Subsequent Aflatoxin Contamination Research Articles

Inhibitory effects of epiphytic Kluyveromyces marxianus from Indian senna (Cassia angustifolia Vahl.) on growth and aflatoxin production of Aspergillus flavus

Aspergillus flavus infection and subsequent aflatoxin contamination are considered the major constraints in senna (Cassia angustifolia Vahl.) export. Using native epiphytic yeast to control phytopathogens is a successful strategy for managing plant diseases. In the present investigation, we exploited the antagonistic potential of epiphytic yeast isolates obtained from senna against A. flavus growth and aflatoxin B1 (AFB1) production. Four Kluyveromyces marxianus strains (YSL3, YSL16, YSP12, and YSF9) exhibited vigorous antagonistic activity with a maximum inhibition of 64 %. In vivo evaluation of senna pods showed that K. marxianus strains effectively reduced A. flavus colonization with a population range of 5.87 to 7.08 log10 CFU/g. In contrast, the untreated senna pods were found to have severe fungal colonization with a population of 7.84 log10 CFU/g. In addition, HPLC analysis showed that aflatoxin B1 in senna pods was drastically reduced upon yeast treatment up to 14 DAI. Furthermore, we demonstrated the antifungal action mechanisms of K. marxianus, such as surface colonizing ability on pods, production of antifungal volatiles (VOCs), siderophores, extracellular lytic enzymes, and cell wall binding ability to AFB1. All four strains of K. marxianus showed rapid colonization on the senna pod, and YSP12 reached the maximum population of 7.18 log10 CFU/pod at 9 days after inoculation (DAI). The exposure of A. flavus to K. marxianus VOCs significantly reduced the growth by up to 99 and 93.2 % at 7 and 14 DAI, respectively. Scanning electron microscopic images demonstrated severe mycelial damage and hyphal deformities of A. flavus. In addition, yeast VOCs can reduce aflatoxin biosynthesis in A. flavus by up to 99 and 93.2 % at 7 and 14 DAI, respectively. Gas chromatography–mass spectrometry analysis confirmed the presence of antimicrobial compounds such as dimethyl trisulfide, ethyl acetate, ethanol, 3-methyl butanal, 2-methyl-1-butanol, and 3-methyl-1-butanol in the volatiles. K. marxianus strains produced siderophores and hydrolytic enzymes such as chitinase and β-1,3-glucanase. A higher AFB1 binding ability was observed in the heat-killed cells (47.5 to 70.65 %) than in the viable cells (43.16 to 60.98 %) of K. marxianus. The current study demonstrated that epiphytic K. marxianus isolated from senna could be a successful biocontrol source to reduce aflatoxin contamination in senna pods.

Spatial and Temporal Population Dynamics of Aspergillus flavus in Commercial Pistachio Orchards in Arizona

Aspergillus flavus infects a wide range of crops, including pistachio, and subsequent aflatoxin contamination results in significant economic losses. Application of biocontrol products based on nonaflatoxigenic (atoxigenic) strains of A. flavus is one of the most effective tactics for controlling aflatoxins in crops. Both risk of aflatoxin contamination and effectiveness of biocontrol are influenced by the extent to which A. flavus spores move into pistachio tree canopies during periods of nut development. Thus, the purpose of this study was to evaluate spatial and temporal population dynamics of A. flavus, including the applied biocontrol strain AF36, in canopies of pistachio orchards in Arizona. Propagule densities of A. flavus were quantified on leaf samples collected from lower, middle, and upper canopies from spring through harvest in 2018 and 2019. A. flavus propagule densities peaked during periods of high temperature and rainfall in 2018 (up to 600 CFU/g) and 2019 (up to 23 CFU/g), which coincided with nut development and maturation. The applied biocontrol strain AF36 was detected at all canopy heights but overall propagule densities were greater in the upper and middle canopy (mean = 70 CFU/g) compared with the lower canopy (mean = 47 CFU/g). Results suggest that June to August is the period during which A. flavus inoculum increases in Arizona pistachio orchards and, to most effectively displace aflatoxin-producing fungi in tree canopies, biocontrol applications should precede this period. In addition, this study demonstrates that soil-applied biocontrol strains can successfully disperse throughout the canopies of commercial tree nut orchards.

Aflatoxin contamination in groundnut (Arachis hypogaea L.); its causes and management

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The Development of a qPCR Assay to Measure Aspergillus flavus Biomass in Maize and the Use of a Biocontrol Strategy to Limit Aflatoxin Production.

