Biological Control of Aflatoxin Contamination of Crops

Aflatoxin contamination of crops compromises the safety of food and feed supplies and causes significant economic losses each year. Of the many research approaches being studied to reduce and, ultimately, eliminate aflatoxin contamination, biological control is one of the more promising, particularly for the near‐term. Numerous organisms have been tested for biological control of aflatoxin contamination including bacteria, yeasts, and nontoxigenic strains of the causal organisms, Aspergillus flavus and A. parasiticus. Most of the field successes to date have been achieved by applying certain nontoxigenic strains of A. flavus and A. parasiticus to soil of susceptible crops, such as peanuts, cotton, and corn. The applied strains occupy the same niche as the naturally occurring toxigenic strains and competitively exclude them when crops are susceptible to infection. Various formulations have been used to apply the nontoxigenic strains to soil, but the most effective methods have been to combine the desired strain with a carrier/substrate, such as a small grain. This was done either by minimally growing the desired strain on sterilized grain or by coating the surface of the grain with conidia of the strain. After application to the field and uptake of moisture, the fungus completely colonizes the grain, and abundant sporulation provides inoculum levels sufficient to achieve a competitive advantage for the nontoxigenic strain. In several years of field studies, particularly with peanuts and cotton, significant reductions in aflatoxin contamination in the range of 70–90% have been achieved consistently. Two separate products have recently received EPA registration as biopesticides to control aflatoxin contamination in cotton (AF36) and peanuts (afla‐guard®).     

New perspectives for the application of bioplastic materials in the biocontrol of Aspergillus flavus in corn

Mycotoxins are secondary metabolites produced by certain filamentous fungi that can contaminate a large variety of agricultural commodities before and after harvest. Among different mycotoxins, aflatoxins and especially aflatoxin B1 are of particular concern because they are potent natural carcinogens. Aflatoxin-producing fungi, mainly Aspergillus flavus and A. parasiticus, are ubiquitous, being commonly isolated from agricultural soil and crop debris. Although many aspects of the ecology of aflatoxin-producing fungi have been elucidated, control of aflatoxin contamination of agricultural crops remains a difficult task. Agronomical practices promoting general plant health have shown variable and more frequently limited success in preharvest control of aflatoxin contamination. Competitive replacement of indigenous toxigenic soil isolates is considered a more promising and effective approach. This biocontrol strategy is based on field application of a large number of propagules of nontoxigenic strains of A. flavus. Biocontrol strains are typically formulated as inoculated or spore-coated grain seeds. More recently, efforts to explore new approaches and technologies have resulted in the development of other practical solutions, including a bioplastic-based formulation. This formulation originally developed in 2008, consists of bioplastic granules entrapping spores of the nontoxigenic biocontrol strain, A. flavus NRRL 30797. Laboratory and field studies that have been conducted until now have clearly shown that granules of the starch-based bioplastic Mater-Bi^® are effective in delivering this biocontrol strain. In addition to having a satisfactory shelf life, the granules are easy to prepare, handle, and apply to agricultural fields. More importantly, this novel bioplastic formulation is capable of efficiently reducing aflatoxin contamination of corn. The bioplastic Mater-Bi^® can also have other applications. For instance, rods or granules prepared using a slightly modified Mater-Bi^® bioplastic matrix can be used to selectively isolate A. flavus from soil and corn kernels.     

Effects of Light on the Ochratoxigenic Fungi Aspergillus ochraceus and A. carbonarius

Ochratoxin A (OTA) usually contaminates agricultural products such as grapes, oatmeal, coffee and spices. Light was reported as an effective strategy to control spoilage fungi and mycotoxins. This research investigated the effects of light with different wavelengths on the growth and the production of OTA in Aspergillus ochraceus and Aspergillus carbonarius. The results showed that the growth of both fungi were extremely inhibited by UV-B. Short-wavelength (blue, violet) significantly inhibited the production of OTA in both fungi, while the inhibitory effect of white was only demonstrated on A. ochraceus. These results were supported by the expression profiles of OTA biosynthetic genes of A. ochraceus and A. carbonarius. To clarify, the decrease in OTA production is induced by inhibition or degradation; therefore, the degradation of OTA under different wavelengths of light was tested. Under UV-B, the degradation rate of 10 μg/mL OTA standard pure-solution samples could reach 96.50% in 15 days, and the degradation effect of blue light was relatively weak. Furthermore, infection experiments of pears showed that the pathogenicity of both fungi was significantly decreased under UV-B radiation. Thus, these results suggested that light could be used as a potential target for strategies in the prevention and control of ochratoxigenic fungi.     

