

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.

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.

This study was carried out to examine the efficacy of two biocontrol agents (Clonostachys rosea 016, BCA1; Gram-negative bacterium, BCA5) for control of FUM1 gene expression and fumonisin B1 (FB1) production by F. verticillioides FV1 on maize cobs of different ripening stages: R3, Milk (0.985 aw); R4, Dough (0.976 aw); R5, Dent (0.958 aw). Initially, temporal studies on FUM1 gene expression and FB1 production were performed on maize kernels for up to 14 days. This revealed that day 10 was optimum for both parameters, and was used in the biocontrol studies. Maize cobs were inoculated with 50:50 mixtures of the pathogen:antagonist inoculum and incubated in environmental chambers to maintain the natural aw conditions for ten days at 25 and 30 °C. The growth rates of F. verticillioides FV1, the relative expression of the FUM1 gene and FB1 production were quantified. It was found that, aw × temp had significant impacts on growth, FUM1 gene expression and FB1 production by F. verticillioides FV1 on maize cobs of different maturities. The fungal antagonist (BCA1) significantly reduced FB1 contamination on maize cobs by > 70% at 25 °C, and almost 60% at 30 °C regardless of maize ripening stage. For the bacterial antagonist (BCA5) however, FB1 levels on maize cobs were significantly decreased only in some treatments. These results suggest that efficacy of antagonists to control mycotoxin production in ripening maize cobs needs to take account of the ecophysiology of the pathogen and the antagonists, as well as the physiological status of the maize during silking to ensure effective control.

This paper discusses the relationship between biocontrol agents (BCAs) and mycotoxigenic fungi and mycotoxin control. In most cases BCAs are examined for control of growth of fungal pathogens and disease symptoms. However, for mycotoxin control the approach and focus needs to be different. The mechanism of action and the inoculum dose necessary for control of toxin production by Aspegillus, Penicillium and Fusarium species may be different from that for traditional fungal plant pathogens. The mechanisms of action, the relative inoculum potential and the impact that interacting environmental conditions have on control of key components of the life cycle of mycotoxigenic fungi are considered. The practical aspects of production and formulation hurdles are discussed and potential future approaches and strategies which may need to be considered for more effective biocontrol of mycotoxigenic fungi and mycotoxins are presented.

This paper discusses the relationship between biocontrol agents (BCAs) and mycotoxigenic fungi and mycotoxin control. In most cases BCAs are examined for control of growth of fungal pathogens and disease symptoms. However, for mycotoxin control the approach and focus needs to be different. The mechanism of action and the inoculum dose necessary for control of toxin production by Aspegillus, Penicillium and Fusarium species may be different from that for traditional fungal plant pathogens. The mechanisms of action, the relative inoculum potential and the impact that interacting environmental conditions have on control of key components of the life cycle of mycotoxigenic fungi are considered. The practical aspects of production and formulation hurdles are discussed and potential future approaches and strategies which may need to be considered for more effective biocontrol of mycotoxigenic fungi and mycotoxins are presented.

This study was carried out to examine the efficacy of two biocontrol agents (Clonostachys rosea 016, BCA1; Gram-negative bacterium, BCA5) for control of FUM1 gene expression and fumonisin B1 (FB1) production by F. verticillioides FV1 on maize cobs of different ripening stages: R3, Milk (0.985 aw); R4, Dough (0.976 aw); R5, Dent (0.958 aw). Initially, temporal studies on FUM1 gene expression and FB1 production were performed on maize kernels for up to 14 days. This revealed that day 10 was optimum for both parameters, and was used in the biocontrol studies. Maize cobs were inoculated with 50:50 mixtures of the pathogen:antagonist inoculum and incubated in environmental chambers to maintain the natural aw conditions for ten days at 25 and 30 °C. The growth rates of F. verticillioides FV1, the relative expression of the FUM1 gene and FB1 production were quantified. It was found that, aw × temp had significant impacts on growth, FUM1 gene expression and FB1 production by F. verticillioides FV1 on maize cobs of different maturities. The fungal antagonist (BCA1) significantly reduced FB1 contamination on maize cobs by > 70% at 25 °C, and almost 60% at 30 °C regardless of maize ripening stage. For the bacterial antagonist (BCA5) however, FB1 levels on maize cobs were significantly decreased only in some treatments. These results suggest that efficacy of antagonists to control mycotoxin production in ripening maize cobs needs to take account of the ecophysiology of the pathogen and the antagonists, as well as the physiological status of the maize during silking to ensure effective control.

