Aspergillus Flavus In Peanuts Research Articles

Biocontrol of volatile organic compounds obtained from Bacillus subtilis CL2 against Aspergillus flavus in peanuts during storage

One of the saprophytic fungi that contaminates crops with carcinogenic aflatoxins is Aspergillus flavus. During pre-harvest and post-harvest, microbial produced bioactive compounds are regarded to be efficient biocontrol strategies for preventing contamination caused by foodborne pathogens and mycotoxins. The inhibitory effects on Aspergillus flavus of volatile organic compounds (VOCs) generated by Bacillus subtilis CL2 and the synthetic volatile 2,3-butanedione were investigated in this study. The results showed that strains CL2 and 2,3-butanedione reduced the disease severity caused by A. flavus infestation and suppressed the generation of A. flavus spores on peanut kernels under storage circumstances. Scanning electron microscopy revealed severely deformed conidia and hyphae of A. flavus after exposure to various doses of 2,3-butanedione. The cell content release and ergosterol content revealed that 2,3-butanedione harmed both the A. flavus cell wall and cell membrane. In conclusion, A. flavus is effectively inhibited by B. subtilis CL2 and its metabolic product 2,3-butanedione during peanut preservation. This research will look for novel strategies to use microbial and bioactive chemicals to combat fungal diseases in food and grain storage.

Cuminaldehyde in cumin essential oils prevents the growth and aflatoxin B1 biosynthesis of Aspergillus flavus in peanuts

The contamination of food with Aspergillus flavus has caused serious economic losses and posed a great threat to human and animal health. Cumin, a condiment and flavoring in many dishes, was showed to have great potential to prevent A. flavus contamination in the present study. By using cumin essential oil (CEO), the occurrence of A. flavus contamination on peanuts was prevented during the storage period even up to 9 months. We further identified cuminaldehyde as the predominant component in CEO with great inhibitory activity to both the growth of A. flavus and aflatoxin B1 (AFB1) biosynthesis. The data showed that the minimum inhibitory concentration (MIC) of cuminaldehyde to conidial growth was 0.64 μL/mL, and the mycelia exposed to 0.80 μL/mL cuminaldehyde reduced AFB1 production by more than 99% in comparison to the control. RNA-Seq profiles showed that some genes, involving the synthesis pathway of ergosterol and AFB1, were significantly down-regulated in the transcription by cuminaldehyde. Further investigation confirmed a significantly reduced cellular ergosterol content in the presence of cuminaldehyde, and the qRT-RCR results were also in agreement with that by RNA-Seq, indicating down-regulated expressions of those genes involving ergosterol and AFB1 synthesis. Taken together, our study strongly supported cuminaldehyde as a potential agent for the prevention of A. flavus contamination.

Investigation of the Antifungal and Anti-Aflatoxigenic Potential of Plant-Based Essential Oils against Aspergillus flavus in Peanuts.

Aspergillus species are known to cause damage to food crops and are associated with opportunistic infections in humans. In the United States, significant losses have been reported in peanut production due to contamination caused by the Aspergillus species. This study evaluated the antifungal effect and anti-aflatoxin activity of selected plant-based essential oils (EOs) against Aspergillus flavus in contaminated peanuts, Tifguard, runner type variety. All fifteen essential oils, tested by the poisoned food technique, inhibited the growth of A. flavus at concentrations ranging between 125 and 4000 ppm. The most effective oils with total clearance of the A. flavus on agar were clove (500 ppm), thyme (1000 ppm), lemongrass, and cinnamon (2000 ppm) EOs. The gas chromatography-mass spectrometry (GC-MS) analysis of clove EO revealed eugenol (83.25%) as a major bioactive constituent. An electron microscopy study revealed that clove EO at 500 ppm caused noticeable morphological and ultrastructural alterations of the somatic and reproductive structures. Using both the ammonia vapor (AV) and coconut milk agar (CMA) methods, we not only detected the presence of an aflatoxigenic form of A. flavus in our contaminated peanuts, but we also observed that aflatoxin production was inhibited by clove EO at concentrations between 500 and 2000 ppm. In addition, we established a correlation between the concentration of clove EO and AFB1 production by reverse-phase high-performance liquid chromatography (HPLC). We demonstrate in our study that clove oil could be a promising natural fungicide for an effective bio-control, non-toxic bio-preservative, and an eco-friendly alternative to synthetic additives against A. flavus in Georgia peanuts.

Identification of Two Novel Peanut Genotypes Resistant to Aflatoxin Production and Their SNP Markers Associated with Resistance.

