Peanut gene expression profiling in developing seeds at different reproduction stages during <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis>infection

Background  

Peanut (Arachis hypogaea L.) is an important crop economically and nutritionally, and is one of the most susceptible host crops to colonization of Aspergillus parasiticus and subsequent aflatoxin contamination. Knowledge from molecular genetic studies could help to devise strategies in alleviating this problem; however, few peanut DNA sequences are available in the public database. In order to understand the molecular basis of host resistance to aflatoxin contamination, a large-scale project was conducted to generate expressed sequence tags (ESTs) from developing seeds to identify resistance-related genes involved in defense response against Aspergillus infection and subsequent aflatoxin contamination.     

Aflatoxin-producing Aspergillus species from saffron field soils in the South Khorasan province of Iran

The aim of the present study was to isolate and identify Aspergillus species associated with saffron plants in the city of Birjand (South Khorasan Province, Iran) as well as to assess their aflatoxin B1 production. Sampling was performed during 2013–2014 crop season. Aspergillus species were isolated and purified using general and specific culture media. Growth rates and macroscopic and microscopic characteristics of the isolates were determined using yeast extract, Czapek yeast extract, malt extract and creatine sucrose agar media at 25 and 37 °C. DNA was extracted by the modified CTAB method and beta-tubulin, calmodulin and internal transcribed spacer genes were amplified and sequenced. Phylogenetic position of the isolates was determined against other Aspergillus species. Thin layer chromatography was used to investigate the production of aflatoxin B1 by Aspergillus isolates. Based on the morphological characteristics, shape and colour of the colonies, and sequencing results, the isolates belonged to Aspergillus terreus, A. flavus, A. flavipes and A. niger species. Only A. flavus isolates were aflatoxin B1 producers. We concluded that the soil of the studied saffron fields contained several species of Aspergillus, with A. flavus significantly affecting crop production through contamination of the crop by aflatoxin.     

Survey of Vietnamese Peanuts,Corn and Soil for the Presence of <Emphasis Type="Italic">Aspergillus flavus</Emphasis> and <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis>

Aspergillus flavus and Aspergillus parasiticus cause perennial infection of agriculturally important crops in tropical and subtropical areas. Invasion of crops by these fungi may result in contamination of food and feed by potent carcinogenic aflatoxins. Consumption of aflatoxin contaminated foods is a recognised risk factor for human hepatocellular carcinoma (HCC) and may contribute to the high incidence of HCC in Southeast Asia. This study conducted a survey of Vietnamese crops (peanuts and corn) and soil for the presence of aflatoxigenic fungi and used microsatellite markers to investigate the genetic diversity of Vietnamese Aspergillus strains. From a total of 85 samples comprising peanut (25), corn (45) and soil (15), 106 strains were isolated. Identification of strains by colony morphology and aflatoxin production found all Vietnamese strains to be A. flavus with no A. parasiticus isolated. A. flavus was present in 36.0% of peanut samples, 31.1% of corn samples, 27.3% of farmed soil samples and was not found in virgin soil samples. Twenty-five per cent of the strains produced aflatoxins. Microsatellite analysis revealed a high level of genetic diversity in the Vietnamese A. flavus population. Clustering, based on microsatellite genotype, was unrelated to aflatoxin production, geographic origin or substrate origin.     

Mycoflora of mangrove mud

Summary Thirty-nine species and four varieties of fungi are reported from mangrove mud collected from Kagh Island, Port Canning and Diamond Harbour, West Bengal, India. Of these forms twelve species and two varieties belonged to genusAspergillus and three species belonged, each toMucor, Penicillium andPreussia. Two species both ofFusarium andTrichoderma were also isolated. Two new species,Cladosporium indicum andPaecilomyces indicus and five other interesting fungi are described in detail.     

Understanding the genetics of regulation of aflatoxin production and Aspergillus flavus development

