Flavus NRRL Research Articles

Locust Pathogen Aspergillus oryzae XJ1 Is Different from Aspergillus oryzae and Aspergillus flavus Based on Genomics Comparisons

Fungi play an increasingly important role in the biological control of insect pests. Aspergillus oryzae XJ1 is highly virulent to locust adults and nymphs, which are a destructive economic pest worldwide. Because of its host association with locusts, which is unique in Aspergillus, in this study, we examined the genetic relationships of A. oryzae XJ1 within Aspergillus. We sequenced the genome of A. oryzae XJ1 and compared it with the genomes of other Aspergillus species. The complete genome of A. oryzae XJ1 is 37.9 Mb, comprising 11,720 putative genes, assembled into eight chromosomes. The genome size is similar to that of other A. oryzae strains. Phylogenomic analysis indicated that A. oryzae XJ1 was most closely related to A. flavus NRRL3357, not A. oryzae RIB40. Core/pan-genome analysis of A. oryzae XJ1 and other Aspergillus species revealed that A. oryzae XJ1 had 704 strain-specific genes, whereas A. flavus NRRL3357, A. oryzae KDG 21, and A. parasiticus NRRL 2999 had 646, 955, and 779 unique genes, respectively. The A. oryzae XJ1 genome showed structural differences compared with the genomes of A. oryzae RIB40 and A. flavus NRRL3357 in genomic synteny analysis. These results indicate that A. oryzae XJ1 is genetically distinct at the genome level from other Aspergillus species, including A. oryzae and A. flavus, and may be as a distinct species. This will provide new insight into the classification of Aspergillus based on genomics.

Investigating the Impact of Flavonoids on Aspergillus flavus: Insights into Cell Wall Damage and Biofilms.

Aspergillus flavus, a fungus known for producing aflatoxins, poses significant threats to agriculture and global health. Flavonoids, plant-derived compounds, inhibit A. flavus proliferation and mitigate aflatoxin production, although the precise molecular and physical mechanisms underlying these effects remain poorly understood. In this study, we investigated three flavonoids-apigenin, luteolin, and quercetin-applied to A. flavus NRRL 3357. We determined the following: (1) glycosylated luteolin led to a 10% reduction in maximum fungal growth capacity; (2) quercetin affected cell wall integrity by triggering extreme mycelial collapse, while apigenin and luteolin caused peeling of the outer layer of cell wall; (3) luteolin exhibited the highest antioxidant capacity in the environment compared to apigenin and quercetin; (4) osmotic stress assays did not reveal morphological defects; (5) flavonoids promoted cell adherence, a precursor for biofilm formation; and (6) RNA sequencing analysis revealed that flavonoids impact expression of putative cell wall and plasma membrane biosynthesis genes. Our findings suggest that the differential effects of quercetin, luteolin, and apigenin on membrane integrity and biofilm formation may be driven by their interactions with fungal cell walls. These insights may inform the development of novel antifungal additives or plant breeding strategies focusing on plant-derived compounds in crop protection.

Production of Single Cell Protein (SCP) from the Peel Waste of Pea, Potato, and Banana by Aspergillus Flavus NRRL 21882 as an Efficient Organic Poultry Supplement.

Food protein deficit has become a major issue worldwide, particularly in underdeveloped countries. Scientists are searching for a variety of less expensive solutions to this issue. One of these less expensive methods is to create single cell protein as a substrate from leftover fruit and vegetable waste, which is typically thrown away. In this regard, the fungal strain Aspergillus flavus (NRRL 21882) was used for the synthesis of SCP (single cell protein) from the waste of banana, potato, and pea. In this manner, 30 samples were collected from the whole substrate with a share of 10 samples each from banana, potato, and pea peels, which were in turn dried and powdered finely. The fermentation process was done by the process of solid state fermentation. Aspergillus flavus (NRRL 21882) generated the highest percentage, i.e. 60.67%, of crude protein from the pea peels. The composition of amino acids in crude proteins was also investigated. The findings demonstrated that the highest percentage of aspartic acid (13.34 ± 0.80%) and glutamic acid (14.92 ± 0.69%) was found in A. flavus single cell protein produced from pea peels. Soybean was supplemented with single cell protein in the boilers' diet. Compared to all treated groups, there was a substantial (p ≤ 0.05) increase in the level of antibody titer against the Newcastle disease vaccine. The supplementation of single cell protein with soybean meal had no effect on the levels of liver enzymes. The liver enzymes found in all four groups (A, B, C, and D) were within normal limits. None of the examined groups experienced any change in the feed conversion ratio, with all groups exhibiting an average FCR of 1.6. The current study concludes that broiler health and immunity is increased by supplementing poultry feed with single cell protein.

