Progress 02/15/19 to 02/14/20
Outputs
Target Audience:The target audience has been primarily the scientific community working on mycotoxins and microbial ecology.The Postdoctoral Associate (Milton Drott) supported on this project presented a poster at the Fungal Genetic Conference at Asilomar, Pacific Grove California in March 2019. No other outreach was done for this project. Changes/Problems:As described in accomplishments for Objective 3, we did not find any differences in the survival of sclerotia from aflatoxigenic and non-aflatoxigenic isolates. Because of the laborious nature of this study, we have abandoned this objective. We tried to obtain CRISPR mutants of non-aflatoxigenic isolates of A. flavus. We did not succeed, but also gave up because of problems associated with non-target mutations induced by CRISPR methods. What opportunities for training and professional development has the project provided?This project funded a full-time PhD candidate through to August 31, 2019. How have the results been disseminated to communities of interest?The postdoctoral researcher (Milton Drott) supported on this project presented a poster on the results of the Added Objective at the Fungal Genetics Conference at Asilomar, Pacific Grove, CA, in March 2019. We submitted a paper on population genomics (Added Objective) for publication in mBio. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1: Nothing to report since last year. Objective 2: Nothing to report since last year. Objective 3: We tested the viability of a subset of sclerotia (dormant structures of A. flavus) that were buried in the field and recovered one year later. Overall survival, as indicated by germination, was extremely low (3%). This finding is lower than expected but consistent with some reports of sclerotial survival under adverse environmental conditions. As overall germination rates were too low to be informative of fitness differences between aflatoxigenic and non-aflatoxigenic isolates, we assessed the overall damage to sclerotia under the assumption that more damaged sclerotia would reflect lower fitness. We scored approximately 700 sclerotia representing 29 of 144 replicates buried in the field (inside metal tea balls) and 11 of 22 isolates (6 aflatoxigenic and 5 non-aflatoxigenic isolates). Processing the sclerotia in this way requires physical assessment of each single sclerotium and is thus extremely laborious. As we put exactly 30 sclerotia into each tea ball, we interpreted missing sclerotia as completely destroyed. However, we also analyzed data using the actual number of recovered sclerotia. No significant differences were found from either data set in the fitness of aflatoxigenic and non-aflatoxigenic isolates. Although we only processed about 20% of the sclerotia we concluded it is unlikely that further observations will show different results. Therefore, we stopped these assessments because of the inordinate investment of labor it would require. We conclude that if aflatoxin has an impact on the survival of sclerotia under field conditions it is very small, or may not be detectable in the experimental conditions we used. Objective 4: Nothing to report since last year. Added Objective: Two years ago we proposed to conduct a population genetics analysis of A. flavus in the U.S. after we abandoned Objective 2. Last year we reported findings based on microsatellite genotyping of a large sample of A. flavus from two north-south transects in the U.S. We pursued this objective further in the past year by performing whole-genome sequencing on a subset of 94 isolates used in our initial microsatellite study. With the higher resolution added by this data, we found that the overall population is subdivided into three genetically differentiated populations (A, B, and C) that differ greatly in their extent of recombination, diversity, and aflatoxin-producing ability. Estimates of the number of recombination events and linkage disequilibrium decay suggest relatively frequent sex only in population A. Population B is sympatric with population A but produces significantly less aflatoxin and is the only population where the inability of non-aflatoxigenic isolates to produce aflatoxin was explained by multiple gene deletions. Population expansion evident in B suggests a recent introduction or range expansion. Population C is largely non-aflatoxigenic and restricted mainly to northern sampling locations through restricted migration and/or selection. Despite differences in the number and types of mutations in the aflatoxin gene cluster, codon optimization and site-frequency differences in synonymous and nonsynonymous mutations suggest that low levels of recombination in some A. flavus populations are sufficient to purge deleterious mutations. The finding of population C raises suggests that some populations are in fact northern and less aflatoxigenic. While we previously showed that the frequency of aflatoxin-producing A. flavus isolates has no association with latitude, the presence of a largely non-aflatoxigenic population in the north, where A. flavus population density is remarkably low, raises questions about the potential role of aflatoxin in latitude-associated adaptation.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2020
Citation:
Drott M.T., Satterlee T. R., Skerker J. M., Pfannenstiel B. T. ,Glass N. L. , Keller N. P., Milgroom M. G. 2020. The frequency of sex inferred from recombination and population structure using population genomics of the aflatoxin- producing fungus Aspergillus flavus. mBio
|
Progress 02/15/16 to 02/14/20
Outputs