Aflatoxins are polyketide-derived, toxic, and carcinogenic secondary metabolites produced primarily by two fungal species, Aspergillus flavus and A. parasiticus, on crops such as corn, peanuts, cottonseed, and treenuts. Regulatory guidelines issued by the U.S. Food and Drug Administration (FDA) prevent sale of commodities if contamination by these toxins exceeds certain levels. The biosynthesis of these toxins has been extensively studied. About 15 stable precursors have been identified. The genes involved in encoding the proteins required for the oxidative and regulatory steps in the biosynthesis are clustered in a 70 kb portion of chromosome 3 in the A. flavus genome. With the characterization of the gene cluster, new insights into the cellular processes that govern the genes involved in aflatoxin biosynthesis have been revealed, but the signaling processes that turn on aflatoxin biosynthesis during fungal contamination of crops are still not well understood. New molecular technologies, such as gene microarray analyses, quantitative polymerase chain reaction (PCR), and chromatin immunoprecipitation are being used to understand how physiological stress, environmental and soil conditions, receptivity of the plant, and fungal virulence lead to episodic outbreaks of aflatoxin contamination in certain commercially important crops. With this fundamental understanding, we will be better able to design improved non-aflatoxigenic biocompetitive Aspergillus strains and develop inhibitors of aflatoxin production (native to affected crops or otherwise) amenable to agricultural application for enhancing host-resistance against fungal invasion or toxin production. Comparisons of aflatoxin-producing species with other fungal species that retain some of the genes required for aflatoxin formation is expected to provide insight into the evolution of the aflatoxin gene cluster, and its role in fungal physiology. Therefore, information on how and why the fungus makes the toxin will be valuable for developing an effective and lasting strategy for control of aflatoxin contamination.     

Occurrence of Aspergillus section Flavi and aflatoxin B1 in corn genotypes and corn meal in Argentina

A study has been carried out in Argentina on samples of corn genotypes from a breeding station as well as in commercially available corn meal. All samples were analyzed for fungal infection and aflatoxin B₁.Mycological analysis of corn genotypes showed the presence of three principal genera of filamentous fungi Fusarium (100%), Penicillium (67%) and Aspergillus (60%). In the genus Fusarium three species were identified, F. moniliforme (42%), F. nygamai (56%) andF. proliferatum (1.8%). Eight species ofPenicillium were identified, the predominant species isolated were P. minioluteum, P. funiculosum and P. variabile. In the genus ranked third in isolation frequency, two species were identified, A. flavus and A. parasiticus, the percentage of infection was 78% and 21%, respectively. Only one corn genotype was contaminated with aflatoxin B₁ at a level of 5 ppb. The cornmeal samples showed great differences in fungal contamination, the values ranging from 1 × 10¹ to 7 × 10⁵ cfu g^–1. Fusarium (68%), Aspergillus (35%) and Penicillium (21%) were the most frequent genera isolated. Among the genus, Aspergillus, A. parasiticus (38%) was the most frequent species isolated. All the samples of corn meal were negative to aflatoxin B₁. These results indicate a low degree of human exposure to aflatoxins in Argentina through the ingestion of maize or corn meal.This revised version was published online in October 2005 with corrections to the Cover Date.     

Environmental and developmental factors influencing aflatoxin production by <Emphasis Type="Italic">Aspergillus flavus</Emphasis> and <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis>

Aflatoxins are carcinogenic mycotoxins formed by a number of fungi in the genus Aspergillus. The major fungi responsible for aflatoxin formation in crop seeds in the field and in storage are Aspergillus flavus and A. parasiticus. This review emphasizes developmental, environmental, biological, and chemical factors that influence aflatoxin formation by A. flavus and A. parasiticus.     

Incidence of seed-borne fungi and aflatoxins in Sudanese lentil seeds

Thirteen seed samples of lentil (Lens esculenta) were incubated on agar plate and moist filter papers (Moist Chambers) at 28 ± 2 °C for determination of the incidence of seed-borne fungi. Aflatoxins content of the seeds was measured using the bright greenish-yellow fluorescence test (BGYF) and thin-layer chromatography (TLC). Sixty-nine species and seven varieties, which belong to 24 genera of fungi, were isolated from this crop. Of these fungi, 51 species and two varieties are considered new for this crop, whereas seven genera and 13 species are new to the mycoflora of the Sudan. The genus Aspergillus (13 species and 6 varieties) which comprising 44% of the total colony count was the most prevalent genus followed by Rhizopus (2 species, 19%), Penicillium (6 species) and Fusarium (8 species) (12%), Chaetomium (3 species) and Cladosporium (5 species) (6%), where the 18 genera (1–4 species) showed very low level of incidence (19%). Of the possible pathogens of lentil plants, F. oxysporum the main cause of vascular wilt was recovered from seeds of this crop. Thin layer chromatographic analysis of chloroform extracts of 13 seed samples showed that only one samples was naturally contaminated with aflatoxins B₁, B₂, G₁ and G₂ (14.3 μg/kg). This revised version was published online in June 2006 with corrections to the Cover Date.     