Controlling Aflatoxin and Fumonisin in Maize by Crop Management

Maize is a vital food and feed grain worldwide. Aflatoxin and fumonisin, mycotoxins produced primarily by the fungi Aspergillus flavus and Aspergillus parasiticus Speare, and Fusarium moniliforme J. Sheld, respectively, are very potent carcinogens in both humans and livestock and can readily contaminate maize grain in the field and in storage. Stress on developing maize, particularly during reproductive growth, facilitates infection by the fungi, production of mycotoxins and contamination of the grain. Drought, excessive heat, inadequate plant nutrition, insect feeding on developing kernels, weeds, excessive plant populations, and other plant diseases can produce plant stress and facilitate the infection of maize grain by mycotoxin producing fungi. Timely planting of adapted hybrids, proper plant nutrition, irrigation, and insect control either by insecticides or the use of transgenic hybrids all assist in curbing mycotoxin contamination. Production practices that produce high yields are basically the same ones that help control mycotoxins. Care must also be exercised in harvesting and handling grain in transport and storage to reduce kernel breakage and prevent contamination. Harvesting early and artificial drying helps reduce the incidence of mycotoxins as well as preventing kernel breakage and stored‐grain insect infestations.     

Inflammatory cytokine gene expression in THP-1 cells exposed to Stachybotrys chartarum and Aspergillus versicolor

Very little is known about the mechanisms that occur in human cells upon exposure to fungi as well as their mycotoxins. A better understanding of toxin‐regulated gene expression would be helpful to identify safe levels of exposure and could eventually be the basis for establishing guidelines for remediation scenarios following a water intrusion event. In this research, cytokine mRNA expression patterns were investigated in the human monocytic THP‐1 cell line exposed to fungal extracts of various fragment sizes obtained from Stachybotrys chartarum RTI 5802 and/or Aspergillus versicolor RTI 3843, two common and well‐studied mycotoxin producing fungi. Cytokine mRNA expression was generally upregulated 2–10 times following a 24 h exposure to fungal extracts. Expression of the proinflammatory interleukin‐1β, interleukin‐8, and tumor necrosis factor‐α genes increased while the anti‐inflammatory gene interleukin‐10 also increased albeit at very low level, suggesting that negative feedback regulation mechanism of production of proinflammatory cytokines initiated upon 24 h of incubation. In addition, submicron size extracts of A. versicolor caused significant death of THP‐1 cells, whereas extracts of S. chartarum caused no cell death while the mixture of the two fungi had an intermediate effect. There was no general correlation between gene expression and fragment sizes, which suggests that all submicron fragments may contribute to inflammatory response. © 2011 Wiley Periodicals, Inc. Environ Toxicol, 2013.     

Study of fungi and their toxigenic potential isolated from wheat and wheat bran

The present study was designed to investigate the fungi and their toxigenic potentials isolated from the wheat and wheat brans. A total of 67 samples of wheat and 17 samples of wheat bran were collected from Faisalabad district of Pakistan. Forty-five (67.16%) samples of wheat yielded fungi. Frequency distribution based on total samples, Aspergillus was the highest (44.77%) genus followed by Penicillium, Fusarium and Alternaria. Penicillium verucosum (30.64%) was the most frequently isolated species followed by A. niger aggregates, A. flavus, A. parasiticus, P. chrysogenum, A. ochraceous, A. carbonarius and A. fumigatus. Among Aspergilli, A. niger aggregates (46.67%) were most frequently isolated species. Out of 30 Aspergilli isolates from wheat samples, 17 (56.66%) were found toxigenic. AFB1 produced by aflatoxigenic Aspergilli varied from 1.44 to 836.3?ng/g, while ochratoxin A levels varied from 0.037 to 15 045?ng/g. Among Penicillium species, P. verrucosum (63.15%) were found ochratoxigenic and OTA levels were varied from 7.31 to 8400?ng/g. In wheat bran, 10 (58.82%) samples yielded fungi. Based upon total samples, frequency distribution of Aspergillus (35.28%) was the highest followed by Penicillium and Fusarium. Similar pattern was observed in relative density of isolates. A. niger aggregates and P. verrucosum were predominant species (23.07%) isolated from wheat bran. Among Aspergilli, A. niger aggregates (50%) were the most frequently isolated species followed by A. flavus, A. fumigatus and A. ochraceous (16.67%) each. The OTA levels of fungi isolated from wheat bran varies from 0.292 to 2500?ng/g. Isolation of toxigenic A. niger aggregates from wheat indicates that these species should be considered as possible contributors of OTA contamination in wheat and its by-products in Pakistan.     