Aspergillus flavus colonisation of maize can produce mycotoxins that are detrimental to both human and animal health. Screening of maize lines, resistant to A. flavus infection, together with a biocontrol strategy, could help minimize subsequent aflatoxin contamination. We developed a qPCR assay to measure A. flavus biomass and showed that two African maize lines, GAF4 and KDV1, had different fungal loads for the aflatoxigenic isolate (KSM014), fourteen days after infection. The qPCR assay revealed no significant variation in A. flavus biomass between diseased and non-diseased maize tissues for GAF4, while KDV1 had a significantly higher A. flavus biomass (p < 0.05) in infected shoots and roots compared to the control. The biocontrol strategy using an atoxigenic isolate (KSM012) against the toxigenic isolate (KSM014), showed aflatoxin production inhibition at the co-infection ratio, 50:50 for both maize lines (KDV1 > 99.7% and GAF ≥ 69.4%), as confirmed by bioanalytical techniques. As far as we are aware, this is the first report in Kenya where the biomass of A. flavus from maize tissue was detected and quantified using a qPCR assay. Our results suggest that maize lines, which have adequate resistance to A. flavus, together with the appropriate biocontrol strategy, could limit outbreaks of aflatoxicoses.

English

Groundnut (Arachis hypogaea L.) is an important cash crop in the lowland areas of Ethiopia. However, prevalence of Aspergillus invasions and subsequent aflatoxin contamination compromises the quality of groundnut kernels. This study was conducted to evaluate the effect of farm yard manure (FYM) and seed treatments against Aspergillus species pod colonization and aflatoxin accumulation under field conditions. The inhibitory efficacy of Trichoderma species as biocontrol agents was also assessed. A total of 20 treatment combinations including pre-planting applications of FYM at 0, 2.5, 5, and 7.5 tons/ha and seed treatments with carbendazim at 2 g/kg and mancozeb at 3 g kg-1, and Trichoderma harzianum and Trichoderma viride each at 5 g/kg as well as untreated seed as control were used. Treatments were laid out in a randomized complete block design (RCBD) in three replications. The experiment was conducted in two consecutive seasons (2014 and 2015) at Babile Haramaya University sub-Research Station. The highest pod and seed yields (1901.5 and 1281.5 kg/ha, respectively) were recovered from plots treated with T. harzianum at 5 g/kg seed. A. flavus was abundantly recorded in control plots, which could be responsible for the high aflatoxin B1 (5704.4 µg/kg) and B2 (2219.0 µg/kg) contamination. However, plots treated with T. harzianum at 5 g/kg seed and FYM at 5 tons/ha + T. harzianum at 5 g/kg were free from aflatoxin. Integrations of T. harzianum as biocontrol seed treatment and soil amendment with FYM were effective in the pre-harvest management of Aspergillus spp. and aflatoxins contamination. Key words: Aflatoxin, Aspergillus, carbendazim, farm yard manure, groundnut, mancozeb, Trichoderma species.

Effect of Storage Time on Nutrient, Biodeteriorating Fungi and Aflatoxin Contents of Bitter Leaf (Vernonia amygadalina) and JUTE (Corchorus olitorius)

Unhygienic handlings of several African food products during storage have been reported to promote fungal biodeterioration and subsequent aflatoxin contamination. Therefore, this study was aimed to investigate the effect of storage time on nutrient, commonly associated bio-deteriorating fungi and aflatoxin contents of selected vegetables; Bitter leaf (Vernonia amygdalina) and Jute (Corchorus olitorius) in Nigeria. Fresh and stored vegetable samples (24) were collected from different markets in Ibadan, Nigeria, they were analyzed for proximate (% Moisture, Carbohydrate, Crude Protein, Fat and Fibre), nutrient (Potassium, Calcium, Phosphorus, Sodium Iron, Zinc and Magnesium), fungal and aflatoxin contents (AFB1, AFB2, AFG1 and AFG2). Most dominantly associated fungal strains were identified as Aspergillus niger, Aspergillus flavus, Aspergillus terreus, Aspergillus tamari, Aspergillus parasiticus, Aspergillus fumigatus, Penicillium chrysogenum and Rhizopus nigricans. The fungal incidence was more pronounced in the stored samples as compared to the fresh samples. The storage time also significantly affected the nutrient and aflatoxin contents of the vegetables; most nutrients decreased with increasing storage period while the aflatoxin contents increased. The study affirms that the storage time has effect on the nutrient, fungal contamination and consequently on the associated aflatoxin, although the detected aflatoxin levels were below the tolerance limit but these may increase with the storage time. It is therefore advisable to take hygienic measures during storage period and also, prolong storage should be discouraged.