Aflatoxin B1 (AFB1) and aflatoxin B2 (AFB2) are the most common aflatoxins produced by Aspergillus flavus in peanuts, with high carcinogenicity and teratogenicity. Identification of DNA markers associated with resistance to aflatoxin production is likely to offer breeders efficient tools to develop resistant cultivars through molecular breeding. In this study, seeds of 99 accessions of a Chinese peanut mini-mini core collection were investigated for their reaction to aflatoxin production by a laboratory kernel inoculation assay. Two resistant accessions (Zh.h0551 and Zh.h2150) were identified, with their aflatoxin content being 8.11%–18.90% of the susceptible control. The 99 peanut accessions were also genotyped by restriction site-associated DNA sequencing (RAD-Seq) for a genome-wide association study (GWAS). A total of 60 SNP (single nucleotide polymorphism) markers associated with aflatoxin production were detected, and they explained 16.87%–31.70% of phenotypic variation (PVE), with SNP02686 and SNP19994 possessing 31.70% and 28.91% PVE, respectively. Aflatoxin contents of accessions with “AG” (existed in Zh.h0551 and Zh.h2150) and “GG” genotypes of either SNP19994 or SNP02686 were significantly lower than that of “AA” genotypes in the mean value of a three-year assay. The resistant accessions and molecular markers identified in this study are likely to be helpful for deployment in aflatoxin resistance breeding in peanuts.

Effects of edible coating containing Williopsis saturnus var. saturnus on fungal growth and aflatoxin production by Aspergillus flavus in peanuts

AbstractProduction of aflatoxin by Aspergillus flavus in peanuts is both a health hazard and major problem. This study investigated the ability of a whey protein concentrate (WPC)‐based edible coating containing Williopsis saturnus var. saturnus to prevent growth of A. flavus and aflatoxin production in peanuts. WPC with/without W. saturnus (7 log colony‐forming unit [CFU]/g) or W. saturnus alone was sprayed on peanuts inoculated with A. flavus (3 log CFU/g) and stored for 84 days at 25°C. Application of coating with W. saturnus reduced both growth of A. flavus and aflatoxin level by 82 and 69.5% compared with the control, respectively. Thiobarbituric acid values were around 60% lower in peanuts coated with WPC compared with the control. Sensory and chemical properties of peanuts were not significantly affected by coating treatment (p < .05). Based on results, coating with W. saturnus appears useful in preventing both growth of A. flavus and aflatoxin production in peanuts.Practical ApplicationsImproved WPC based coating containing W. saturnus can be applied on surface of roasted peanuts to prevent growth of A. flavus and aflatoxin production.

Co-overexpression of Brassica juncea NPR1 (BjNPR1) and Trigonella foenum-graecum defensin (Tfgd) in transgenic peanut provides comprehensive but varied protection against Aspergillus flavus and Cercospora arachidicola.

Coexpression of two antifungal genes ( NPR1 and defensin ) in transgenic peanut results in the development of resistance to two major fungal pathogens, Aspergillus flavus and Cercospora arachidicola. Fungal diseases have been one of the principal causes of crop losses with no exception to peanut (Arachis hypogeae L.), a major oilseed crop in Asia and Africa. To address this problem, breeding for fungal disease resistance has been successful to some extent against specific pathogens. However, combating more than one fungal pathogen via breeding is a major limitation in peanut. In the present study, we demonstrated the potential use of co-overexpression of two genes, NPR1 and defensin isolated from Brassica juncea and Trigonella foenum-graecum respectively; that offered resistance towards Aspergillus flavus in peanut. The transgenic plants not only resisted the mycelial growth but also did not accumulate aflatoxin in the seeds. Resistance was also demonstrated against another pathogen, Cercospora arachidicola at varied levels; the transgenic plants showed both reduction in the number of spots and delay in the onset of disease. PCR, Southern and Western blot analysis confirmed stable integration and expression of the transgenes in the transgenic plants. The combinatorial use of the two pathogen resistance genes presents a novel approach to mitigate two important fungal pathogens of peanut.

Comparative transcript profiling of resistant and susceptible peanut post-harvest seeds in response to aflatoxin production by Aspergillus flavus.