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.

A mycotoxigenic strain of Fusarium verticillioides previously isolated from Malaysian maize kernels and identified morphologically and molecularly was used in the present work. The objectives were (1) to screen the competitiveness of three potential antagonists isolated from Malaysian maize and three other known candidates for control of growth of F. verticillioides in vitro based on interaction scores, growth rates and hyphal area of F. verticillioides, and (2) to examine the best candidates using different spore/cell ratios on milled maize agar at different water activity conditions on relative control of fumonisin B1 (FB1). Three fungi and three bacteria (BCAs 1-6) were examined for antagonistic effects against F. verticillioides in dual-culture assays. These showed that all fungal candidates intermingled with F. verticillioides while all bacterial candidates inhibited F. verticillioides on contact or at a distance, which in turn decreased the growth rates and hyphal area of F. verticillioides significantly. Although BCA1 (Clonostachys rosea 016) did not inhibit growth or hyphal area of F. verticillioides, it was included in FB1 inhibition studies with other bacterial candidates (BCA4, Streptomyces sp. AS1; BCA5, Gram-negative rod; BCA6, Enterobacter hormaechei) because of its established mycoparasitism. The FB1 inhibition studies were conducted on milled maize agar with different spore/cell ratios of pathogen:antagonist mixtures at 0.95/0.98 water activity (aw) and 25 °C for 14 days. FB1 biosynthesis for all treatments was significantly higher at 0.95 than 0.98 aw. Of the four antagonists tested, the best was BCA1 which inhibited FB1 biosynthesis by 73 and 100% at 0.95 and 0.98 aw, respectively. BCA5 was the next best, resulting in 38 and 78% FB1 inhibition at 0.95 and 0.98 aw, respectively. The pathogen:antagonist ratios for BCA1 and BCA5 showed best results at 50:50 and 25:75. These results were discussed in context of using biocontrol agents to minimise fumon

A mycotoxigenic strain of Fusarium verticillioides previously isolated from Malaysian maize kernels and identified morphologically and molecularly was used in the present work. The objectives were (1) to screen the competitiveness of three potential antagonists isolated from Malaysian maize and three other known candidates for control of growth of F. verticillioides in vitro based on interaction scores, growth rates and hyphal area of F. verticillioides, and (2) to examine the best candidates using different spore/cell ratios on milled maize agar at different water activity conditions on relative control of fumonisin B1 (FB1). Three fungi and three bacteria (BCAs 1-6) were examined for antagonistic effects against F. verticillioides in dual-culture assays. These showed that all fungal candidates intermingled with F. verticillioides while all bacterial candidates inhibited F. verticillioides on contact or at a distance, which in turn decreased the growth rates and hyphal area of F. verticillioides significantly. Although BCA1 (Clonostachys rosea 016) did not inhibit growth or hyphal area of F. verticillioides, it was included in FB1 inhibition studies with other bacterial candidates (BCA4, Streptomyces sp. AS1; BCA5, Gram-negative rod; BCA6, Enterobacter hormaechei) because of its established mycoparasitism. The FB1 inhibition studies were conducted on milled maize agar with different spore/cell ratios of pathogen:antagonist mixtures at 0.95/0.98 water activity (aw) and 25 °C for 14 days. FB1 biosynthesis for all treatments was significantly higher at 0.95 than 0.98 aw. Of the four antagonists tested, the best was BCA1 which inhibited FB1 biosynthesis by 73 and 100% at 0.95 and 0.98 aw, respectively. BCA5 was the next best, resulting in 38 and 78% FB1 inhibition at 0.95 and 0.98 aw, respectively. The pathogen:antagonist ratios for BCA1 and BCA5 showed best results at 50:50 and 25:75. These results were discussed in context of using biocontrol agents to minimise fumon