Exploration, antifungal and antiaflatoxigenic activity of halophilic bacteria communities from saline soils of Howze-Soltan playa in Iran.

In the present study, halophilic bacteria communities were explored in saline soils of Howze-Soltan playa in Iran with special attention to their biological activity against an aflatoxigenic Aspergillus parasiticus NRRL 2999. Halophilic bacteria were isolated from a total of 20 saline soils using specific culture media and identified by 16S rRNA sequencing in neighbor-joining tree analysis. Antifungal and antiaflatoxigenic activities of the bacteria were screened by a nor-mutant A. parasiticus NRRL 2999 using visual agar plate assay and confirmed by high-performance liquid chromatography. Among a total of 177 halophilic bacteria belonging to 11 genera, 121 isolates (68.3%) inhibited A. parasiticus growth and/or aflatoxin production. The most potent inhibitory bacteria of the genera Bacillus, Paenibacillus and Staphylococcus were distributed in three main phylogenetic clusters as evidenced by 16S rRNA sequence analysis. A. parasiticus growth was inhibited by 0.7-92.7%, while AFB1 and AFG1 productions were suppressed by 15.1-98.9 and 57.0-99.6%, respectively. Taken together, halophilic bacteria identified in this study may be considered as potential sources of novel bioactive metabolites as well as promising candidates to develop new biocontrol agents for managing toxigenic fungi growth and subsequent aflatoxin contamination of food and feed in practice.

Unraveling the mode of antifungal action of Bacillus subtilis and Bacillus amyloliquefaciens as potential biocontrol agents against aflatoxigenic Aspergillus parasiticus

Some filamentous fungi are plant pathogens that cause serious economic losses in foodstuffs and agricultural commodities. In the present study, the antifungal effects of culture filtrates of soil strains of Bacillus subtilis and Bacillus amyloliquefaciens were studied against some common field-pathogenic fungi with special reference to the possible mode of actions against Aspergillus parasiticus NRRL2999. The fungal growth and aflatoxin production were determined by microbioassay technique. Changes in membrane ergosterol content, mitochondrial dehydrogenase activity, and chitin and β-1,3-glucan content of the fungal cell wall were evaluated as the different possible targets in the presence of various concentrations of bacterial culture filtrates. According to the results, both bacteria showed strong antifungal activity against the broad spectrum of filamentous fungi. They were able to suppress A. parasiticus growth (up to 92%) and aflatoxin production (up to 100%) and showed good proteolytic activities (up to 10.5 ± 0.4 mm clear zones on gelatin agar). Both antagonistic bacteria decreased ergosterol content of the fungal cell membrane of A. parasiticus (9.0–80%) in a dose-dependent manner. They also affected dehydrogenase activity of fungal cell mitochondria. Interestingly, by using 2500 μl of bacterial culture filtrate, the slight resume of fungal growth and cell wall compositions, referred as the paradoxical effect, was exhibited. Scanning and transmission electron microscopy showed drastic changes in structure and shapeof the treated fungal hyphae and vesicles including folding, wrinkling, cell depletion and vacuolization. Altogether, our results show B. subtilis and B. amyloliquefaciens target different sites in the fungal cells and thus, they could act as biological control agents to combat A. parasiticus growth and subsequent aflatoxin contamination of crops and agricultural commodities.

Biocontrol of toxigenic strain of Aspergillus flavus isolated from the root tubers of safed musli (Chlorophytum borivilianum Sant. F) using its rhizospheric mycoflora