Comparative Genome Analysis of Japanese Field-Isolated Aspergillus for Aflatoxin Productivity and Non-Productivity.

Aspergillus flavus produces aflatoxin, a carcinogenic fungal toxin that poses a threat to the agricultural and food industries. There is a concern that the distribution of aflatoxin-producing A. flavus is expanding in Japan due to climate change, and it is necessary to understand what types of strains inhabit. In this study, we sequenced the genomes of four Aspergillus strains isolated from agricultural fields in the Ibaraki prefecture of Japan and identified their genetic variants. Phylogenetic analysis based on single-nucleotide variants revealed that the two aflatoxin-producing strains were closely related to A. flavus NRRL3357, whereas the two non-producing strains were closely related to the RIB40 strain of Aspergillus oryzae, a fungus widely used in the Japanese fermentation industry. A detailed analysis of the variants in the aflatoxin biosynthetic gene cluster showed that the two aflatoxin-producing strains belonged to different morphotype lineages. RT-qPCR results indicated that the expression of aflatoxin biosynthetic genes was consistent with aflatoxin production in the two aflatoxin-producing strains, whereas the two non-producing strains expressed most of the aflatoxin biosynthetic genes, unlike common knowledge in A. oryzae, suggesting that the lack of aflatoxin production was attributed to genes outside of the aflatoxin biosynthetic gene cluster in these strains.

Investigating safe storage conditions to mitigate aflatoxin contamination in rice

Mold contamination, especially with aflatoxin-producing Aspergillus species, poses a notable issue for rice, particularly in ready-to-eat (RTE) items, turning them inedible for both humans and animals due to the possible presence of aflatoxins (AFs). Understanding accumulation of the molds and their resulting toxic metabolites is critical in designing quality control strategies in the production of RTE rice-based products such as germinated instant rice, extruded rice cakes and crispies, rice-derived malted beer and flour. There is no clear understanding of the growth of A. flavus and AF accumulation on various fractions of hybrid rice cultivars utilized in the manufacture of the RTE rice-derived food products. In the present work, different experiments were conducted to identify the extent to which temperature (20°C, 25°C, and 30°C), relative humidity (RH) (75%, 85%, and 95%), storage period, storage moisture content (MC), and initial spore concentration impacted the growth of AF-producing A. flavus NRRL 3357. The study also examined how these factors affected ergosterol content and aflatoxin B1 (AFB1) production in different hybrid rice fractions including rough, brown, and milled rice, at different MCs (12%, 15%, and 18% w.b.). The samples, both autoclaved and non-autoclaved rice fractions, were inoculated with different A. flavus spore concentrations (1 × 104 and 1 × 106 spores/mL) and incubated at different temperatures and RHs. A. flavus plate count, ergosterol and AFB1 production were measured over time for up to 60 days. The maximum A. flavus growth was observed in brown rice (autoclaved) at 30°C and 95% RH. Brown rice fraction (non-autoclaved) had the highest AFB1 concentration (9.20 μg/g) which was determined after 15 days of incubation at 30°C and 75% RH. Aflatoxin B1 concentration in brown rice (non-autoclaved) fraction inoculated with the highest concentration of A. flavus and stored at 30°C and 95% RH initially increased after 20 days and then decreased towards the end of the storage period. The findings from this research provide important data related to the growth behavior of A. flavus on various rice fractions. The results contribute to understanding of the safety of RTE foods derived from various fractions of rice that is utilized by the food industry.