Phlomis purpurea (Lamiaceae), found in Quercus suber and Quercus ilex ssp. rotundifolia forest habitats in southern Portugal, is a non-host for the oomycete Phytophthora cinnamomi, the main biotic factor involved in cork oak and holm oak decline in the Iberian Peninsula. The effect of P. purpurea crude ethanol root extract was evaluated in vitro on P. cinnamomi mycelial growth, sporangial production, zoospore release and germination as well as on chlamydospore production and viability. The protection of cork oak against infection by the pathogen was also evaluated in planta. At 10 mg ml(-1), in vitro inhibition of the pathogen structures was 85-100 %. In addition, P. purpurea plants were shown to protect Q. suber and Q. ilex from P. cinnamomi infection and to reduce the inoculum potential in glasshouse trials, indicating the ability to reduce root infection by the pathogen. The results suggest that P. purpurea has the potential to reduce disease spread and that their root extracts could provide candidate substances for control of the important pathogen, P. cinnamomi., AuthorCount:10

A study was undertaken to investigate the role of water activity (aw) and fungicides on the competitiveness of two Fusarium verticillioides strains against other spoilage fungi commonly present in maize (F. proliferatum, Aspergillus niger, A. flavus, A. ochraceus and Penicillium verrucosum). Fungal strains were inoculated on artificial media containing maize flour. The effect of three aw levels (0.99, 0.98 and 0.95) and 3 fungicides (Tebuconazole, Procloraz and Prothioconazole) on fungal interactions, the Index of Dominance (ID) of isolates and fumonisin B1+B2 (FBs) production were determined. The two strains of F. verticillioides showed similar behaviour in conditions where water was freely available (0.99 aw); at 0.98 and 0.95 aw both F. verticilliodes strains had the lowest total ID scores (8-6 and 10-12, respectively). They showed the same ability to compete against other fungi having the highest ID scores against P. verrucosum and A. ochraceus and the lowest against A. niger and A. flavus. Interestingly, 0.95 aw was the most conducive condition for fumonisin production with significant differences respect to 0.98 and 0.99 aw. In co-inoculation experiment, only FBs production obtained with P. verrucosum resulted significantly higher from those obtained in the presence of the other fungi. The use of fungicides reduced both F. verticilliodes strains Index of Dominancy (ID). In particular, a significant reduction in F. verticilloides strain growth was observed when combining water stress x fungicide treatments. This information could be useful for a better understanding of the colonisation patterns of F. verticillioides in relation to other mycobiota and to both environmental and chemical stresses, also in future climate change scenarios.

A key step in the infection cycle by Aspergillus flavus in maize is sporulation of sclerotia present in soil or in crop debris. However, little information is available on this critical and important phase. This study included experiments on artificial (CZ agar) and natural (maize stalks) substrates under different conditions of temperature (T; from 5 to 45°C) and water activity (aw; from 0.50 to 0.99) levels to quantify sporulation from sclerotia. The mean numbers of spores was higher on defined nutritional medium in vitro on CZ agar than on maize stalks (4.5 x 106 versus 4.2 x 104) with production initiated after 6 and 24 hours, respectively. Surprisingly, optimal temperature was found at 30-35°C for CZ agar (9.23 x 106 spores/sclerotium) and at 20-25°C for maize stalks (6.26 x 104 spores/sclerotium). Water stress imposition only reduced sporulation at ≤ 0.90 aw. With more available water no significant differences were found between 0.90-0.99 aw. This type of data is critical in the development of a mechanistic model to predict the infection cycle of A. flavus in maize in relation to meteorological conditions.