Mycotoxins in food systems in Sub Saharan Africa: A review

Mycotoxins, toxic secondary metabolites of fungi are now recognised as major cause of food intoxications in Sub Saharan Africa (SSA). Aflatoxins, the most important of the group have been implicated in acute aflatoxicoses, carcinogenicity, growth retardation, neonatal jaundice and immunological suppression in SSA. The hot and humid tropical climate provides ideal condition for growth of toxigenicAspergillus spp, making food contamination to be widespread in SSA, with maize and groundnuts being the most contaminated. The available data suggests that cassava products (the most important African food) are not prone to aflatoxin contamination. Recent data on ochratoxin A produced by species ofAspergillus on grains have indicated the necessity for it to be monitored in SSA. Fumonisins represent the most importantFusarium mycotoxins in SSA, and surveillance data indicate very high contamination rates of almost 100% in maize samples from West Africa. Limited information exists on the occurrence of trichothecenes, while the data currently available suggest that zearalenone contamination seems not to be a problem in SSA. The strategies under investigation to mitigate the mycotoxin problem in SSA include education of the people on the danger of consuming mouldy foods, pre and post harvest management strategies with emphasis on biological control, use of plant products to arrest fungal growth during storage, enterosorbent clay technology, and the search for traditional techniques that could reduce/detoxify mycotoxins during food processing.     

Ecology of aflatoxin producing fungi and biocontrol of aflatoxin contamination

Summary  Aflatoxins, highly toxic and carcinogenic compounds that frequently contaminate foods and feeds, are produced by several genera in the genusAspergillus. Aspergillus flavus, the most common species causing crop contamination, is a common inhabitant of the Sonoran desert of North America where it resides in complex communities composed of diverse individuals. This diversity reflects divergent adaptation to various ecological niches. SomeA. flavus isolates that are well adapted to plant associated niches do not produce aflatoxins yet have the capacity to competitively exclude aflatoxin producers. These atoxigenic strains can serve as biological control agents for management of aflatoxins in crops. Detailed knowledge of the ecology of aflatoxin-producing fungi may lead to novel practical methods for limiting contamination. Presented at the EU-USA Bilateral Workshop on Toxigenic Fungi & Mycotoxins, New Orleans, USA, July 5–7, 2005.     

Survey of the mycoflora and mycotoxins of cotton seeds and cotton seed products in Egypt

Thirty-nine species and 16 fungal genera were isolated from Egyptian cotton seeds, cotton seed meal and cotton seed cake on 1% glucose-Czapek's agar medium incubated at 28 °C. Aspergillus was the most frequent genus and it emerged in 87–100% of the samples contributing 70–98% of total fungi in the three substrates tested. The most common species were A. niger, A. flavus, A. fumigatus, A. terreus and Rhizopus stolonifer; A. niger, A. fumigatus and Penicillium corylophilum; and A. niger, A. flavus, A. terreus, A. nidulans and Rhizopus stolonifer, respectively. Cotton seeds and cotton seed products were naturally contaminated by aflatoxin B₁ and B₂. About 16% of the different substrates tested were positive for aflatoxin contamination. No citrinin, ochratoxin A, patulin, sterigmatocystin, diacetoxyscirpenol, T-2 toxin or zearalenone were detected in the samples assayed.     

Impact of Aspergillus section Flavi community structure on the development of lethal levels of aflatoxins in Kenyan maize (Zea mays)

Aims: To evaluate the potential role of fungal community structure in predisposing Kenyan maize to severe aflatoxin contamination by contrasting aflatoxin‐producing fungi resident in the region with repeated outbreaks of lethal aflatoxicosis to those in regions without a history of aflatoxicosis. Methods and Results: Fungi belonging to Aspergillus section Flavi were isolated from maize samples from three Kenyan provinces between 2004 and 2006. Frequencies of identified strains and aflatoxin‐producing abilities were assessed, and the data were analysed by statistical means. Most aflatoxin‐producing fungi belonged to Aspergillus flavus. The two major morphotypes of A. flavus varied greatly between provinces, with the S strain dominant in both soil and maize within aflatoxicosis outbreak regions and the L strain dominant in nonoutbreak regions. Conclusions: Aspergillus community structure is an important factor in the development of aflatoxins in maize in Kenya and, as such, is a major contributor to the development of aflatoxicosis in the Eastern Province. Significance and Impact of the Study: Since 1982, deaths caused by aflatoxin‐contaminated maize have repeatedly occurred in the Eastern Province of Kenya. The current study characterized an unusual fungal community structure associated with the lethal contamination events. The results will be helpful in developing aflatoxin management practices to prevent future outbreaks in Kenya.     