Target Audience:The target audience has been primarily the scientific community working on mycotoxins and microbial ecology. Changes/Problems:As in most research projects, we had to adjust our objectives as we got results. In this project, we found that the original Objective 2 was unrealistic because we could not detect any differences in fitness in aflatoxigenic and non-aflatoxigenic isolates of A. flavus growing in maize kernels, even though we could detect differences in the same isolates in soil or in the presence of insects. (The rationale for dropping this objective is given in the Accomplishments section.) Similarly, we could not detect any differences in fitness in aflatoxigenic and non-aflatoxigenic isolates of A. flavus based on survival of their sclerotia buried in field soil for one year. Overall survival was very low, much lower than expected from pilot studies, making it unrealistic to put additional time and resources into this objective. Because of the difficulties with Objectives 2 and 3, we substituted an "Additional Objective", as reported in Accomplishments. We instead conducted a study on the population genetics and population genomics of A. flavus in the U.S. What opportunities for training and professional development has the project provided?The most significant training and development in this project was in supporting a PhD student (Milton Drott) for 2.5 years. After finishing his PhD, he continued this project as a postdoctoral associate at the University of Wisconsin-Madison on a subaward to Dr. Nancy Keller. We also trained several undergraduate students on this project in many different aspects of microbiology, fungal biology and molecular biology. One was a summer intern who concentrated on studies with insect/fungus interactions--he later went on to a PhD program in ecology at Stanford University. How have the results been disseminated to communities of interest?The target audience has been primarily the scientific community working on mycotoxins and microbial ecology. To this end, the PD attended two NIFA project directors' meetings in Washington DC in June 2016 and December 2017 where he presented posters as progress reports. In addition, the graduate student and later postdoctoral associate (Milton Drott) supported on this project presented oral talks and posters at the International Congress of Plant Pathology in Boston in August 2018, and at the Fungal Genetics Conference at Asilomar, Pacific Grove, CA, in March 2019. Results for Objective 1 were published in mBio in February 2019. Results for Objective 4 were published in Proceedings of the Royal Society of London in December 2017. Results for the Added Objective were published in Phytopathology in April 2019; the population genomics follow up to this objective are currently under review at mBio. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Accomplishments for Objective 1: We tested the role of aflatoxin production on the fitness of A. flavus in both artificial media and in soil. We did not observe any differences in fitness on artificial media. This result is consistent with other systems in which fitness effects of environmental stressors are only observable in nutrient-poor conditions like soils. Therefore, we tested the growth of aflatoxigenic and non-aflatoxigenic isolates of A. flavus under sub-optimal, optimal, and stressful temperatures, in both sterile and non-sterile soil. Interaction between temperature and soil community (sterile and non-sterile) differentially affected growth of aflatoxigenic and non-aflatoxigenic isolates as follows. First, we demonstrated that while the fungus grows similarly in sterile soils at the lower two temperatures, it achieves only about one fifth the growth at suboptimal temperatures as it does at optimal temperature in non-sterile soil. This suggests a suppressive effect of soil microbial communities on A. flavus. Second, at optimal temperatures, the production of aflatoxin resulted in decreased fitness of A. flavus when soil microbes are present, but not in sterile soils. This is contrary to expected results that aflatoxin would benefit A. flavus by inhibiting soil microbes that compete for resources. Neither aflatoxigenic isolates nor addition of pure aflatoxin to soil had any effect on the composition of soil microbial communities as determined by metagenomic sequencing. We did not see any difference in fitness between aflatoxigenic and non-aflatoxigenic isolates in sterile soil. We speculate that the metabolic costs of producing aflatoxin are great enough to reduce the fitness of A. flavus in natural soils where competition for nutrients is strong. Accomplishments for Objective 2: This objective was aimed at finding differences in fitness between aflatoxigenic and non-aflatoxigenic isolates of A. flavus when grown in maize kernels. However, we did not observe any differences in fitness, even though we could detect differences in more stressful environments, as described in Accomplishments for Objectives 1 and 4. Results from this objective are consistent with those of Objective 1 in the fact that maize kernels are nutrient-rich substrates. Together these results suggest that aflatoxin production has no effect on fitness when A. flavus colonizes maize kernels--the "pathogenic" phase we tested--because they are nutrient-rich, whereas we did detect a fitness cost of aflatoxin production using the same fungal isolates in the saprophytic phase in nutrient-poor soil. Accomplishments for Objective 3: For this objective, we buried sclerotia (dormant structures) of A. flavus in soil in the field inside metal mesh tea balls. We recovered tea balls a year later and tested the viability of a subset of the sclerotia in them. Overall survival, as indicated by germination, was