The temporal efficacy of three different chemical fungicides (Folicur®, Proline®, Sportak 45EW®) and a biocontrol bacterium (Serenade, B. subtilis) in reducing growth and toxin production by isolates of F. verticillioides and A. flavus was studied in vitro under different water activity regimes (0.99, 0.98 and 0.95). All the fungicides significantly inhibited mycelial growth compared with the control; the most effective treatment, both against F. verticillioides and A. flavus, was Sportak 45EW® (approx. 99%). The inhibitory effect of all fungicides generally improved with increasing concentration. Serenade always decreased fungal growth, with optimal results at concentrations of 104 and 106 (70‒75% reduction). All the fungicide treatments resulted in a significant reduction in both FB1+FB2 and AFB1 production when compared to the control, at the end of the incubation period and with the 2 concentrations used (approx. 99%). A threshold concentration inoculum of at least 104 CFUs of B. subtilis per g was required to achieve a significant control of mycotoxin production. Sportak 45EW® and Serenade gave the best control of mycotoxin production with a reduction of 95% compared to the controls. Use of Serenade in the field should include due consideration to its sensitivity to low water activities, when compared to the target pathogens.

Basaltic glasses (hyaloclastite) are a widespread habitat for life in volcanic environments, yet their interior physical conditions are poorly characterized. We investigated the characteristics of exposed weathered basaltic glass from a surface outcrop in Iceland, using microprobes capable of continuous sensing, to determine whether the physical conditions in the rock interior are hospitable to microbial life. The material provided thermal protection from freeze-thaw and rapid temperature fluctuations, similar to data reported for other rock types. Water activity experiments showed that at moisture contents less than 13% wet weight, the glass and its weathering product, palagonite, had a water activity below levels suitable for bacterial growth. In pore spaces, however, these higher moisture conditions might be maintained for many days after a precipitation event. Gas exchange between the rock interior and exterior was rapid (<10 min) when the rocks were dry, but when saturated with water, equilibration took many hours. During this period, we demonstrated the potential for low oxygen conditions within the rock caused by respiratory stimulation of the heterotrophic community within. These conditions might exist within subglacial environments during the formation of the rocks or in micro-environments in the interior of exposed rocks. The experiments showed that microbial communities at the site studied here could potentially be active for 39% of the year, if the depth of the community within the outcrop maintains a balance between access to liquid water and adequate protection from freezing. In the absence of precipitation, the interior of weathered basaltic glass is an extreme and life-limiting environment for microorganisms on Earth and other planets.

Basaltic glasses (hyaloclastite) are a widespread habitat for life in volcanic environments, yet their interior physical conditions are poorly characterized. We investigated the characteristics of exposed weathered basaltic glass from a surface outcrop in Iceland, using microprobes capable of continuous sensing, to determine whether the physical conditions in the rock interior are hospitable to microbial life. The material provided thermal protection from freeze-thaw and rapid temperature fluctuations, similar to data reported for other rock types. Water activity experiments showed that at moisture contents less than 13% wet weight, the glass and its weathering product, palagonite, had a water activity below levels suitable for bacterial growth. In pore spaces, however, these higher moisture conditions might be maintained for many days after a precipitation event. Gas exchange between the rock interior and exterior was rapid (<10 min) when the rocks were dry, but when saturated with water, equilibration took many hours. During this period, we demonstrated the potential for low oxygen conditions within the rock caused by respiratory stimulation of the heterotrophic community within. These conditions might exist within subglacial environments during the formation of the rocks or in micro-environments in the interior of exposed rocks. The experiments showed that microbial communities at the site studied here could potentially be active for 39% of the year, if the depth of the community within the outcrop maintains a balance between access to liquid water and adequate protection from freezing. In the absence of precipitation, the interior of weathered basaltic glass is an extreme and life-limiting environment for microorganisms on Earth and other planets.