Aspergillus Colonization and Aflatoxin Contamination of Maize and Sesame Kernels in Two Agro‐ecological Zones in Senegal

Aflatoxin contamination of major food crops is a serious problem in Senegal. Maize and sesame samples were collected during a survey in five districts located in two agro‐ecological zones in Senegal to determine levels of aflatoxin contamination and the distribution and toxigenicity potential of members of Aspergillus section Flavi. Maize samples from the Guinea Savannah zone (SG) exhibited lower aflatoxin content and colony‐forming units (cfu) than those collected from the Sudan Savannah (SS) zone. In maize, aflatoxin concentration and cfu of A. flavus varied with cultivars, shelling practices and storage methods. The maize variety ‘Jaune de Bambey’ had high aflatoxin levels in both agro‐ecological zones. Aflatoxin content in machine‐shelled maize (120 ng/g) was more than 10‐fold higher than that in manually shelled (8 ng/g) or unshelled maize. Aflatoxin content (between 0.1 and 1.2 ng/g) and cfu values (between 13 and 42 000 cfu/g) of sesame were low, suggesting a low susceptibility to A. flavus. In both agro‐ecological zones, and in all storage systems, aflatoxin contamination was lower in sesame than in maize. In this study, only three species of Aspergillus section Flavi (A. flavus, A. tamarii and the unnamed taxon S_BG) were observed with the frequency of toxigenic strains remaining below 50% in maize from the SG zone compared with 51% of isolates from samples collected in Sedhiou district in SS zone. The proportion of toxigenic strains isolated from sesame was variable. For both crops, L‐strains were the most prevalent in the two agro‐ecological zones. Some of the atoxigenic strains collected could be valuable microbial resources for the biological control of aflatoxin in Senegal.     

Aspergillus on tree nuts: incidence and associations

California exports tree nuts to countries where they face stringent standards for aflatoxin contamination. Trade concerns have stimulated efforts to eliminate aflatoxins and Aspergillus flavus from almonds, pistachios and walnuts. Incidence of fungi on tree nuts and associations among fungi on tree nuts were studied. Eleven hundred pistachios, almonds, walnuts and brazil nuts without visible insect damage were plated on salt agar and observed for growth of fungi. Samples came both from California nut orchards and from supermarkets. To distinguish internal fungal colonization of nuts from superficial colonization, half the nuts were surface-sterilized before plating. The most common genera found were Aspergillus , Rhizopus and Penicillium . Each species of nut had a distinct mycoflora. Populations of most fungi were reduced by surface sterilization in all except brazil nuts, suggesting that they were present as superficial inoculum on (rather than in) the nuts. In general, strongly positive associations were observed among species of Aspergillus ; nuts infected by one species were likely to be colonized by other species as well. Presence of Penicillium was negatively associated with A. niger and Rhizopus in some cases. Results suggest that harvest or postharvest handling has a major influence on nut mycoflora, and that nuts with fungi are usually colonized by several fungi rather than by single species. This revised version was published online in June 2006 with corrections to the Cover Date.     

Drought Stress and Preharvest Aflatoxin Contamination in Agricultural Commodity: Genetics, Genomics and Proteomics

Throughout the world, aflatoxin contamination is considered one of the most serious food safety issues concerning health. Chronic problems with preharvest aflatoxin contamination occur in the southern US, and are particularly troublesome in corn, peanut, cottonseed, and tree nuts. Drought stress is a major factor to contribute to preharvest afiatoxin contamination. Recent studies have demonstrated higher concentration of defense or stress-related proteins in corn kernels of resistant genotypes compared with susceptible genotypes, suggesting that preharvest field condition (drought or not drought) influences gene expression differently In different genotypes resulting in different levels of "end products": PR(pathogenesis-related) proteins in the mature kernels. Because of the complexity of Aspergillus-plant interactions, better understanding of the mechanisms of genetic resistance will be needed using genomics and proteomics for crop improvement. Genetic Improvement of crop resistance to drought stress is one component and will provide a good perspective on the efficacy of control strategy. Proteomic comparisons of corn kernel proteins between resistant or susceptible genotypes to Aspergillus flavus infection have identified stress-related proteins along with antifungal proteins as associated with kernel resistance. Gene expression studies in developing corn kernels are In agreement with the proteomic studies that defense-related genes could be upregulated or downregulated by abiotic stresses.     