Current Research on Reducing Pre‐ and Post‐harvest Aflatoxin Contamination of U.S. Almond,Pistachio, and Walnut

Aflatoxins are considered to be potent carcinogens and teratogens to humans and farm animals. A variety of species of the fungal genus Aspergillus (mainly A. flavus and A. parasiticus) synthesize aflatoxins. Spores of these fungi are common in air and soil of agricultural areas of temperate and tropical environments. Because aflatoxigenic fungi are ubiquitous and opportunistic, aflatoxin contamination has become a food safety concern. The chief U.S. crops affected by the threat of contamination with aflatoxin include corn, peanuts, cottonseed, and certain tree nuts. Additionally, aflatoxin contamination has also become an international trade issue. Major trading partners of U.S. agricultural products have set total aflatoxin action threshold levels at four ng/g (ppb). This action level is far below the 20 ppb level recommended by the U.S. Food and Drug administration for domestic foods. Almonds, pistachios and walnuts are one of the major food commodities affected by food safety and trade issues associated with aflatoxin contamination. Commercial domestic production of these tree nuts in the U.S. is entirely in California. Moreover, 50 to 75% of domestically produced tree nuts are exported, chiefly to countries of the European Union (EU), which adhere to the four ppb action threshold level. Scientists at the USDA's Western Regional Research Center and the University of California, Davis' Department of Pomology and Kearney Agricultural Center have developed products and methods to reduce aflatoxin contamination of tree nuts. Control of insect pests in tree nut orchards is a major strategy to curtail aflatoxin contamination. Insect feeding damage can lead to fungal infection and concomitant aflatoxin contamination. This is especially the case with navel orangeworm on pistachio and almond. A new and potent lure has been developed to control codling moth, a major insect pest of walnuts whose feeding damage potentially leads to fungal infection. Through breeding and genetic engineering, new varieties of almonds and walnuts have been developed which are resistant to insect attack. New orchard management strategies have been prescribed to reduce reservoirs of A. flavus in tree nut orchards. A number of saprophytic yeasts, natural to tree nut orchards, have been discovered which show promise as biological control agents of A. flavus, in vitro, and are awaiting field testing. New and improved risk assessment models have been developed for sampling and measuring aflatoxin contamination through the processing stream and in bulk shipping lots of tree nuts. An automated sorter that detects and removes aflatoxin contaminated nuts from a processing stream in real time was developed. It was also concluded that methods currently used for hand‐cracking of closed shell pistachios result in a higher risk of aflatoxin contamination. Perhaps the foremost breakthrough to date, however, is that constituents of walnut seed coat, especially from the cultivar ‘Tulare’, are potent inhibitors of aflatoxin biosynthesis, capable of rendering aflatoxigenic A. flavus virtually atoxigenic.     

Use of a microbial toxicity test (Microtox) to determine the toxigenicity of Aspergillus fumigatus strains isolated from different sources

The toxic activity of Aspergillus fumigatus is attributable to substances secreted by its cells. Specific toxic compounds synthesized by the fungi such as gliotoxin, can be detected by sensitive chemical procedures like TLC or HPLC. Measuring the total toxigenicity of a strain extract, however, requires a bioassay. In the present study, we evaluated the possibility of using the Microtox^® bioassay to determine the toxigenicity of A. fumigatus, using 32 strains from different sources. The Microtox^® method is based on the ability of Vibrio fischeri to produce luminescence, and their sensitivity to toxins. A. fumigatus strains, grouped according to their original sources, showed differences in toxigenicity. Strains isolated from invasive aspergillosis patients proved to be more toxigenic than environmental strains, or strains from colonized patients. Since the strains that were more toxigenic were isolated from sick patients, it is not surprising they showed more virulence than the other strains, and as expected, virulence could be correlated with high toxigenicity. The Microtox bioassay could be a useful tool in the study of toxigenicity of the mycelial fungi and their possible pathogenic roles, and for rapid assessment of secreted toxic compounds.     

Antifungal and anti-aflatoxigenic effect of probiotics against Aspergillus flavus and Aspergillus parasiticus

Aflatoxins are carcinogenic mycotoxin, produced by Aspergillus species. These molds infect food crops in warm humid conditions causing economic losses and affecting the consumers' health adversely. In this study, antifungal activity and aflatoxin inhibiting ability of four probiotic strains against Aspergillus flavus and Aspergillus parasiticus were studied. The aflatoxin secreted was analyzed and quantified by both UV spectrophotometer and HPLC. It was found that Lactobacillus delbrueckii subsp. lactis showed maximal antifungal (67.43% reduction) and anti-aflatoxigenic (94.33% reduction) activity against A. flavus whereas A. parasiticus was inhibited by Lactobacillus brevis with the antifungal reduction of 69.38% and anti-aflatoxigenic reduction of 96.12%.     