The aim of this study was to define quantitative relationships between temperature and water activity (aw), fungal growth and aflatoxin B1 (AFB1) production. A strain of A. flavus isolated from maize in North Italy, and previously tested and found positive for AFB1 production, was used for these experiments. The optimum temperature for AFB1 production was at 25°C, slightly lower with respect to results obtained in other countries. 0.83 aw was the limit for growth of this strain of A. flavus after 60 days incubation at the optimum temperature. The solutes used to modify aw, glycerol and NaCl, influenced both growth and secondary metabolite production. Media modified with glycerol resulted in more AFB1 production when compared to the non-ionic solute NaCl added media. Maize based media, prepared with flour obtained from kernels at different ripening stages, only slightly influenced growth rates of A. flavus. The quantitative relationship obtained between fungal growth and AFB1 production in diverse temperature and aw levels were used to develop a valid predictive model for A. flavus presence and AFB1 production in the field.

Aims: To evaluate the potential use of volatile fingerprints produced by Penicillium nordicum to discriminate ochratoxin A (OTA) producers or non-producers strains on a ham-based medium. Methods and Results: Experiments were carried out on a 3% ham medium at two water activities (0.995, 0.95 aw) inoculated with P. nordicum spores and incubated at 25°C for up to 14 days. Agar discs were sampled after 1, 2, 3, 7 and 14 days, placed in 30 ml vials, sealed and the head space analysed using e-nose. The effect of environmental conditions on growth and OTA production was evaluated based on the qualitative response. PCA showed that after 72 hrs volatile fingerprints did not help discriminate between treatments, after 7 days, they were able to discriminate between strains grown at 0.995 aw and after 14 days the OTA producer and non-producer strain and the control could be discriminated at both aw. Conclusions: This study suggests that volatile fingerprints produced by P. nordicum strains may differ and be used to predict the presence of toxigenic contaminants in ham. Significance and impact of the study: This approach could be utilised in ham production as part of a quality assurance system for preventing OTA contamination.

Several preventive and corrective measures to minimize toxin contamination in agricultural commodities particularly by mycotoxins have been attempted. These can be divided into three broad categories: plant breeding, good agronomic practice and detoxification. . The focus of this current review is the evaluation of agronomic practises aimed to modulate “natural toxins”. In order to identify what is known about the agronomic effects and how they might potentially be manipulated to reduce toxin levels, it is necessary to analyse the relationship between toxins, crops and agricultural practises. In this discussion paper we will deal first with two major food crops (cereals and potatoes) and their food safety. We will then deal with agronomic factors and their effects on different crops, and also on fungal strains, and the occurrence of mycotoxins or the occurrence of inherent toxins on different food crops. Detoxification is considered outside the scope of this review. As starting point the two key food crops in Europe, cereals and potatoes, were selected .The intention was to develop an inventory of the current agronomic practices applied in order to reduce the concentration of inherent plant toxins and mycotoxins. With respect to food processing only those closely related to harvesting, storage, or domestic food preparation will be considered. Industrial food processing is outside the remit of this paper. Other topics considered include masked mycotoxins, mycotoxins boundaries and the environmental conditions that influence fungal growth. This review considers different fungal strains, the influence of climate (temperature), growth conditions, toxicity, data gaps and research needs, toxin interaction with other compounds, kinetics, sensitivity of the fungal population and possible polymorphism. Additional topics include the definition and comparison of agronomic factors, the application of plant protection products, hereafter referred to as pesticides, and the occurr