Miraculous herb safed musli (Chlorophytum borivilianum Sant. F), family liliaceae, is well recognized for its immense potential as an aphrodisiac. The root tubers of this herbal drug were found to be invested with Aspergillus flavus during field and storage. Therefore, the present study was designed to explore the ability of 26 co-existing rhizospheric mycoflora to inhibit A. flavus invasion and subsequent aflatoxin contamination of safed musli. The interaction of these moulds with highly toxigenic strain (CB55) of A. flavus was evaluated by dual culture method and type of interaction was graded. Most likely antagonistic effects were shown by fifteen (15) moulds, out of which Type ‘C’ interaction was evidenced in the case of six moulds; A. clavatus, A. terreus, Botryotrichum piluliferum, Candida albicans, Cephalosporium acremonium, and Cunninghamella sp. Further, ‘D’ type interaction was displayed by seven moulds which include A. niger, Colletotrichum sp., Drechslera sp., Mucor haemalis, Mycelia sterilia, Rhizopus arrhizus and Stachybotrys atra and ‘E’ type interaction was noted in the case of Trichoderma viride and Trihcothecium roseum. Regarding human health it is critical to use an ecofriendly approach to control the invasion of toxigenic moulds with root tubers of safed musli.

Efficacy of Bacillus subtilis and Bacillus amyloliquefaciens in the control of Aspergillus parasiticus growth and aflatoxins production on pistachio

Pistachio (Pistacia vera) is an important nut for its economic, nutritional and health aspects but it can be contaminated by aflatoxigenic fungi in the field and during storage. Biological control could be considered as an alternative to chemical treatment. In this study, we evaluated the antifungal and anti-mycotoxigenic capability of two Bacillus spp. both in vitro and on pistachio kernels. In in vitro conditions, both strains were able to reduce the mycelial growth and they were able to degrade the four aflatoxins during the first three days after inoculation. AFG1 and AFG2 were rapidly degraded within two days of incubation with the bacterial strains. No aflatoxin was found in the bacterial cell walls, permitting exclusion of mycotoxin adsorption and hypothesis of an in vitro biodegradation as a mode of action. The cultivar of pistachio most susceptible to fungal colonization was ‘Ahmad-Aghaei’, selected among four main Iranian cultivars. A. parasiticus was able to grow and produce aflatoxins on pistachios, but at longer inoculation periods, a natural decrease of aflatoxins was registered. Both strains were able to reduce the fungal incidence and number of spores on pistachio with a stronger effect during the first 5dpi. The effect on aflatoxin content in vivo was less pronounced than in vitro, with a maximum effect at 8dpi. At longer times, there was a contrasting effect due to the lower activity of Bacillus spp. in stationary phase and higher growth of Aspergillus species. This consideration could explain the lack of aflatoxin reduction at 12dpi. Both bacterial strains showed good antifungal activity and aflatoxin reduction in in vitro conditions and on pistachio kernels. Altogether, these results indicate that Bacillus species could be considered as potential biocontrol agents to combat toxigenic fungal growth and subsequent aflatoxin contamination of nuts and agricultural crops in practice.

Production of the 14 kDa trypsin inhibitor protein is important for maize resistance againstAspergillus flavus infection/aflatoxin accumulation

Maize (Zea mays L.) is one of the major crops susceptible toAspergillus flavus Link ex. Fries infection and subsequent aflatoxin contamination. Previous studies found the production of an antifungal 14 kDa trypsin inhibitor (TI) was associated with maize aflatoxin resistance. To further investigate whether the TI plays any direct role in resistance, a TI gene silencing vector was constructed and transformed into maize. Mature kernels were produced from 66 transgenic lines representing 18 independent events. A final total of twelve lines representing four independent events were confirmed positive for transformation, five of which showed significant reduction (63 to 88%) in TI transcript abundance in seedling leaf tissue and seven of which showed significant TI protein reduction (39-85%) in mature kernels. Six of the seven silenced transgenic lines supported higher levels of aflatoxin production compared to negative controls. To further confirm the role of TI in field resistance to aflatoxin accumulation, DNA sequence polymorphisms from within the gene or linked simple sequence repeats were tested in four quantitative trait loci (QTL) mapping populations for QTL effect, and three QTL with log of the odds scores of 11, 4.5, and 3.0 and possibly caused by the TI protein encoding gene were found. Sequence polymorphisms were also tested for association to aflatoxin levels in an association mapping panel, and three single nucleotide polymorphisms were found associated with aflatoxin accumulation (P&lt;0.01). The data from both RNAi and genetic mapping studies demonstrated that production of the TI in maize is important for its resistance toA. flavus infection and/or aflatoxin production.