Bacillus amyloliquefaciens A-1 inhibiting fungal spoilage in agricultural products is improved by metabolic engineering of enhancing surfactin yield

Fungi and subsequent mycotoxins contamination in agricultural products have caused enormous losses and great harm to human and animal health. Biological control has attracted the attention of researchers due to its advantages, including mild conditions, low cost, high efficiency and low nutrient loss. In this study, a newly isolated strain Bacillus amyloliquefaciens A-1 (A-1), was screened for its ability to inhibit the growth and Aflatoxin B1 (AFB1) production of Aspergillus flavus NRRL 3357. Electron microscopy results revealed that mycelium and conidia of A. flavus were destroyed by A-1, affecting hyphae, cell walls, cell membranes and organelles. RNA-seq analysis indicated disturbance in gene expression profiles of A. flavus, including amino acid degradation and starch and sucrose metabolism pathways. Importantly, the biosynthesis of AFB1 was significantly inhibited by the down-regulation of key regulatory genes, aflR and aflS, and the simultaneous down-regulation of most structural genes. Genome analysis predicted six secondary metabolites biosynthetic gene clusters. Then, four surfactin synthesized by cluster C were identified as the main active substance of A-1 using HPLC-Q-TOF-MS. The addition of alanine, threonine, Fe2+ increased surfactin production. Notably, the overexpression of comX also improved surfactin production. The vivo test results indicated that A-1 could significantly inhibit the decay of pear by Aspergillus westerdijkiae, and the mildew of maize and peanuts. Especially, the overexpression of comX in A-1 could enhance the inhibitory activity. In conclusion, the inhibition mechanism of A-1 was revealed, and comX was found can improve the production of surfactin and subsequent activities, which provides the scientific basis for the development of biocontrol agents to reduce spoilage in agricultural products.

Quantification of Antimicrobial Compounds Produced by Lactic Acid Bacteria Isolated from Pulses

In this study, 11 LAB isolates were isolated from the pulses (green gram and black gram) were tested for biochemical characteristics (catalase, MR-VP, citrate utilization, nitrate reduction) based on these tests four isolates were evaluated for their antimicrobial activity against three prominent pathogens: Escherichia coli, Pseudomonas aeruginosa, and Aspergillus flavus NRRL3357 under different pH 9 (5.5, 6.5, 7.5 and 8.5) and temperature (17ºC, 27ºC, and 40ºC). Among the four isolates, one isolate LB4 exhibited notable antimicrobial efficacy against all three pathogens under pH (6.5) 8.1± 0.6 mm, 8.3 ± 0.5 mm, and 6.6 ± 0.8 mm respectively. Optimum antimicrobial activity showed at temperature 27º against same pathogens the zone of inhibition mean values 10± 0.9 mm, 5.0± 0.6 mm, 8.3± 1.2 mm respectively. LB4 isolate was tested for quantification of key metabolites after three days of incubation, lactic acid, acetic acid, and hydrogen peroxide. Lactic acid (4.6 g/l) and acetic acid (4.8 g/l) were consistently produced in higher amounts, while hydrogen peroxide production remained relatively low (0.58 g/l) by the LB4 isolate. The results indicated the intricate relationship between antimicrobial activity, environmental factors, and metabolite production, emphasizing the promising role of this isolate in combating microbial infections under diverse conditions.

Identification of volatile organic compounds (VOCs) by SPME-GC-MS to detect Aspergillus flavus infection in pistachios

Aflatoxin contamination poses a serious problem for the pistachio industry as aflatoxin B1 (AFB1), the most harmful aflatoxin, can be produced by some species of the Aspergillus and Penicillium. Like any other living organism, Aspergillus flavus (A. flavus) releases specific volatile organic compounds (VOCs) as a result of its metabolic reactions. In this study, we extracted and analyzed the VOCs produced by toxigenic A. flavus using solid-phase microextraction (SPME) in combination with gas chromatography-mass spectroscopy (GC-MS). The study was conducted on culture media and pistachio inoculated samples over a period of 16 days to observe the production of VOCs and aflatoxin over time. Various chemicals from different classes (such as organic acids, alcohols, ketones, and hydrocarbons) were detected during the growth of A. flavus on pistachios. Agglomerative hierarchical clustering was performed on the GC-MS spectra to identify VOCs corresponding to AFB1. We have identified ten VOCs with the potential to function as biomarkers for monitoring the growth of A. flavus NRRL 3357 on in-shell pistachios, and α,β-dimethylbenzenepropanoic acid was identified as the primary early marker for detecting A. flavus infection in pistachios.