Several preventive and corrective measures to minimize toxin contamination in agricultural commodities particularly by mycotoxins have been attempted. These can be divided into three broad categories: plant breeding, good agronomic practice and detoxification. . The focus of this current review is the evaluation of agronomic practises aimed to modulate “natural toxins”. In order to identify what is known about the agronomic effects and how they might potentially be manipulated to reduce toxin levels, it is necessary to analyse the relationship between toxins, crops and agricultural practises. In this discussion paper we will deal first with two major food crops (cereals and potatoes) and their food safety. We will then deal with agronomic factors and their effects on different crops, and also on fungal strains, and the occurrence of mycotoxins or the occurrence of inherent toxins on different food crops. Detoxification is considered outside the scope of this review. As starting point the two key food crops in Europe, cereals and potatoes, were selected .The intention was to develop an inventory of the current agronomic practices applied in order to reduce the concentration of inherent plant toxins and mycotoxins. With respect to food processing only those closely related to harvesting, storage, or domestic food preparation will be considered. Industrial food processing is outside the remit of this paper. Other topics considered include masked mycotoxins, mycotoxins boundaries and the environmental conditions that influence fungal growth. This review considers different fungal strains, the influence of climate (temperature), growth conditions, toxicity, data gaps and research needs, toxin interaction with other compounds, kinetics, sensitivity of the fungal population and possible polymorphism. Additional topics include the definition and comparison of agronomic factors, the application of plant protection products, hereafter referred to as pesticides, and the occurr

The objective of this study was to evaluate (a) the effect of water availability on growth and ochratoxin A (OTA) production by Penicillium nordicum in a ham-based medium and (b) the potential for discriminating between producing (MPVP 1669) and non-producing strains (MPVP 1446) of P.nordicum using volatile production patterns with a hybrid sensor array system ((NST 3220 Lab Emission Analyser, Applied Sensors, Linköping, Sweden). Initial studies using a 3% ham-based medium were used to identify the water activity (aw) range for growth and OTA production at 25°C. This showed that growth occurred over the range 0.99 to 0.90 aw and was able to produce OTA after 7 and 14 days. Subsequent studies were carried out with both the producing and non-producing strains of P.nordicum to examine the potential for differentiation based on qualitative volatile production patterns. Thus the 3% ham media were inoculated with spores by spread plating onto the surface of 5 replicate treatment at 0.995 and 0.95 aw at 25°C for up to 14 days. Plates were destructively sampled after 1, 2, 3, 7 and 14 days. Agar discs (3 x 2 cm) were placed in vials and sealed, placed in the autosampler of the sensor array unit and the head space analysed after 1 hrs incubation. The maximum response of the sensor array (metal ion and metal oxide arrays) were used in the PCA statistical analyses of the data sets. Up to 72 hrs high associations were observed based on aw more than the capacity for the strains to produce OTA. In fact, the strain were grouped at aw 0.95 and 0.995. However, after 7 days incubation the e-nose was able to discriminate the two strains of P. nordicum grown at aw of 0.995, while strains at aw of 0.95 were associated. The most interesting results were obtained after 14 days incubation. The e-nose was able to discriminate between the producer and non-producer strains at both aw levels. This suggests that qualitative volatile production patterns may differ between these two strains beca

The objective of this study was to evaluate (a) the effect of water availability on growth and ochratoxin A (OTA) production by Penicillium nordicum in a ham-based medium and (b) the potential for discriminating between producing (MPVP 1669) and non-producing strains (MPVP 1446) of P.nordicum using volatile production patterns with a hybrid sensor array system ((NST 3220 Lab Emission Analyser, Applied Sensors, Linköping, Sweden). Initial studies using a 3% ham-based medium were used to identify the water activity (aw) range for growth and OTA production at 25°C. This showed that growth occurred over the range 0.99 to 0.90 aw and was able to produce OTA after 7 and 14 days. Subsequent studies were carried out with both the producing and non-producing strains of P.nordicum to examine the potential for differentiation based on qualitative volatile production patterns. Thus the 3% ham media were inoculated with spores by spread plating onto the surface of 5 replicate treatment at 0.995 and 0.95 aw at 25°C for up to 14 days. Plates were destructively sampled after 1, 2, 3, 7 and 14 days. Agar discs (3 x 2 cm) were placed in vials and sealed, placed in the autosampler of the sensor array unit and the head space analysed after 1 hrs incubation. The maximum response of the sensor array (metal ion and metal oxide arrays) were used in the PCA statistical analyses of the data sets. Up to 72 hrs high associations were observed based on aw more than the capacity for the strains to produce OTA. In fact, the strain were grouped at aw 0.95 and 0.995. However, after 7 days incubation the e-nose was able to discriminate the two strains of P. nordicum grown at aw of 0.995, while strains at aw of 0.95 were associated. The most interesting results were obtained after 14 days incubation. The e-nose was able to discriminate between the producer and non-producer strains at both aw levels. This suggests that qualitative volatile production patterns may differ between these two strains becau

