Progress 12/17/14 to 09/30/17
Outputs
Target Audience:Scientists working on the biology and ecology of the aflatoxin-producing fungus, Aspergillus flavus, and others interested in biology of mycotoxins. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In this reporting period, one graduate student, one undergraduate student and a half-time technician contributed to this project. How have the results been disseminated to communities of interest?Dissemination has been primarily through a recent publication in the Proceedings of Royal Society B-Biological Sciences. This publication generated broader interest from the scientific community. Here are links to two news articles about this work: Science http://www.sciencemag.org/news/2017/12/bugs-may-be-causing-common-crop-mold-produce-deadly-toxin Phys.org https://m.phys.org/news/2017-12-dangerous-crop-fungus-toxic-chemical.html What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The overall goal of this project was to understand how fungi that produce aflatoxin benefit from producing it. Humans think of aflatoxin as a serious contaminant of moldy food, which can have severe health effects when ingested by humans or livestock. Aflatoxin can cause suppressed immunity, stunted growth, liver cancer and death from acute exposures. However, damage to humans/livestock does not explain how fungi that produce aflatoxin benefit from its production. In this study, we have shown that aflatoxin production benefits the fungus Aspergillus flavus when it interacts with insects, but is detrimental to the fungus when it interacts with microbes in soil. These positive and negative effects on the growth of A. flavus help to explain why strains of A. flavus that do not produce aflatoxin can be found in approximately 30% of all strains isolated from nature. Strains that do not produce aflatoxin are being exploited for biological control of aflatoxin in some places, which is the reason we wanted to learn more about their biology and ecology. We also found that A. flavus produces greater quantities of aflatoxin when insects are present than when they are absent, and when the fungus is mechanically disturbed to simulate insect feeding. This latter results suggests that insect control during storage of grains such as corn might be an essential component of aflatoxin management. Objective 1: The goal for this objective was to test whether aflatoxin would favor the growth and survival of A. flavus in some environments, and be detrimental in others. We originally hypothesized that aflatoxin would be beneficial in the saprotrophic phase of its lifecycle in soil, but detrimental while it was interacting with plants. Because of difficulties working with A. flavus and aflatoxin production in corn kernels (as originally proposed) we shifted emphasis to looking at how aflatoxin affects the interaction of A. flavus with insects. We found that aflatoxin reduces the growth and feeding of some insects (Drosophila melanogaster larvae), and allows the fungus to utilize nutrient resources better. Therefore, when the fungus produces aflatoxin, it can grow and reproduce at higher rates than strains of fungus that do not produce aflatoxin. Furthermore, the fungus produces significantly more aflatoxin when it interacts with insects than with no insects present. This effect was confirmed by mechanically simulating feeding by repeatedly stabbing the fungal tissues with a sterile toothpick. Mechanical damage stimulated even greater aflatoxin production than feeding by fruit fly larvae. We repeated similar experiments with maize weevils (Sitophilus zeamais) and found that they are not sensitive to aflatoxin. Therefore, weevils were not studied further. In contrast to insects, strains of fungus that produce aflatoxin (aflatoxigenic) grow and reproduce more slowly than strains that do not produce aflatoxin (non-aflatoxigenic) when they are present in soil. This result was unexpected since we were testing the hypothesis that aflatoxin would benefit the fungus in soil by reducing antagonistic interactions with the soil microbial community (see Objective 2 below). We speculate that there are metabolic costs to producing aflatoxin that reduce growth relative to fungi that do not produce it. The significance of results lies in providing a plausible explanation for the balancing selection that maintains both aflatoxigenic and non-aflatoxigenic strains in most populations in the US. Objective 2: Because A. flavus is thought to spend much of its lifecycle living in soil, we wanted to determine if aflatoxin would affect the fitness of the fungus by reducing antagonistic interactions with soil microbes. We used a metagenomics approach in which we incubated strains of aflatoxigenic and non-aflatoxigenic strains in soil, sampled the total DNA from the soil, then amplified bacterial 16S genes and fungal ITS sequences for next-generation sequencing. The microbial community composition determined by 16S and ITS sequences were nearly identical between the two treatments. Therefore, contrary to expectations, aflatoxin appears to have no effect on microbial communities in soil, and does not benefit the fungus in the soil environment.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Drott, M.T., Lazzaro, B.P., Brown, D.L., Carbone, I., Milgroom, M.G. 2018. Balancing selection for aflatoxin in Aspergillus flavus is maintained through interference competition with, and fungivory by insects. Proc. R. Soc. B (in press).