Non-Aflatoxigenic Aspergillus flavus: A Promising Biological Control Agent against Aflatoxin Contamination of Corn.

Aflatoxins (AFs) are a family of mycotoxins produced by molds in agricultural products. To deal with this problem, one of the control methods is the biological solution using a non-pathogenic strain Aspergillus flavus NRRL 21882 (Afla-Guard). This study was conducted to evaluate the potential of A. flavus NRRL 21882 to control the AF contamination of corn in the field and during storage in 2018 and 2019. The experimental design consists of treatment at different vegetative stages of infested corn in the field trial. After the field has been harvested, half the corn kernels from both treated and control plots were treated with biopesticide; the other half of the kernels from each group were not treated and used as the control of the storage. Consequently, storage applications consisted of kernels: (1) not treated at all; (2) treated prior to storage; (3) field-treated; and (4) treated both in the field and prior to storage. After field trials, the AF content was very low in the treated plots, ranging from 0.50 to 1.04 μg/kg and from 0.50 to 0.73 μg/kg in 2018 and 2019, respectively, while the AF content in the control was 98.3 and 73.9 μg/kg in 2018 and 2019, respectively. After storage, corn kernels from field plots that were treated with the biopesticide (treated/control) showed low levels of AFs, even after they have been stored under conditions conducive to AF contamination. The biopesticide effect ranged from 98 to 99% and from 69 to 99% in the field and during storage, respectively. This paper has provided the first indications on AF biocontrol based on a competitive exclusion in the corn-growing region of Turkey. The data showed that spraying during the storage period did not provide any further prevention of AF contamination, and only treatment in the field had a significant effect on AFs that occurred in storage.

Flavonoids Modulate Aspergillus flavus Proliferation and Aflatoxin Production.

Aflatoxins are carcinogenic mycotoxins produced by Aspergillus flavus. They contaminate major food crops, particularly corn, and pose a worldwide health concern. Flavonoid production has been correlated to resistance to aflatoxin accumulation in corn. The effects of flavonoids on fungal proliferation and aflatoxin production are not well understood. In this study, we performed bioassays, fluorescence and scanning electron microscopy, and total antioxidant analysis to determine the effects of three flavonoids (apigenin, luteolin, and quercetin) on proliferation and aflatoxin production in A. flavus NRRL 3357. Results showed that concentrations of apigenin and luteolin modulated fungal proliferation and aflatoxin production in a dose-dependent manner, leading to inhibition or promotion of proliferation and toxin production. Microscopy studies of fungi exposed to flavonoids showed mycelial cell wall disruption, abnormal cell wall invaginations, and tears. Fluorescent enhancement of apigenin and luteolin using Naturstoff reagent A showed that these chemicals localized in sphere-like structures on the mycelia surface. Fungi exposed to low concentrations of apigenin, luteolin, and quercetin lowered the total antioxidant capacity in the environment compared to controls. Our results indicate that flavonoids disrupt cell wall integrity and may localize in vesicle-like structures. We hypothesize that flavonoids could act as potential signaling molecules at low concentrations and change the oxidative state of the microenvironment, either or both of which may lead to reduction of fungal proliferation and aflatoxin production.