Discovery and confirmation of genes/proteins associated with maize aflatoxin resistance

Maize (Zea mays L.) is one of the major crops susceptible toAspergillus flavus infection and subsequent aflatoxin contamination. Many earlier studies indicated the roles of kernel proteins, especially constitutively expressed proteins, in maize resistance toA. flavus infection and aflatoxin production. In this review, we examined the past and current efforts in identifying maize genes and proteins from kernel, rachis, and silk tissues that may play an important role in resistance toA. flavus infection and aflatoxin contamination, as well as the efforts in determining the importance or involvement of them in maize resistance through biochemical, molecular and genetics studies. Through these studies, we gained a better understanding of host resistance mechanism: resistant lines appear to either express some stress-related and antifungal proteins at higher levels in endosperm, embryo, rachis and silk tissues beforeA. flavus infection or induce the expression of these proteins much faster compared to susceptible maize lines. In addition, we summarised several recent efforts in enhancing maize resistance to aflatoxin contamination using native genes from maize or heterologous and synthetic genes from other sources as well as fromA. flavus. These efforts to either suppressA. flavus growth or aflatoxin production, have all shown some promising preliminary success. For example, maize plants transformed with an ?-amylase inhibitor protein fromLablab purpurea showed reduced aflatoxin levels by 56% in kernel screening assays. The antifungal potentials of transgenic maize plants expressing synthetic lytic peptides, such as cecropin-based D4E1 or tachyplesin-based AGM peptides with demonstrated anti-flavus activity (IC50 = 2.5 to 10 ?M), are yet to be assayed. Further investigation in these areas may provide a more cost-effective alternative to biocontrol in managing aflatoxin contamination in maize and other susceptible crops.

Global health issues of aflatoxins in food and agriculture: challenges and opportunities.

Global health issues of aflatoxins in food and agriculture: challenges and opportunities.

Antiaflatoxigenic activity of Carum copticum essential oil

Plants are unique sources of useful metabolites. Plant essential oils display a wide range of antimicrobial effects against various pathogens. Here, we studied the essential oil from the seeds of Carum copticum. We monitored aflatoxin by high-performance liquid chromatography. Results show that Carum copticum essential oil inhibits Asergillus parasiticus growth and prevents aflatoxin production. The half-maximal inhibitory concentration (IC50) is 127.5 μg mL−1 for aflatoxin B1 and 23.22 μg mL−1 for aflatoxin G1. Our findings show that Carum copticum essential oil is a potential candidate for the protection of foodstuff and feeds from toxigenic fungus growth and their subsequent aflatoxin contamination.

Overexpression of ARAhPR10, a Member of the PR10 Family, Decreases Levels of &amp;lt;i&amp;gt;Aspergillus flavus&amp;lt;/i&amp;gt; Infection in Peanut Seeds

Peanut (Arachis hypogaea L.) is one of the most susceptible host crops to Aspergillus flavus invasion and subsequent aflatoxin contamination. In this report, a new member of PR10 family putative resistant gene (designated as ARAhPR10, No. EU661964.1) encoding a PR10 protein was isolated and characterized. Analysis of qRT-PCR showed that the expression of ARAhPR10 was induced by pre-harvested A. flavus infection, but no significant difference was observed between resistant genotype “GT-C20” and susceptible genotype “Yueyou 7”. Seven transgenic peanut lines expressing the ARAhPR10 gene under the control of 35S promoter were obtained using the Agrobacterium tumefaciens-mediated method. Real time RT-PCR results showed that the expression level of the ARAhPR10 was significantly higher and the A. flavus infection and aflatoxin content were significantly lower in seeds of transgenic lines than that of the wild type. A significant negative correlation between ARAhPR10 expression at transcript level and seeds aflatoxin production was observed. Combining the previous results, it is suggested that ARAhPR10 expression play an important role in peanut host resistance to A. flavus infection and aflatoxin producing.

Search for novel antifungals from 49 indigenous medicinal plants: Foeniculum vulgare and Platycladus orientalis as strong inhibitors of aflatoxin production by Aspergillus parasiticus

