Aspergillus flavus

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1. Classification

a. Higher order taxa

Eukaryota; Ascomycota; Eurotiomycetes; Eurotiales; Trichocomaceae

b. Species

Aspergillus flavus

2. Description and significance

Aspergillus flavus is a pathogenic fungus in the phylum Ascomycota. This species is known primarily for its ability to produce a potent toxin and carcinogen known as aflatoxin (1). Aflatoxin is known to contaminate many types of crop seeds, but in the field it is predominantly problematic for maize, peanuts, cotton seed, and tree nuts (1). A. flavus also has a great impact on human health, in which immunosuppressed people are most susceptible to infection by this fungus (1). A. flavus may be found in any type of climate, but it is most common in warm temperate zones and environments with low water levels and higher temperatures (1).

3. Genome structure

The genus Aspergillus consists of 250 recognizable species. These species are further divided into different groups (2). A. flavus is part of the Flavi group, which is notable for its aflatoxin-producing fungi. This section also includes A. parasiticus. The A. flavus genome consists of 37 million base pairs arranged into eight different chromosomes (4). It was found that the genome contains 13,485 predicted protein coding regions, including several secondary metabolite biosynthetic gene clusters (3). Further research on the secondary metabolite biosynthetic gene clusters of this species revealed that there were more than 56 biosynthetic gene clusters (4). The secondary metabolite biosynthetic gene clusters responsible for the production of aflatoxin consists of 25 genes, spanning 70 kb DNA sections located near the telomere of the third chromosome (4).

4. Cell structure

The microscopic characteristics of A. flavus align with the other organisms in the Aspergillus genus. The hyphae of A. flavus are partitioned by a septum and are hyaline, giving them a glossy appearance (2). The organism is a circular vesicle, with protruding filamentous extensions (2). In cell cultures, A. flavus are known to grow as yellow-green colonies and are 65-70 mm in diameter on Czapek yeast extract (1). As the spores mature, they transition into a darker green color (1).

5. Metabolic processes

A. flavus grows and produces aflatoxin in the presence of oil-rich agricultural crops including maize, peanuts, and cotton seeds. The biosynthesis of aflatoxin involves a regulatory mechanism mediated by pathway-specific genes aflR and aflS. The aflR gene expresses the DNA binding promoter AflR (4). Expression of aflatoxin is upregulated when AflR is bound to the promoter of the aflatoxin gene (4). This binding is not required for aflatoxin production, however. Aflatoxin will still be expressed in low levels without any AflR present in the cell (4). Moreover, AflS, a regulatory protein expressed by aflS, takes an important role in aflatoxin biosynthesis by acting as a transcriptional enhancer or co-activator of AflR (4). The exact role of AflS is still unknown, but recent research has shown that AflS was required to properly transport AflR to and from the nucleus (4).

Additional gene clusters have been shown to regulate the production of aflatoxin, one of which is veA (5). A deletion of this gene causes a down regulation of aflatoxin, in addition to the prevention of the formation of sclerotia, a part of the cell membrane that helps the fungal cell to survive environmental extremes for long periods (5). Since veA regulates both aflatoxin and sclerotia, it is presumed that the protein encoded by this gene is involved in controlling both processes, but the exact mechanism of how veA regulates these is still unknown (5). In addition to veA, laeA has also been shown to be required for aflatoxin and sclerotial formation (6). LaeA is shown to be involved in the conversion of heterochromatin, where gene expression is suppressed, to euchromatin, where gene expression is promoted (5). Both veA and laeA are known to be global regulators since they regulate several gene clusters in the cell (4). VeA, LaeA, and VelB form a heterotrimer, known as the Velvet Complex, in the nucleus to control fungal development and secondary metabolite production (4).

6. Ecology

A. flavus inhabits a broad range of environments. The prevalence of A. flavus has led some researchers to deem it ubiquitous, as scientists have found species of Aspergillus every time they sought to look for them (7). This claim was supported by a meta-analysis that revealed that A. flavus was found at all latitudes studied. Despite the capacity to colonize a wide range of environments, A. flavus grows more readily in warm climates and thrives in tropical latitudes (7). Specifically, A. flavus responds best to temperatures between 36 and 38 degrees Celsius. Additionally, A. flavus flourishes in drier environments, and is also represented in desert regions (7).

A. flavus is similarly diverse in the ecological roles it fulfills within an environment. It is primarily a saprophyte in soil, and acts to recycle nutrients (7). A. flavus is also an opportunistic parasite capable of infesting a wide range of hosts. Although it has been documented within avian and mammalian hosts, A. flavus is primarily a parasite that affects plants, some of which include a large number of economically important crops (7).

7. Pathology

A. flavus infections had a sizable impact on peanut and corn crops in the southeast region of the United States in the 1970’s, and were responsible for the yellow-spot diseases on cotton balls (8). States reliant on agriculture, such as Virginia, Arizona, California, and North Carolina were all affected by these infections. Contamination by A. flavus is detrimental to crops because of A. flavus’ ability to produce Aflatoxin B1 (AFB1), a toxin that can cause liver damage in both human and animals (8).