Assessment of mycoflora and infestation of insects,vector of <Emphasis Type="Italic">Aspergillus</Emphasis> section <Emphasis Type="Italic">Flavi</Emphasis>, in stored peanut from Argentina

The occurrence of spoilage fungi and Aspergillus section Flavi populations, the aflatoxins incidence, the role of insects as vectors of mycotoxin-producing fungi and the AFs-producing ability of the isolated species throughout the peanut (Arachis hypogaea L.) storage period were evaluated. Analyses of fungal populations from 95 peanut seed samples did not demonstrate significant differences between the incidences in each sampling period. Aspergillus section Flavi were isolated during all incubation periods. Cryptolestes spp. (Coleoptera: Cucujidae) were collected in August, September and October with 18, 16 and 28% of peanut samples contaminated, respectively. Insects isolated during August showed 69% of Aspergillus section Flavi contamination. A. flavus was the most frequently isolated (79%) from peanut seeds and from insect (59%). The greater levels of AFB₁ were detected in September and October with a mean of 68.86 μg/kg and 69.12 μg/kg respectively. The highest proportion of A. flavus toxigenic strains (87.5%) was obtained in June. The presence of Aspergillus section Flavi and insect vectors of aflatoxigenic fungi presented a potential risk for aflatoxin production during the peanut storage period. Integrated management of fungi and insect vectors is in progress.     

Mixed Herbs Drugs: Inhibitory Effect on Growth of the Endogenous Mycoflora and Aflatoxin Production

Twenty commercial mixed herbal drugs were examined for mycological profile. Aspergillus species were the predominant fungi found in the drugs. Other fungi harboured in the drugs with less frequency were Paecilomyces species, Eurotium species, Monascus species, Acremonium species, Penicillium species, Cladosporium species, Scopulariopsis species, Phialophora species and Fonseceae species. Fungal count was between 1.0 log₁₀ CFU and 2.4 log₁₀ CFU per gram of sample. When the drugs were incubated in 85% humidity at 25°C, fungal colonies grew on only two of the drugs. The mixed herbal drugs were extracted with water and the extracts were used to grow Aspergillus parasiticus. All extracts reduced aflatoxin B₁ and aflatoxin G₁ production by 62–97%. All but two of the extracts reduced aflatoxin B₂ and aflatoxin G₂ production by 39–95%. It can be concluded that the commercial powdered mixed herbal drugs contained low number of endogenous fungi, and these drugs are inhibitory to the growth of its endogenous fungi and aflatoxins production by aflatoxigenic fungi.     

An isolate of <Emphasis Type="Italic">Aspergillus flavus</Emphasis> used to reduce aflatoxin contamination in cottonseed has a defective polyketide synthase gene

Contamination of certain foods and feeds with the highly toxic and carcinogenic family of Aspergillus mycotoxins, the aflatoxins, can place a severe economic burden on farmers. As one strategy to reduce aflatoxin contamination, the non-aflatoxin-producing A. flavus isolate AF36 is currently being applied to agricultural fields to competitively exclude aflatoxin-producing Aspergillus species. We now show that the polyketide synthase gene (pksA) required for aflatoxin biosynthesis in AF36, and in other members of the same vegetative compatibility group, possesses a nucleotide polymorphism near the beginning of the coding sequence. This nucleotide change introduces a premature stop codon into the coding sequence, thereby preventing enzyme production and aflatoxin accumulation.     

Studies on the biocontrol of aflatoxin in maize in Thailand

Aflatoxins in maize and peanuts remain a major cause of liver cancer and other human and animal health issues. The principal causal fungi are Aspergillus flavus and A. parasiticus. Relatively little attention has been paid to reducing aflatoxin formation before harvest. The most promising approach is biocontrol by competitive exclusion. This project aimed to demonstrate the efficacy of locally isolated strains of A. flavus for biocontrol of aflatoxin in maize in Thailand. After a rigorous process utilising molecular methods was used to select non-toxigenic A. flavus strains, field inoculum was produced by using hulled rice coated with A. flavus spores in molasses. Field experiments were conducted over two years in two districts, one of light sandy soil (Chokchai), the other a heavy, close textured, soil (Pakchong). Postharvest treatments representative of local practice were also undertaken. Crops 1 and 2 were not significantly contaminated with aflatoxin at the time of harvest, so any impact of biocontrol could not be assessed. However, wet shelling plus storage before drying resulted in increased aflatoxin contamination; biocontrol had no impact on this increase. In crops 3 and 4, biocontrol had a beneficial impact in some freshly harvested maize. Biocontrol treatments also significantly reduced aflatoxin contamination in samples from some treatments stored for two or four days after shelling, but had minimal effect in others. These experiments demonstrated that biocontrol can be highly effective in reducing aflatoxin contamination in maize in Thailand, both at harvest and during poor postharvest crop handling. However, results were inconsistent.