In a search for novel antifungals from natural sources, essential oils (EOs) and extracts of 49 medicinal plants were studied against an aflatoxin (AF)-producing Aspergillus parasiticus using a microbioassay technique. AF levels were measured in culture broth by high performance liquid chromatography. The EOs were analyzed by gas chromatography/mass spectrometry (GC/MS). Based on the results obtained, Achillea millefolium subsp. elborsensis, Ferula gummosa, Mentha spicata, Heracleum pubescens and Thymus fedtschenkoi markedly inhibited A. parasiticus growth by IC50 values of 35 to 1,815 μg/ml without affecting AF production by the fungus. The EOs of flowers and roots of Foeniculum vulgare significantly inhibited both fungal growth (∼70.0%) and production of AFs B1 and G1 (∼99.0%). The ethyl acetate extract of Platycladus orientalis leaves suppressed AFB1 (∼90.0%) but not fungal growth and AFG1 production. This work provides evidence for the first time that F. vulgare and P. orientalis are strong inhibitors of aflatoxin biosynthesis in A. parasiticus. The antifungal activities of the bioactive plants introduced in the present study could make an important contribution to explaining the use of these plants as effective antimicrobial candidates to protect foods and feeds from toxigenic fungus growth and subsequent AF contamination.

Identification of seed proteins associated with resistance to pre-harvested aflatoxin contamination in peanut (Arachis hypogaea L)

BackgroundPre-harvest infection of peanuts by Aspergillus flavus and subsequent aflatoxin contamination is one of the food safety factors that most severely impair peanut productivity and human and animal health, especially in arid and semi-arid tropical areas. Some peanut cultivars with natural pre-harvest resistance to aflatoxin contamination have been identified through field screening. However, little is known about the resistance mechanism, which has slowed the incorporation of resistance into cultivars with commercially acceptable genetic background. Therefore, it is necessary to identify resistance-associated proteins, and then to recognize candidate resistance genes potentially underlying the resistance mechanism.ResultsThe objective of this study was to identify resistance-associated proteins in response to A. flavus infection under drought stress using two-dimensional electrophoresis with mass spectrometry. To identify proteins involved in the resistance to pre-harvest aflatoxin contamination, we compared the differential expression profiles of seed proteins between a resistant cultivar (YJ-1) and a susceptible cultivar (Yueyou 7) under well-watered condition, drought stress, and A. flavus infection with drought stress. A total of 29 spots showed differential expression between resistant and susceptible cultivars in response to A. flavus attack under drought stress. Among these spots, 12 protein spots that consistently exhibited an altered expression were screened by Image Master 5.0 software and successfully identified by MALDI-TOF MS. Five protein spots, including Oso7g0179400, PII protein, CDK1, Oxalate oxidase, SAP domain-containing protein, were uniquely expressed in the resistant cultivar. Six protein spots including low molecular weight heat shock protein precursor, RIO kinase, L-ascorbate peroxidase, iso-Ara h3, 50 S ribosomal protein L22 and putative 30 S ribosomal S9 were significantly up-regulated in the resistant cultivar challenged by A. flavus under drought stress. A significant decrease or down regulation of trypsin inhibitor caused by A. flavus in the resistant cultivar was also observed. In addition, variations in protein expression patterns for resistant and susceptible cultivars were further validated by real time RT-PCR analysis.ConclusionIn summary, this study provides new insights into understanding of the molecular mechanism of resistance to pre-harvest aflatoxin contamination in peanut, and will help to develop peanut varieties with resistance to pre-harvested aflatoxin contamination.

Association between aflatoxin contamination and drought tolerance traits in peanut

The current study investigates the association between drought tolerance traits and aflatoxin contamination in peanut grown under long-term drought. Two field experiments were conducted at Khon Kaen University, Thailand using a split–split plot design with three drought stress levels as main plots, 11 genotypes as sub-plots, and two soil inoculations of Aspergillus flavus treatments as sub-sub-plots. The effects of temperature, soil moisture and A. flavus population on kernel colonization and aflatoxin contamination, and drought tolerance traits viz. specific leaf area (SLA) and root length density (RLD) were measured. The results demonstrated that elevated soil temperatures and reduced soil moisture, favored aflatoxin production. Drought in combination with higher levels of A. flavus inoculum load in the soil resulted in an increase in the fungal populations in the soil which in turn resulted in increased kernel colonization and subsequent aflatoxin contamination. A combination of SLA and RLD, and kernel colonization had a significant influence on aflatoxin contamination under drought conditions in both seasons ( r = 0.73** and 0.76**). The results revealed that drought tolerance traits (SLA and RLD) could be contributing to resistance to aflatoxin contamination suggesting that a combination of SLA, RLD and kernel colonization could be used as selection criteria in selecting parents for aflatoxin resistance.