Growth and Aflatoxin B1 biosynthesis rate of model Aspergillus flavus NRRL 3357 exposed to selected infrared wavelengths

Aflatoxin B1 (AFB1) is a highly toxic substance produced mainly by Aspergillus spp., which are known to contaminate a wide variety of foods and feeds. The objective of this study was to understand the growth and AFB1 production rate of Aspergillus flavus NRRL 3357 and investigate the impact of different infrared (IR) heat intensities on the growth and aflatoxin-producing potential of A. flavus. Thirty healthy corn kernels were inoculated with 1 × 106 spores/mL of A. flavus spores and incubated at 25 °C. Then, A. flavus plate count, ergosterol, and AFB1 were measured every 24 h. In another experiment, inoculated corn incubated for 24 h was exposed to different IR heat intensities and heating durations. Afterward, the IR-exposed A. flavus were incubated for additional 3 and 6 days (4 and 7 days of incubation in total), and the ergosterol and AFB1 concentrations were measured. For non-IR exposed A. flavus, the highest AFB1 concentration (32.2 μg/g) was observed after 6 days of incubation. Treatment at high IR heat intensities and heating duration resulted in low AFB1 and ergosterol concentrations. However, IR heat treatment at low intensities (e.g., 1.86 kW/m2) for 20 s resulted in high AFB1 and ergosterol concentrations (97.83 and 34.84 μg/g, respectively). The sublethal IR heat intensity may have increased the potential of the exposed A. flavus NRRL 3357 to grow and produce AFB1. The outcome of this study will contribute to and enhance the continual efforts of mitigating AFB1 contamination of economically important grains.

Identification of long-chain alkane-degrading (LadA) monooxygenases in Aspergillus flavus via in silico analysis.

Efficient degradation of alkanes in crude oil by the isolated Aspergillus flavus MM1 alluded to the presence of highly active alkane-degrading enzymes in this fungus. A long-chain alkane-degrading, LadA-like enzyme family in A. flavus was identified, and possible substrate-binding modes were analyzed using a computational approach. By analyzing publicly available protein databases, we identified six uncharacterized proteins in A. flavus NRRL 3357, of which five were identified as class LadAα and one as class LadAβ, which are eukaryotic homologs of bacterial long-chain alkane monooxygenase (LadA). Computational models of A. flavus LadAα homologs (Af1-Af5) showed overall structural similarity to the bacterial LadA and the unique sequence and structural elements that bind the cofactor Flavin mononucleotide (FMN). A receptor-cofactor-substrate docking protocol was established and validated to demonstrate the substrate binding in the A. flavus LadAα homologs. The modeled Af1, Af3, Af4, and Af5 captured long-chain n-alkanes inside the active pocket, above the bound FMN. Isoalloxazine ring of reduced FMN formed a π–alkyl interaction with the terminal carbon atom of captured alkanes, C16–C30, in Af3–Af5 and C16–C24 in Af1. Our results confirmed the ability of identified A. flavus LadAα monooxygenases to bind long-chain alkanes inside the active pocket. Hence A. flavus LadAα monooxygenases potentially initiate the degradation of long-chain alkanes by oxidizing bound long-chain alkanes into their corresponding alcohol.

First Report of Aspergillus flavus Causing Fruit Rot on Kiwifruit in China.