BackgroundAflatoxin contamination caused by Aspergillus flavus in peanut (Arachis hypogaea) including in pre- and post-harvest stages seriously affects industry development and human health. Even though resistance to aflatoxin production in post-harvest peanut has been identified, its molecular mechanism has been poorly understood. To understand the mechanism of peanut response to aflatoxin production by A. flavus, RNA-seq was used for global transcriptome profiling of post-harvest seed of resistant (Zhonghua 6) and susceptible (Zhonghua 12) peanut genotypes under the fungus infection and aflatoxin production stress.ResultA total of 128.72 Gb of high-quality bases were generated and assembled into 128, 725 unigenes (average length 765 bp). About 62, 352 unigenes (48.43 %) were annotated in the NCBI non-redundant protein sequences, NCBI non-redundant nucleotide sequences, Swiss-Prot, KEGG Ortholog, Protein family, Gene Ontology, or eukaryotic Ortholog Groups database and more than 93 % of the unigenes were expressed in the samples. Among obtained 30, 143 differentially expressed unigenes (DEGs), 842 potential defense-related genes, including nucleotide binding site-leucine-rich repeat proteins, polygalacturonase inhibitor proteins, leucine-rich repeat receptor-like kinases, mitogen-activated protein kinase, transcription factors, ADP-ribosylation factors, pathogenesis-related proteins and crucial factors of other defense-related pathways, might contribute to peanut response to aflatoxin production. Notably, DEGs involved in phenylpropanoid-derived compounds biosynthetic pathway were induced to higher levels in the resistant genotype than in the susceptible one. Flavonoid, stilbenoid and phenylpropanoid biosynthesis pathways were enriched only in the resistant genotype.ConclusionsThis study provided the first comprehensive analysis of transcriptome of post-harvest peanut seeds in response to aflatoxin production, and would contribute to better understanding of molecular interaction between peanut and A. flavus. The data generated in this study would be a valuable resource for genetic and genomic studies on crops resistance to aflatoxin contamination.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0738-z) contains supplementary material, which is available to authorized users.

Aspergillus flavus Population and Aflatoxin B1 Content of Processed Peanut Products in Municipality of Bogor, West Java, Indonesia

The objectives of this study were to get informations on the population of A. flavus and aflatoxin B1 content of five processed peanut products collected from retailers in Kecamatan Bogor Tengah (Subdistrict of Central Bogor), Municipality of Bogor. A total of 129 samples of processed peanut products was collected. They consisted of roasted peanuts with skin pods (33 samples), flour-coated peanuts (33), siomay sauce (18), pecel/gado-gado sauce (33) and satai sauce (12). A sample each of 2 000 g roasted peanuts with skin pods as well as flour-coated peanuts, and a sample each of 1 500 g siomay sauce, pecel/gado-gado sauce as well as satai sauce was mixed homogenously. It was then divided two times manually to obtain working samples to determine A. flavus population, AFB1 content and a reserve sample. Peanut kernels of roasted peanuts with skin pods and flour-coated peanuts were obtained by peeling their skin pods and the batter coat of tapioca flour manually, respectively. Aspergillus flavus in peanut processed products was isolated using a serial dilution method, followed by pour plate method on Aspergillus Flavus and Parasiticus Agar (AFPA). AFB1 content was determined using Thin Layer Chromatography method. Two replicates were used for each sample. The results showed that the population of A. flavus in roasted peanuts with skin pods, flour-coated peanuts, siomay sauce, pecel/gado-gado sauce and satai sauce were 0.3, 0.1, 0.3, 13.2 and 0.4 cfu/g (wet basis), respectively. The highest AFB1 content of processed peanut products (43.2 ppb) was found in roasted peanuts with skin pods, followed by flour-coated peanuts (34.3 ppb), satai sauce (23.2 ppb), pecel/gado-gado sauce (17.1 ppb) and siomay sauce (4.4 ppb).Key words : Aspergillus flavus, aflatoxin B1, processed peanut products, Municipality of Bogor

Evaluation of selected geocarposphere bacteria for biological control of Aspergillus flavus in peanut

Selected bacterial strains isolated from the region of peanut pod development (geocarposphere) and two additional bacterial strains were screened as potential biological control agents against Aspergillus flavus invasion and subsequent aflatoxin contamination of peanut in laboratory, greenhouse, and field trials. All 17 geocarposphere strains tested delayed invasion of young roots and reduced colonization by the fungus in a root-radicle assay used as a rapid laboratory prescreen. In a greenhouse study, seven bacterial strains significantly reduced pod colonization by A. flavus compared to the control. In a field trial, conducted similarly to the greenhouse assay, pods sampled at mid-peg from plants seed-treated with suspensions of either 91A-539 or 91A-550 were not colonized by A. flavus, and the incidence of pods invaded from plants treated with either 91A-539 or 91A-599 was consistently lower than nonbacterized plants at each of five sampling dates. At harvest, 8 geocarposphere bacterial strains significantly lowered the percentage of pods colonized (> 51%) compared to the control. Levels of seed colonization ranged from 1.3% to 45% and did not appear related to aflatoxin concentrations in the kernels.