extremely low (3%). As overall germination rates were too low to be informative of fitness differences between aflatoxigenic and non-aflatoxigenic isolates, we assessed the overall damage to sclerotia under the assumption that more damaged sclerotia would reflect lower fitness. We scored approximately 700 sclerotia from 6 aflatoxigenic and 5 non-aflatoxigenic isolates. No significant differences were found in the fitness of aflatoxigenic and non-aflatoxigenic isolates. Although we only processed about a fifth of the sclerotia overall, we concluded it is unlikely that further observations will show different results. Therefore, we stopped these assessments because of the inordinate investment of labor it would have required. We concluded that if aflatoxin has an impact on the survival of sclerotia under field conditions it is very small, or may not be detectable in the experimental conditions we used. Accomplishments for Objective 4: We demonstrated that the addition of aflatoxin to culture medium dramatically increased the fitness of A. flavus in presence of Drosophila melanogaster larvae. We observed a 26-fold increase in fungal biomass when aflatoxin was added. No differences in fitness were observed due to added aflatoxin when larvae were not present. We observed small but significant differences in fitness between aflatoxigenic and non-aflatoxigenic isolates when larvae were present. Greater fitness of aflatoxigenic isolates was consistent with the effects of added aflatoxin. In addition to demonstrating a fitness advantage of aflatoxin production to the fungus, we also showed that aflatoxin production was induced both by insect feeding and by physical wounding of fungal mycelium that simulated feeding. This last finding raises the possibility that aflatoxin production represents an induced resistance mechanism against insects. Altogether, our results suggest that aflatoxin may benefit the fungus in the presence of insects, but may be costly when insects are absent and resources are scarce. Accomplishments for Added Objective: We added an objective in 2018 when it was clear that we had to abandon Objective 2 (see above). We conducted a population genetics analysis of A. flavus in the U.S. in which we conducted microsatellite genotyping of a large sample of A. flavus from two north-south transects in the U.S. We pursued this objective further by performing whole-genome sequencing on a subset of 94 isolates used in our initial microsatellite study. Contrary to previous claims in the literature, we did not find that aflatoxigenic isolates were more prevalent in southern states, however, this interpretation is subject to determining an underlying population structure that was not previously known. With the high resolution added by genome sequencing, we found that the overall population is subdivided into three genetically differentiated populations (A, B, and C) that differ greatly in their extent of recombination, diversity, and aflatoxin-producing ability. Estimates of the number of recombination events and linkage disequilibrium decay suggest relatively frequent sex only in population A. Population B is sympatric with population A but produces significantly less aflatoxin and is the only population where the inability of non-aflatoxigenic isolates to produce aflatoxin was explained by multiple gene deletions. Population expansion evident from molecular signatures in B suggests a recent introduction or range expansion. Population C is largely non-aflatoxigenic and restricted mainly to northern sampling locations. Despite differences in the number and types of mutations in the aflatoxin gene cluster, codon optimization and site-frequency differences in synonymous and nonsynonymous mutations suggest that low levels of recombination in some A. flavus populations are sufficient to purge deleterious mutations. The finding of population C raises suggests that some populations are in fact northern and less aflatoxigenic. While we previously showed that the frequency of aflatoxin-producing A. flavus isolates has no association with latitude, the presence of a largely non-aflatoxigenic population in the north, where A. flavus population density is remarkably low, raises questions about the potential role of aflatoxin in latitude-associated adaptation.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Drott, M. T., Lazzaro, B. P., Brown, D. L., Carbone, I., and Milgroom, M. G. (2017). Balancing selection for aflatoxin in Aspergillus flavus is maintained through interference competition with, and fungivory by insects. Proceedings of the Royal Society B: Biological Sciences 284, 20172408.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Drott, M. T., Debenport, T., Higgins, S. A., Buckley, D. H., and Milgroom, M. G. (2019). Fitness Cost of aflatoxin production in Aspergillus flavus when competing with soil microbes could maintain balancing selection. mBio 10, e02782-02718.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Drott, M. T., Fessler, L. M., and Milgroom, M. G. (2019). Population subdivision and the frequency of aflatoxigenic isolates in Aspergillus flavus in the United States. Phytopathology 109, 878-886.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2020
Citation:
Drott, M. T., Satterlee, T. R., Skerker, J. M., Pfannenstiel, B. T., Glass, N. L., Keller, N. P., and Milgroom, M. G. (2020). The frequency of sex: inferred from recombination and population structure from whole genome sequences of the aflatoxin-producing fungus Aspergillus flavus sampled in the United States. mBio
|
Progress 02/15/18 to 02/14/19
Outputs