Predicted Aflatoxin B1 Increase in Europe Due to Climate Change: Actions and Reactions at Global Level

Climate change (CC) is predicted to increase the risk of aflatoxin (AF) contamination in maize, as highlighted by a project supported by EFSA in 2009. We performed a comprehensive literature search using the Scopus search engine to extract peer-reviewed studies citing this study. A total of 224 papers were identified after step I filtering (187 + 37), while step II filtering identified 25 of these papers for quantitative analysis. The unselected papers (199) were categorized as “actions” because they provided a sounding board for the expected impact of CC on AFB₁ contamination, without adding new data on the topic. The remaining papers were considered as “reactions” of the scientific community because they went a step further in their data and ideas. Interesting statements taken from the “reactions” could be summarized with the following keywords: Chain and multi-actor approach, intersectoral and multidisciplinary, resilience, human and animal health, and global vision. In addition, fields meriting increased research efforts were summarized as the improvement of predictive modeling; extension to different crops and geographic areas; and the impact of CC on fungi and mycotoxin co-occurrence, both in crops and their value chains, up to consumers.     

Cyclopiazonic acid biosynthesis by Aspergillus flavus

Cyclopiazonic acid (CPA) is an indole-tetramic acid mycotoxin produced by some strains of Aspergillus flavus. Characterization of the CPA biosynthesis gene cluster confirmed that formation of CPA is via a three-enzyme pathway. This review examines the structure and organization of the CPA genes, elucidates the specific roles of functional domains of each enzyme in carrying out catalytic conversions leading to CPA production, and delineates the molecular basis for the lack of CPA production by A. flavus strains currently being used in biocontrol of aflatoxin contamination of crops.     

Selection of Aspergillus flavus isolates for biological control of aflatoxins in corn

The fungus Aspergillus flavus is responsible for producing carcinogenic mycotoxins, the aflatoxins, on corn (maize) and other crops. An additional harmful toxin, cyclopiazonic acid, is produced by some isolates of A. flavus. Several A. flavus strains that do not produce one or both of these mycotoxins are being used in biological control to competitively exclude the toxin-producing strains from the agroecosystem, particularly from seeds, grain and other marketable commodities. Three well-studied non-aflatoxigenic strains, including two that are commercially available, have been compared in side-by-side field trials. The results of that study, together with a growing understanding of A. flavus ecology and new genetic insights, are guiding the selection of biocontrol strains and influencing crop management decisions for safe and sustainable production.     

The echinocandin micafungin: a review of the pharmacology,spectrum of activity,clinical efficacy and safety

Micafungin is a relatively broad-spectrum antifungal agent available for clinical use in the US and Japan. By inhibiting the production of β-1,3-glucan, an essential fungal cell wall component, micafungin has reduced toxicity to mammalian cells while maintaining potent antifungal activity against many pathogenic fungi including polyene- and azole-resistant isolates. Indeed, micafungin has been shown to be efficacious in the treatment of infections caused by Candida and Aspergillus species in clinical trials without the associated toxicities of amphotericin B formulations and drug interactions that occur with the azoles. In this review, the pharmacology, spectrum of activity, clinical efficacy and safety profile of micafungin are discussed.     

Synthesis of aryl aldimines and their activity against fungi of clinical interest

Aldimines are aldehyde‐derived compounds that contain a C=N group. Besides its broad industrial applications, this class of non‐naturally occurring compounds are found to possess antibacterial, antifungal, antimalarial, antiproliferative, anti‐inflammatory, antiviral, and antipyretic properties. Based on this, six aryl aldimines were synthesized from the condensation of aromatic amines with benzaldehydes. The antifungal activities of synthesized compounds were evaluated against nineteen fungal strains that included Candida and Aspergillus species, Cryptococcus neoformans. The aryl aldimines 2‐(benzylideneamino)phenol ( 3 ) and 4‐(benzylideneamino)phenol ( 8 ) were the most active compounds against the fungi studied. Compounds 3 and 8 efficiently inhibited the metabolism of C. neoformans mature biofilm.     