Growth of and aflatoxin production by Aspergillus flavus in peanuts stored under modified atmosphere packaging (MAP) conditions

The combined effects of water activity ( a w), storage temperature, headspace oxygen and carbon dioxide concentrations on the growth of, and aflatoxin production by Aspergillus flavus on sterile peanuts were examined using a process optimization technique termed response surface methodology (RSM). Regression analysis of the data indicated that a w, storage temperature and initial headspace oxygen concentration were all significant factors ( P < 0.001) affecting the growth of, and aflatoxin production by A. flavus. Extensive growth and aflatoxin production occurred during the first week of storage in most treatment combinations. Maximum growth occurred in peanuts with an a w of 0.97, a storage temperature of 25°C and headspace oxygen of 10% (balance 60:40 carbon dioxide: nitrogen), after 21 days of storage while maximum aflatoxin production occurred at a lower a w of 0.94, after 21 days under similar storage/gaseous conditions. In several treatment combinations, where high levels of aflatoxin ( > 20 ng/g) were initially detected, aflatoxin concentration decreased during storage to levels less than the current regulatory limit of 20 ng/g. This study has shown that A. flavus can grow and produce aflatoxin in carbon dioxide enriched atmospheres in the presence of oxygen. It also emphasizes the combined effect of several ‘barriers’ to inhibit and reduce aflatoxin in MAP products containing various levels of residual oxygen.

Effect of gas barrier characteristics of films on aflatoxin production by Aspergillus flavus in peanuts packaged under modified atmosphere packaging (MAP) conditions

The effects of the gas barrier characteristics of three films (ASI, ASII and ASIII) and storage temperature on the growth of, and aflatoxin production by, Aspergillus flavus in peanuts packaged in air and under a modified gas atmosphere of CO 2:N 2 (65:35) were investigated. Mold growth was barely visible in air packaged peanuts using high-medium barrier films (ASI and ASII) and stored at 20°C with more extensive growth occurring in air packaged peanuts stored at 25 and 30°C. Extensive mycelial growth and sporulation occurred in all air packaged peanuts in a low barrier film (ASIII), especially at 30°C. Gas packaging inhibited mold growth in peanuts packaged in a high gas barrier film at 20°C. However, mold growth occurred in gas packaged peanuts packaged in film ASII at higher storage temperatures while extensive mycelial growth was observed in all peanuts packaged in film ASIII irrespective of storage temperature. Levels of aflatoxin greater than the regulatory limit of 20 ng/g were detected in all air packaged peanuts with the highest level of aflatoxin (∼76 ng/g) being detected in peanuts packaged in a high gas barrier film ASI. Aflatoxin production was inhibited in gas packaged peanuts using a high barrier film. However, higher levels of aflatoxin were detected in all gas packaged peanuts in medium-low gas barrier films (ASII and ASIII), particularly at higher storage temperatures. This study has shown that MAP using a CO 2:N 2 (65:35) gas mixture was effective in controlling aflatoxin production by A. flavus in peanuts to levels less than the regulatory limit of 20 ng/g. However, the antimycotic effect of low O 2-high CO 2 atmospheres is dependent on the gas barrier characteristics of the packaging films, especially at higher storage temperatures.

Production of aflatoxin on peanuts under controlled environments

The influence of temperature, relative humidity, nature of the substrate, atmospheric gases, and other factors on growth and aflatoxin production by Aspergillus flavus in peanuts, Arachis hypogaea L. variety ‘Early Runner’, was investigated under controlled environments. Sound or broken mature kernels, immature kernels, and unshelled peanuts were inoculated with spores of A. flavus and incubated in 98 ± 1 per cent r.h. at temperatures ranging from 10 to 45°C, and also 30 ± 0·5°C in relative humidities ranging from 70 to 99 per cent. The substrate was either heat-treated (sterile) cured peanuts, surface-sterilized pods of freshly dug peanuts, or unsterile cured peanuts. Surface-sterilized, sound mature kernels were used in studies with atmospheric gases. Samples were removed after 7, 21, 42 or 84 days and assayed for aflatoxin, free fatty acids, and kernel moisture. The limiting relative humidity for aflatoxin production was 83 ± 1 per cent or higher at 30°C, varying with the substrate and length of the incubation period. The lower limiting temperature was 11–12 ± 1°C, whereas the upper limiting temperature was 40·5 ± 0·5°C at 98 ± 1 per cent r.h. Living peanuts in the shell were slightly less susceptible to invasion and aflatoxin formation than shelled kernels or heat-killed peanuts. Aflatoxin production in sound mature kernels decreased with increasing concentrations of CO 2 from 0·03 to 100 per cent. Reducing O 2 concentrations generally reduced aflatoxin production. Notable decreases in aflatoxin resulted when O 2 was reduced from 5 to 1 per cent in combination with 0, 20 or 80 per cent CO 2. Lowering temperature or relative humidity from optimum also reduced aflatoxin production. Aflatoxin production varied with strain of the fungus, maturity of the peanut, shake or stationary culture, and microbial interactions. Free fatty acid formation paralleled fungus growth rather than aflatoxin synthesis.