In October 2020, fruit rot symptoms were detected on kiwifruit (Actinidia chinensis var. deliciosa 'Xuxiang') in southwestern Shaanxi (Hanzhong municipality; 107.27° E, 33.23° N) in China. Mature kiwifruit, during the harvest period, exhibited soft rot and brown lesions. The symptoms were similar than those reported for Alternaria alternata, Colletotrichum spp., Fusarium avenaceum and Rhizopus oryzae causing fruit rot on kiwifruit (Feng et al. 2019; Li et al. 2017; Kim et al. 2018; Zhao et al. 2020). The symptoms were observed in approximately 15% of the fruit in 6 kiwifruit orchards (31 ha in total). Ten samples of symptomatic tissue, approximately 1 cm2 in size, were sterilized in 2% NaOCl for 30 seconds and washed twice with sterilized water. The pathogen was isolated from all collected samples via culturing on PDA medium, containing 50 µg/mL chloramphenicol, at 28 ºC. Green powdery-like colonies were detected after 5 days (Figure 1). A total of 12 isolates were obtained via single spore isolation. Internal transcribed spacer (ITS), elongation factor 1-α (EF1-α) and RNA polymerase II subunit (RPB2) genes were amplified using ITS5/ITS4, A_EF1_F/A_EF1_R and RPB2-5F/RPB2-7cR (NJC03), or RPB2-7cF/RPB2-11aR (NJC04), primers, respectively. Eleven isolates shared the same sequences (NJC03), MZ801787 (ITS), MZ701709 (EF1-α) and MZ701707 (RPB2), while one of the isolates provided different sequences (NJC04), OK618459 (ITS), OK634020 (EF1-α) and OL331017 (RPB2). The obtained ITS sequences shared >99% homology to the ITS gene from A. flavus KU20018.4 (MT487825), the EF1-α sequences shared 100% homology to the EF1-α gene from A. flavus clinical2342 (KP054370) and the RPB2 sequences shared >99% homology to the RPB2 genes from A. flavus PW3170 (LC000581) and A. flavus NRRL3357 (XM_041293948). Molecular phylogenetic tree was constructed using MEGA7 with reference Aspergillus strains (Figure 2). Microscope observations of all isolates showed the presence of septate mycelium, circular unicellular conidia (2-4 µm diameter) and conidiophores, and agree with the morphology of A. flavus (Horn 2005). The pathogenicity of all isolates was screened using intact and wounded 'Xuxiang' kiwifruits (ten kiwifruits were used for each combination with 3 replicates), which were purchased from a local market. A 1 × 106 spores/mL (10 µL) solution of the isolates was used for the inoculation. Sterilized water was used in the control experiment. Inoculated kiwifruits were storage at 26 °C and 60% relative humidity for 10 days. Rot lesions in the wounded kiwifruits were totally covered by green mycelia, while the lesions on the intact kiwifruits were similar to the symptoms observed in the field. The pathogen was recovered and its identity was confirmed by sequence analysis of ITS, EF1-α and RPB2, fulfilling Koch's postulates. A. flavus is known to be an important fungal pathogen of corn, cotton and peanuts (Zhang et al. 2020). During recent years, A. flavus was reported to cause fruit rot on grapes (Ghuffar et al. 2020), and was identified on almond, fig, organic spelt and pistachio (Krulj et al. 2017; Ortega-Beltran et al. 2019). The presence of A. flavus in food products is an issue of global concern due to A. flavus is able to produce carcinogenic aflatoxin (Maxwell et al. 2021). As far as we know, this is the first report of A. flavus causing fruit rot on kiwifruit. This report will help to understand the distribution of A. flavus in crops and the food safety hazards that are present in China.

Corepressors SsnF and RcoA Regulate Development and Aflatoxin B1 Biosynthesis in Aspergillus flavus NRRL 3357.

Aspergillus flavus is a saprophytic fungus that can be found across the entire world. It can produce aflatoxin B1 (AFB1), which threatens human health. CreA, as the central factor in carbon catabolite repression (CCR), regulates carbon catabolism and AFB1 biosynthesis in A. flavus. Additionally, SsnF-RcoA are recognized as the corepressors of CreA in CCR. In this study, ssnF and rcoA not only regulated the expressions of CCR factors and hydrolase genes, but also positively affected mycelia growth, conidia production, sclerotia formation, and osmotic stress response in A. flavus. More importantly, SsnF and RcoA were identified as positive regulators for AFB1 biosynthesis, as they modulate the AF cluster genes and the relevant regulators at a transcriptional level. Additionally, the interactions of SsnF-CreA and RcoA-CreA were strong and moderate, respectively. However, the interaction of SsnF and RcoA was weak. The interaction models of CreA-SsnF, CreA-RcoA, and SsnF-RcoA were also simulated with a docking analysis. All things considered, SsnF and RcoA are not just the critical regulators of the CCR pathway, but the global regulators involving in morphological development and AFB1 biosynthesis in A. flavus.

Lessons Learned: the Importance of Biological Curation.