Peanut gene expression profiling in developing seeds at different reproduction stages during Aspergillus parasiticusinfection

BackgroundPeanut (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.ResultsWe constructed six different cDNA libraries derived from developing peanut seeds at three reproduction stages (R5, R6 and R7) from a resistant and a susceptible cultivated peanut genotypes, 'Tifrunner' (susceptible to Aspergillus infection with higher aflatoxin contamination and resistant to TSWV) and 'GT-C20' (resistant to Aspergillus with reduced aflatoxin contamination and susceptible to TSWV). The developing peanut seed tissues were challenged by A. parasiticus and drought stress in the field. A total of 24,192 randomly selected cDNA clones from six libraries were sequenced. After removing vector sequences and quality trimming, 21,777 high-quality EST sequences were generated. Sequence clustering and assembling resulted in 8,689 unique EST sequences with 1,741 tentative consensus EST sequences (TCs) and 6,948 singleton ESTs. Functional classification was performed according to MIPS functional catalogue criteria. The unique EST sequences were divided into twenty-two categories. A similarity search against the non-redundant protein database available from NCBI indicated that 84.78% of total ESTs showed significant similarity to known proteins, of which 165 genes had been previously reported in peanuts. There were differences in overall expression patterns in different libraries and genotypes. A number of sequences were expressed throughout all of the libraries, representing constitutive expressed sequences. In order to identify resistance-related genes with significantly differential expression, a statistical analysis to estimate the relative abundance (R) was used to compare the relative abundance of each gene transcripts in each cDNA library. Thirty six and forty seven unique EST sequences with threshold of R > 4 from libraries of 'GT-C20' and 'Tifrunner', respectively, were selected for examination of temporal gene expression patterns according to EST frequencies. Nine and eight resistance-related genes with significant up-regulation were obtained in 'GT-C20' and 'Tifrunner' libraries, respectively. Among them, three genes were common in both genotypes. Furthermore, a comparison of our EST sequences with other plant sequences in the TIGR Gene Indices libraries showed that the percentage of peanut EST matched to Arabidopsis thaliana, maize (Zea mays), Medicago truncatula, rapeseed (Brassica napus), rice (Oryza sativa), soybean (Glycine max) and wheat (Triticum aestivum) ESTs ranged from 33.84% to 79.46% with the sequence identity ≥ 80%. These results revealed that peanut ESTs are more closely related to legume species than to cereal crops, and more homologous to dicot than to monocot plant species.ConclusionThe developed ESTs can be used to discover novel sequences or genes, to identify resistance-related genes and to detect the differences among alleles or markers between these resistant and susceptible peanut genotypes. Additionally, this large collection of cultivated peanut EST sequences will make it possible to construct microarrays for gene expression studies and for further characterization of host resistance mechanisms. It will be a valuable genomic resource for the peanut community. The 21,777 ESTs have been deposited to the NCBI GenBank database with accession numbers ES702769 to ES724546.

Occurrence of aflatoxin in three maize (Zea mays L.) hybrids over 5 years in Northern Mississippi

Aflatoxins are produced as secondary metabolites under conducive climatic conditions by Aspergillus flavus. The incidence of aflatoxin varies with environmental conditions, genotype, and location. An expanded understanding of the interaction of the plant, fungus, and weather conditions is needed to further elucidate the field infection process of maize by A. flavus and subsequent aflatoxin contamination. One of the problems in evaluating maize hybrids for resistance to kernel infection and aflatoxin contamination is identifying a time period and environmental conditions that are most advantageous. Three maize genotypes (Pioneer Brand 3223, Mo18W x Mp313E, and Mp313E x Mp420) were evaluated from 1998 to 2002 in response to A. flavus inoculation and aflatoxin contamination and weather conditions favorable for aflatoxin contamination were identified. The highest aflatoxin levels were observed in 1998 and 2000 (1186 and 901 ng g(-1); P < 0.0001); while the lowest levels were detected in 1999 (39 ng g(-1)). Pioneer 3223 had significantly higher levels (1198 ng g(-1)) than Mp313E x Mp420 (205 ng g(-1)), and Mo18W xMp313E (161 ng g(-1); P < 0.0001). The hybrids had six weather-related variables in common that were positively correlated with aflatoxin accumulation. Four of these occurred during 65-85 days after planting and were temperature-related. These results suggest that regardless of the hybrid's maturity or physiological development, the time from 65 to 85 days after planting may be indicative of a period of stress which leads to greater aflatoxin accumulation at harvest.