DOI: 10.1098/rspb.2017.2408
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Scientists working on the biology and ecology of the aflatoxin-producing fungus, Aspergillus flavus, and others interested in biology of mycotoxins. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In this reporting period, one undergraduate student and a half-time technician contributed to this project. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?The major focus for the next reporting period is to follow up on on-going experiments described in "Accomplishments". We are continuing to collect and analyze data on these projects. We also plan to begin writing publications based on these results, as soon as they are available.
Impacts
What was accomplished under these goals?
We have continued to optimize methods since this is a new experimental system in the PD's lab. Progress this year was made on optimizing environmental conditions and determining growth curves for the fungus, Aspergillus flavus, in microcosm experiments in soil and maize kernels for Objectives 1 and 2. Experiments were conducted under environmental conditions of high stress (hot and dry, 40 C) and optimal conditions (cool and moist, 30 C) for fungal growth, and in sterile and natural soils. An important finding from these experiments is that A. flavus growth is very high at 30 C/moist conditions in sterile soil. In contrast, growth is almost completely inhibited at these same environmental conditions in natural soils--the only difference is whether soil was sterilized or not. Growth in in sterile soil under stressful (40 C/dry) conditions was much less than at optimal conditions in sterile soil. In contrast, fungal growth was significantly greater in natural soils under hot/dry conditions than cool/moist conditions. No differences were observed, however, between strains that produce aflatoxin and those that are nontoxigenic. Our hypothesis is that microbial populations in the soil suppress A. flavus growth, and therefore, as discussed below, we are investigating whether aflatoxin has an antagonistic effect on microbial populations. For Objective 2 an experiment is still in progess consisting of 54 soil microcosms, varying three factors: environment (cool/moist, hot/dry), aflatoxin concentrations (0 ppb, 500 ppb, 2000 ppb) added to the soil, and fungal strains (toxigenic, nontoxigenic, or no fungus). In this experiment we are interested in comparing fungal fitness with and without aflatoxin in the presence of a natural soil microbial community.Fungal growth is quantified by quantitative PCR (qPCR). To test whether aflatoxin affects microbial communities, we have extracted total DNA from the soil microcosms after incubation with A. flavus. DNAs will be sequenced for 16S bacterial genes and ITS fungal genes. These sequences will allow us to detect any shift in the microbial community. We also hope that results from this experiment will allow us to identify candidate microbes that may be antagonistic to A. flavus. Future studies could then culture these microbe for further study. We have also started aflatoxin extractions from the soil samples to quantify additional aflatoxin produced by the toxigenic strains of the fungus to also correlate to the 16S data.Results are not yet ready to report on this experiment. However, from this experiment we will also be able to test the hypothesis whether aflatoxin increases the fitness of A. flavus in the presence of antagonistic microbes. Preliminary results on this hypothesis suggest that aflatoxin added to the soil does increase the growth of nontoxigenic strains. More data and analysis are needed before we can make any definite claims in this regard.
Publications
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Progress 12/17/14 to 09/30/15
Outputs
Target Audience:Scientists working on the biology and ecology of the aflatoxin-producing fungus, Aspergillus flavus, and others interested in biology of mycotoxins. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?So far, one PhD student and two undergraduate students have contributed to this project. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?The next steps for Objective 1 will be run the experiments under different environmental conditions to determine the fitness of toxigenic and nontoxigenic A. flavus isolates in microcosms. We will also sequence 16S sequences amplified from soil when incubated with toxigenic and nontoxigenic isolates of A. flavus to look for differences in microbial communities.
Impacts
What was accomplished under these goals?
The accomplishments in the first year of this project are mainly in the optimization of methods. In particular, for Objective 1 we have optimized environmental conditions for microcosm experiments in soil and maize kernels. These conditions were optimized for temperature, moisture, and nutrients added (for soil). We also conducted pilot studies to determine the time course for the growth of Aspergillus flavus in order to optimize the timing of the experiments. We also developed methods for the extraction and purification of total DNA from soil and maize kernels and quantitative PCR methods for estimating the total amount of fungal biomass accumulating in microcosms. In addition, we are working on methods to quantify aflatoxin accumulation in microcosms. For Objective 2 we have extracted total DNA from soil and are currently developing PCR primers specific for amplifying 16S genes from Bacillus species, which are the targeted microbes in this study. We have conducted some pilot runs with toxigenic and nontoxigenic isolates of A. flavus but have not yet submitted samples for Illumina sequencing.
Publications
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