An in vitro microcalorimetric method for studying the toxic effect of cadmium on microbial activity of an agricultural soil

Using TAM III multi-channel thermocalorimetry combined with direct microorganism counting (bacteria, actinomycetes and fungi) under laboratory conditions, we determined the microbial population count, resistance and activity toward cadmium (Cd) toxicity in soil. The thermokinetic parameters, which can represent soil microbial activity, were calculated from power-time curves of soil microbial activity obtained by microcalorimetric measurement. Simultaneous application of the two methods showed that growth rate constant (k), peak-heat output power (P _max) and the number of living microorganisms decreased with increasing concentration of Cd. Anncrease in Cd concentration resulted in the decrease of the peak-heat output power and increase in the time of the peak of power. However, the relationships between the thermokinetic parameters (k and P _max) and the number of microorganism were not linear, but the trend was similar. Our research also suggests that microcalorimetry is a very sensitive, simple and useful technique for in vitro investigation of the effects of toxic heavy metals on soil microbial activity.     

Insect Management to Facilitate Preharvest Mycotoxin Management

Many species of insects can facilitate the entry of mycotoxin‐producing fungi to commodities such as cotton seed, maize, peanuts, and tree nuts. The mycotoxins most commonly associated with insect damage are aflatoxin and fumonisin. Insecticides will likely remain an important management tool, especially as predictive models for forecasting mycotoxigenic fungi or mycotoxins become available. Plants with high levels of resistance to insects that facilitate mycotoxins are likely to assist in mycotoxin management. Several studies now indicate Bt maize hybrids that express the protein throughout the plant can prevent fumonisin levels rising above guideline levels of 1–2 ppm when European corn borers (Ostrinia nubilalis) are the predominant insect pests.     

Potential Ecotoxicological Implication of Methyl <Emphasis Type="Italic">tert</Emphasis>-butyl ether (MTBE) Spills in the Environment

Streptomyceticidal activity of Methyl tert-butyl ether (MTBE) elucidated for the first time. Adverse effect of MTBE, the gasoline additive, against 11 soil inhabitant Streptomyces spp. isolates was investigated. MTBE, an octane enhancer is added to gasoline to reduce atmospheric concentrations of carbon monoxide and ozone. It contaminates soil and groundwater by fuel leaks and spills. Streptomyces spp. are of the major contributors to the biological buffering of soils by exerting beneficial and antagonistic activity against wide range of bacteria and fungi. To evaluate anti-streptomycetidal activity of MTBE, it was tested against 11 soil isolates of Streptomyces isolates and also a plant-root bacterial pathogen, Erwinia carotovora and a plant-root fungal pathogen, Fusarium solani. MTBE did not reveal any growth inhibitory activity against E. carotovora and F. solani, but showed strong inhibitory effect against Streptomyces isolates. The Minimum Inhibitory Concentration (MIC) on Streptomyces isolates was 1/800 of the original MTBE. Fuel leaks and spills have the potential to suppress or eliminate the Streptomyces role in the soil causing alteration in the balance of soil micro flora. This change can promote the domination of microorganisms with adverse biological or ecotoxicological effects.     

Microbiological characteristics of a sandy loam soil exposed to tebuconazole and λ-cyhalothrin under laboratory conditions

Changes in microbiological properties of a sandy loam soil in response to the addition of different concentrations of fungicide tebuconazole and pyrethroid insecticide λ-cyhalothrin were assessed under laboratory conditions. To ascertain these changes, the potentially active soil microbial biomass, concentrations of ammonium and nitrate ions, numbers of total culturable bacteria, fungi, nitrogen-fixing bacteria, nitrifying and denitrifying bacteria were determined. Substrate-induced respiration (SIR) increased with time in both control (ranged from 13.7 to 23.7 mg/O₂/kg⁻¹/dry soil/h⁻¹) and pesticide treated soil portions. For both pesticides, SIR values ranged from 12–13 to 23–25 mg/O₂/kg⁻¹/dry soil/h⁻¹ on days 1 and 28, respectively. Also, concentrations of nitrate and ammonium ions, numbers of total culturable bacteria, denitrifying bacteria, nitrogen-fixing bacteria (for the insecticide) and fungi (for the insecticide) were either unaffected or even stimulated by the pesticide treatments. The adverse impacts of the pesticides were observed for nitrate concentrations (on days 1 or 7), numbers of nitrifying bacteria (on day 1), denitrifying bacteria (for the insecticide on days 1 and 14), nitrogen-fixing bacteria (for tebuconazole on day 1) as well as numbers of fungi in tebuconazole-treated soil (on days 1 and 14).