The contamination caused by A. flavus can occur at numerous points during the process of planting, harvesting, and postharvesting, although specific crops and environmental conditions can make contamination more likely (8). A. flavus spores remain in soil and can be spread by insect carriers. Although A.flavus does not propagate in the air often, a form of conidia is often carried by host insects onto damaged plants where further infection takes place (8). Kernels and fruit which have been damaged by insects can be easily infected by A.flavus. The infected parts of plants promote the formation of sclerotia, which returns to soil during harvest. This in turn leads to the production of a new generation of spores (8). Plant and insect fragments are also sites of inoculation during postharvest, which further help A.flavus survive throughout the winter and results in recurring infection. Regions near rye plantations often have more propagules relative to other plantations. Crops subject to water deprivation before harvest exhibit increased rates of A. flavus contamination due to increased germination and proliferation. Drought conditions are also correlated with infection by A. flavus, although infection was not observed at lower growing temperatures, and was far less common in undamaged seeds and kernels (8).

Two strains of A. flavus, L and S were isolated from cotton seeds in Arizona due to the significant agricultural impacts arising from A.flavus infections (9). The sizes of two strains' sclerotia, 150 -250 micrometers for the S strain, and 400 micrometers for the L strain, depends on the given temperature and types of agar in vitro cultures (9). The S strain is capable of growth under a 14 degrees Celsius temperature change, and the L strain exhibits half of the original sclerotia production rate at 25 degrees Celsius. In in vivo cultures, the L strain has a greater infection rate than the S strain (8). Production of aflatoxins B1 and B2 occurs primarily in plants, especially B1 within cotton seeds and corn kernels (9). Aflatoxin G1 and G2 were mainly produced on peanut plants (9).

8. Current Research

Much of the current research into A. flavus focuses on preventing crop damage and other diseases caused by the fungus. In one set of experiments, researchers sought to minimize harm caused by A. flavus aflatoxin production by introducing non-toxic strains of the organism (10) These atoxic strains isolated from Arizona soil were introduced to both corn kernels meant for planting before the growing season and the harvested crop. As a control, some plants were left untreated while others were inoculated with both toxigenic and atoxic strains (10). These treatments significantly reduced the concentration of aflatoxin in any cases where an atoxigenic strain was introduced (10).

Another study explored the effects of essential oils derived from Chenopodium ambrosioides on A. flavus and other fungal populations. C. ambrosioides was chosen as a counteragent for the fungi because of its prevalence as a weed throughout India and elsewhere (11). Treatment with these oils over a period of seven days at a concentration of 100 micrograms per milliliter inhibited the growth of all specimens studied (11). Additionally, levels of aflatoxin in treated crops dropped significantly. Furthermore, treatment with C. ambrosioides prevented fungus-related spoilage in wheat crops for up to a year (11).

9. References

1. Klich, M. A. "Aspergillus flavus: the major producer of aflatoxin". Molecular Plant Pathology. 2007. Volume 8. p. 713–722.

2. Bowman S.M. and S.J. Free. "The structure and synthesis of the fungal cell wall". Bioessays: Ideas that Push the Boundaries. 2006. Volume 28. p. 799-808.

3. Nierman, W. C., J. Yu, N. D. Fedorova-Abrams, L. Losada, T. E. Cleveland, D. Bhatnagar, J. W. Bennett, R. Dean, and G. A. Payned. "Genome Sequence of Aspergillus flavus NRRL 3357, a Strain That Causes Aflatoxin Contamination of Food and Feed". Genome Announcements. 2015. Volume 3. p. 1-2.

4. Amare, M. G. and N. P. Keller. "Molecular mechanisms of Aspergillus flavus secondary metabolism and development". Fungal Genetics and Biology. 2014. Volume 66. p. 11–18.

5. Duran, R.M., J. W. Cary, and A. M. Calvo. "Production of cyclopiazonic acid, aflatrem, and aflatoxin by Aspergillus flavus is regulated by veA, a gene necessary for sclerotial formation". Applied Microbiology Biotechnology. 2007. Volume 73. p. 1158–1168.

6. Kale, S. P., L. Milde, M. K. Trapp, J. C. Frisvad, N. P. Keller, and J. W. Bok. "Requirement of LaeA for secondary metabolism and sclerotial production in Aspergillus flavus". Fungal Genetics and Biology. 2008. Volume 45. p. 1422–1429.

7. Scheidegger, K.A., and Payne, G. A. "Unlocking the Secrets Behind Secondary Metabolism: A Review of Aspergillus flavus from Pathogenicity to Functional Genomics". Journal of Toxicology: Toxin Reviews. 2003. Volume 22. p. 423-459.

8. Diener, U., Cole, R., Sanders, T., Payne, G., Lee, L., & Klich, M. "Epidemiology of Aflatoxin Formation by Aspergillus flavus". Annual Review of Phytopathology. 1987. Volume 25. p. 249-270.

9. Cotty, P.J. "Virulence and cultural characteristics of two Aspergillus flavus strains pathogenic on cotton". Phytopathology. 1989. Volume 79. p. 808-814.

10. Brown R.L., Cotty P.J., and Cleveland T.E. "Reduction in Aflatoxin Content of Maize by Atoxigenic Strains of Aspergillus flavus". Journal of Food Protection. 1991. Volume 54. p. 623-626.

11. Kumar R., Mishra A. K., Dubey N.K., and Tripathi Y.B. "Evaluation of Chenopodium ambrosioides oil as a potential source of antifungal, antiaflatoxigenic and antioxidant activity". International Journal of Food Microbiology. 2007. Volume 115. p. 159–164.

Edited by [Eric Cui, Jun Bai Park Chang, Jordan Newman, and Meiheng Liang], students of Jennifer Talbot for BI 311 General Microbiology, 2017, Boston University.