This study examined the utilization patterns of key carbon sources (CS, 24: including key sugars, amino acids and fatty acids) in maize by strains of Aspergillus flavus and Fusarium verticillioides under different water activity (aw, 0.87-0.98 aw) and temperature (20-35 degrees C) values and compared the niche overlap indices (NOI) that estimate the in vitro CS utilization profiles [Wilson, M., Lindow, S.E., 1994. Coexistence among epiphytic bacterial populations mediated through nutritional resource partitioning. Applied and Environmental Microbiology 60, 4468-4477.]. The ability to grow in these key CS in minimal media was studied for 120 h in 12 h steps. The NOI was calculated for inter-species (F. verticillioides-A. flavus) and for intra-species (A. flavus-A. flavus) using CS utilization patterns over the range of interacting environmental conditions. 30 degrees C, over the whole aw range examined, was found to be optimal for utilization of the maximum number of CS by A. flavus. In contrast, for F. verticillioides this was more so at 20 degrees C; 25 degrees C allowed a suboptimal usage of CS for both species. NOIs confirmed the nutritional dominance of A. flavus at 30 degrees C, especially at lower aw levels and that of F. verticillioides at 20 degrees C, mainly at 0.95 aw. In other conditions of aw, based on CS utilization patterns, the data indicated that A. flavus and F. verticillioides occupied different ecological niches. The variability in nutritional sources utilization between A. flavus strains was not related to their ability to produce aflatoxins (AFs). This type of data helps to explain the nutritional dominance of fungal species and strains under different environmental conditions. This could be useful in trying to find appropriate natural biocontrol microorganisms to compete with these mycotoxigenic species.

The potential for using modified atmospheres of 25-75% CO2 (balanced with N2) and water activity (aw, 0.95, 0.92) to control Aspergillus flavus development and aflatoxin B1 production has been evaluated (a) on synthetic medium and (b) on maize grain during storage for up to 21 days at 25 °C. On agar medium up to 75% CO2 at both 0.95 and 0.92 aw significant inhibition of growth was obtained (P < 0.05). In stored grain inoculated with spores of A. flavus there was significantly higher populations of the species at 0.95 aw than 0.92 aw. Up to 75% CO2 resulted in an inhibition of the populations of A. flavus isolated from the grain. Contrasting aflatoxin B1 production was obtained on agar and in stored maize grain. On agar, greatest amounts were produced at 0.92 aw, while more was produced at 0.95 aw on maize grain. Overall, the efficacy of controlled atmospheres × aw showed that treatment with 25% CO2 could be sufficient to efficiently reduce A. flavus development but at least 50% CO2 was required to obtain a significant reduction of aflatoxin synthesis.

This special issue provides state of the art reviews on the status of OTA contamination in different continents combined with detailed research results from the EU: Risk assessment and integrated ochratoxin A management in grape and wine (WINE OCHRA-RISK, Project no. QLK1-CT-2001-01761. Website http://www.ochra-wine.com). We believe that this special issue will significantly contribute to the scientific knowledge in this field and the development of effective prevention strategies to minimise consumer exposure to OTA in grapes, grape-derived products and wine.