Proper species identification of sequenced fungal isolates or strains is imperative for the interpretation of genomics data. Through this letter (1), and through another recently published research note (2), it has come to our attention that one of the isolates presented in our previous publication (3) was misidentified. The presumptive Aspergillus parasiticus isolate NRRL 2999 has been shown to be an Aspergillus flavus isolate, a clonal derivative of A. flavus NRRL 3357 (2). In our recent publication presenting two new reference genomes for A. flavus (4), we performed a phylogenomics analysis of several publicly available Aspergillus assemblies and noted the sequence similarity between A. flavus NRRL 3357 and A. parasiticus NRRL 2999. We also noted a close relationship between A. flavus WRRL 1519 and A. oryzae RIB40 and that A. oryzae RIB40 grouped among A. flavus isolates, not as an outgroup. Based on these analyses, we concluded that the A. flavus species is polyphyletic. The revelation that NRRL 2999 is indeed an A. flavus isolate clonal to NRRL 3357 (2) does point toward A. flavus as a distinct species from A. parasiticus but does not change the conclusion of the potential polyphyletic nature of A. flavus based on the position of A. oryzae in the analysis (4). All additional conclusions were based on phylogenetic relationships defined within the scope of the manuscript (4) and were therefore independent of species labels.

Selection of Atoxigenic Aspergillus flavus for Potential Use in Aflatoxin Prevention in Shandong Province, China.

Aspergillus flavus is a common filamentous fungus widely present in the soil, air, and in crops. This facultative pathogen of both animals and plants produces aflatoxins, a group of mycotoxins with strong teratogenic and carcinogenic properties. Peanuts are highly susceptible to aflatoxin contamination and consumption of contaminated peanuts poses serious threats to the health of humans and domestic animals. Currently, the competitive displacement of aflatoxin-producers from agricultural environments by atoxigenic A. flavus is the most effective method of preventing crop aflatoxin contamination. In the current study, 47 isolates of A. flavus collected from peanut samples originating in Shandong Province were characterized with molecular methods and for aflatoxin-producing ability in laboratory studies. Isolates PA04 and PA10 were found to be atoxigenic members of the L strains morphotype. When co-inoculated with A. flavus NRRL3357 at ratios of 1:10, 1:1, and 10:1 (PA04/PA10: NRRL3357), both atoxigenic strains were able to reduce aflatoxin B1 (AFB1) levels, on both culture media and peanut kernels, by up to 90%. The extent to which atoxigenic strains reduced contamination was correlated with the inoculation ratio. Abilities to compete of PA04 and PA10 were also independently verified against local aflatoxin-producer PA37. The results suggest that the two identified atoxigenic strains are good candidates for active ingredients of biocontrol products for the prevention of aflatoxin contamination of peanuts in Shandong Province.

Chromosome assembled and annotated genome sequence of Aspergillus flavus NRRL 3357.

Aspergillus flavus is an opportunistic pathogen of crops, including peanuts and maize, and is the second leading cause of aspergillosis in immunocompromised patients. A. flavus is also a major producer of the mycotoxin, aflatoxin, a potent carcinogen, which results in significant crop losses annually. The A. flavus isolate NRRL 3357 was originally isolated from peanut and has been used as a model organism for understanding the regulation and production of secondary metabolites, such as aflatoxin. A draft genome of NRRL 3357 was previously constructed, enabling the development of molecular tools and for understanding population biology of this particular species. Here, we describe an updated, near complete, telomere-to-telomere assembly and re-annotation of the eight chromosomes of A. flavus NRRL 3357 genome, accomplished via long-read PacBio and Oxford Nanopore technologies combined with Illumina short-read sequencing. A total of 13,715 protein-coding genes were predicted. Using RNA-seq data, a significant improvement was achieved in predicted 5’ and 3’ untranslated regions, which were incorporated into the new gene models.

A Novel Site-Specific Integration System for Genetic Modification of Aspergillus flavus.