European wine production represents about 70% of world production and thus is an important export commodity. Ochratoxin A (OTA) was first detected as a wine contaminant in 1996 and the role of Aspergillus section Nigri and A. carbonarius in OTA production discovered in Europe in 1999. Subsequently Europe-wide surveys have shown that A. carbonarius is predominantly responsible for OTA contamination of grapes, wine and vine fruits. Analyses of wine samples throughout Europe have shown that there is a gradient in OTA concentration with a decrease from red, to rose and to white wines. The latitude of production is an important factor in determining risk from OTA contamination. Some geographic regions in Southern Europe are more prone to contamination with the toxigenic species and OTA. Ochratoxin A has also been found in much higher concentrations (max. 53 micro g/kg) in dried vine fruit than in wine suggesting that A. carbonarius can dominate the drying vine fruit ecosystem. There is a significant lack of knowledge in Europe on conducive climatic conditions preharvest and their relationship with levels of risk from OTA contamination in grapes and their fate in wine production. This needs to be integrated with cultivation system to maximise the prevention of OTA entering this food chain.

Geostatistical analysis was applied to the incidence of Aspergillus Section Nigri and A. carbonarius in Southern Europe and Israel for the 3-year period 2001-2003 to facilitate identification of regions of high risk from contamination with these fungi and production of ochratoxin. The highest incidence of black aspergilli was normally observed at harvesting. At this grape growth stage, spatial variability of black aspergilli was significantly related to latitude and longitude, showing a positive West-East and North-South gradient. Predictive maps of infected berries incidence were drawn and showed the same trend in the 3 years, but incidence was highest in 2003, followed by 2001 and 2002. The highest incidence was always observed in Israel, Greece and Southern France, associated with the highest incidence of A. carbonarius. Southern Spain and Southern Italy also had relevant incidence of black aspergilli. The thermo-wetness maps for the 3 years showed a trend similar to the incidence of black aspergilli. The coldest and wettest year was 2002, while 2003 was the hottest and driest, particularly during August, with Israel being the hottest and driest country, followed by Greece and Southern Italy. This indicates that meteorological conditions can contribute to explain spatial distribution variation of black aspergilli within the Mediterranean basin.

Rapid developments in sensor technology have facilitated the production of devices known as electronic noses - that can detect and discriminate the production profiles of volatile compounds from microbial infections in situ. Such qualitative and semi-quantitative approaches could have a significant role in the early diagnosis and detection of microbial diseases. Using artificial intelligence and web-based knowledge systems, electronic noses might also have a valuable role in monitoring disease epidemiology

The use of volatile production patterns produced by Mycobacterium tuberculosis and associated bacterial infections from sputum samples were examined in vitro and in situ using an electronic nose based on a 14 sensor conducting polymer array. In vitro, it was possible to successfully discriminate between M. tuberculosis (TB) and control media, and between M. tuberculosis and M. avium, M. scrofulaceum and Pseudomonas aeruginosa cultures in the stationary phase after 5-6 h incubation at 37degreesC based on 35 samples. Using neural network (NN) analysis and cross-validation it was possible to successfully identify 100% of the TB cultures from others. A second in vitro study with 61 samples all four groups were successfully discriminated with 14 of 15 unknowns within each of the four groups successfully identified using cross-validation and discriminant function analysis. Subsequently, lipase enzymes were added to 46 sputum samples directly obtained from patients and the head space analysed. Parallel measurements of bacterial contamination were also carried out for confirmation using agar media. NN analysis was carried out using some of the samples as a training set. Based on the NN and genetic algorithms of up to 10 generations it was possible to successfully cross-validate 9 of 10 unknown samples. PCA was able to discriminate between TB infection alone, the controls, M. avium, P. aeruginosa and a mixed infection. These findings will have significant implications for the development of rapid qualitative systems for screening of patient samples and clinical diagnosis of tuberculosis.