Aspergillus flavus is a fungus that produces aflatoxin B1, one of the most carcinogenic secondary metabolites. Understanding the regulation mechanism of aflatoxin biosynthesis in this fungus requires precise methods for genomic integration of mutant alleles. To avoid the disadvantage of DNA integration into the genome by non-homologous or ectopic recombination, we developed a novel strategy for site-specific integration of foreign DNA by using a carboxin-resistant sdh2R allele (His 249 Leu). Our results demonstrated that the transformants were generated with a high efficiency (>96%) of correct integration into the sdh2-lcus of the genome of A. flavus NRRL 3357. The advantage of this method is that introduction of the eGFP expression cassette into the sdh2-locus had little effect on fungal growth and virulence while also being rapid and efficient. This system will be a valuable tool for genetic manipulation in A. flavus. To the best of our knowledge, this is the first report on the efficient site-specific integration at the sdh2-locus in the genome of Aspergillus.

English

Seafood is part of a healthful diet, but seafood consumption is not risk-free because of sea contaminations by anthropogenic activities and environmental factors. Their microbiological quality is of major concern to the food processors, consumers and public health authorities hence the need for proper assessment of the microbial quality. This study therefore characterizes pathogenic microorganisms isolated from fresh sea snail and shrimp in Ugbo nla, Igbokoda, Ondo State, Nigeria. Isolation and molecular identification of microorganisms from snail and shrimp using 16sRNA and 18sRNA gene sequence for bacteria and fungi respectively, effects of salt concentration and temperature on isolated microorganisms were carried out using standard microbiological methods. Molecular identification revealed the presence different pathogens namely; Proteus mirabilis LZPM-03, Pantoea agglomerans BJCP2, Pseudomonas aeruginosa R8-589, Klebsiella pneumoniae 2N3, Serratia marcescens, Alcaligenes faecalis ACBC1, Yersinia. pseudotubercules YPIII, Escherichia coli 2A, Lysinibacillus sphaericus C3-41, Listeria monocytogen J1776, Staphylococcus aureus and Proteus mirabilis LCX20, Fusarium proliferatum FproStRINI, Aspergillus flavus NRRL3357, Aspergillus oryzae RIB40 and Aspergillus peniciliodes. Also, 4% salt concentration at 45°C effectively inhibited the pathogens isolated from seafood after 18 h. The presence of pathogenic microorganisms in the sea foods could make them a potential health-risk when consumed without proper cooking. Salting, a traditional technique could be used to eliminate these pathogens before consumption.   Key words: Seafood, molecular identification and salt concentration. &nbsp

Comparison of aflatoxin production of Aspergillus flavus at different temperatures and media: Proteome analysis based on TMT

Aflatoxin production of Aspergillus flavus is affected by abiotic factors such as temperature, water activity, oxidative stress, etc. These factors likely affect different metabolic pathways and result in altered aflatoxin production. Aflatoxin was determined in liquid media at 28 °C, solid media at 28 °C and solid media at 37 °C. The proteomic method was used to elucidate the mechanism of aflatoxin production in A. flavus in liquid media at 28 °C, solid media at 28 °C and solid media at 37 °C. Potential factors affecting aflatoxin production were found by GO and KEGG analysis. A. flavus produces more aflatoxin at 28 °C compared to 37 °C. Our study also found that A. flavus cultured on solid media produced more aflatoxin than in liquid media. In this study, we identified 5029 proteins from A. flavus NRRL3357, in which 1547 differential proteins were identified between liquid media and solid-state media, while 546 differential proteins were identified between 28 °C and 37 °C. Biological informatics analysis showed that these differential proteins were widely involved in a variety of biological processes, molecular functions, and cellular components, and were associated with multiple metabolic pathways. Compared to the liquid media, extracellular hydrolase for nutrient uptake and proteins related to sclerotia development were differentially expressed on solid media (p < 0.05). Enzymes involved in oxidative stress showed significantly down-regulated in liquid media and up-regulated at 28 °C (p < 0.05). Furthermore, our research also revealed aflatoxin synthesis is a complex process that is affected by a variety of factors such as nutrient uptake, oxidative stress, sclerotia development, G protein signaling pathways and valine, leucine and isoleucine degradation, and a speculative model summarizing the regulation of aflatoxin biosynthesis in A. flavus is presented.