Target Audience:The graduate student (Milton Drott) supported on this project presented an oral talk and poster at the International Congress of Plant Pathology in Boston in August 2018. No other outreach was done for this project. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project funded a full-time PhD candidate through to August 30, 2018. His research on this project made up the bulk of his PhD dissertation. He is now continuing this project as a Postdoc at the University of Wisconsin-Madison on a subaward to Dr. Nancy Keller. In 2018, we are also hired one undergraduate student on this project for a semester to help with microsatellite genotyping. How have the results been disseminated to communities of interest?Results for Objective 1 are awaiting publication in mBio; this paper was accepted in January 2019. Results for Objective 4 were published in The Proceedings of the Royal Society B in December 2017. Results for genotyping A. flavus with microsatellites are awaiting publication in Phytopathology; this paper was accepted in November 2018. The graduate student (Milton Drott) supported on this project presented an oral talk and a poster at the International Congress of Plant Pathology in Boston in August 2018. What do you plan to do during the next reporting period to accomplish the goals?This project will continue with a no-cost extension to tie up loose ends and to push forward with plans outlined last year in the section on major changes. As explained in accomplishments, we have already recovered sclerotia that were left out in natural soils for more than a year (Objective 3). These sclerotia will be evaluated for damage and viability in the near future. The other remaining project is to continue with the added objective of creating CRISPR/cas9 mutants that are deficient in aflatoxin production. If we succeed in this objective, we will use replicate isogenic strains to test whether non-aflatoxigenic mutants experience greater fitness than their aflatoxigenic parent strains.
Impacts
What was accomplished under these goals?
Objective 1: Progress in the past year has been in repeating some of the soil microcosm experiments and demonstrating that aflatoxigenic isolates of A. flavus have lower fitness than non-aflatoxigenic isolates in the presence of soil microbial communities. This result is contrary to expected results that aflatoxin would benefit A. flavus by inhibiting soil microbes that compete for resources in the soil. On the contrary, neither aflatoxigenic isolates nor addition of pure aflatoxin to soil had any effect on the composition of soil microbial communities. We could not see any difference in fitness between aflatoxigenic and non-aflatoxigenic isolates in sterile soil. We speculate that the metabolic costs of producing aflatoxin are great enough to reduce the fitness of A. flavus in natural soils where competition for nutrients is strong. As reported last year, growth of A. flavus was markedly reduced in the presence of soil microbes, regardless of aflatoxin-producing ability. Objective 2: Nothing to report since last year. Objective 3: Sclerotia of A. flavus have been recovered from the soil and awaiting assessment for damage and viability to test whether aflatoxin production is associated with survival in dormancy. Objective 4: Nothing to report since last year. Added Objective: Last year we proposed to conduct a population genetics analysis of A. flavus in the U.S. because we had to abandon our original Objective 2. The main focus was to test the hypothesis proposed in the literature that aflatoxigenic individuals occur more frequently in more southerly latitudes because aflatoxin production is favored in hotter climates. To test this hypothesis, we sampled A. flavus from soil from corn fields in two north/south transects, one in the East and the other in the Midwest. We found no differences in the frequency of aflatoxigenic isolates with respect to latitude. However, we found that A. flavus population density (number of isolates recovered per gram of soil) was significantly greater in the south than the north in both transects. We also genotyped a large sample of isolates using microsatellite markers and showed that the population is structured into two distinct subpopulations. Both populations contain aflatoxigenic and non-aflatoxigenic isolates at similar frequencies.
Publications
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2019
Citation:
Drott, M.T., Fesler, L.M., and Milgroom, M.G., 2019. Population subdivision and the frequency of aflatoxigenic isolates in Aspergillus flavus in the United States. Phytopathology (accepted for publication) https://doi.org/10.1094/PHYTO-07-18-0263-R
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2019
Citation:
Drott MT, Debenport T, Higgins SA, Buckley DH, Milgroom MG (2019). Fitness cost of aflatoxin production in Aspergillus flavus when competing with soil microbes could maintain balancing selection. mBio (accepted for publication).
|
Progress 02/15/17 to 02/14/18
Outputs
Target Audience:The PD attended the NIFA-AFRI project directors' meeting in Washington DC in December 2017 and presented a poster on the progress to date. No other outreach was done for this project. Changes/Problems:The original proposal was based on comparing aflatoxigenic and non-aflatoxigenic isolates of A. flavus from sexual crosses made in the laboratory to control for the effects on fitness of background genotypes. We have changed this approach after observing small numbers of isolates available and large variances in fitness among these sexual progeny. We are now working with field isolates, in addition to sexual progeny, because these may better represent the types of nontoxigenic strains found in nature. In addition, we are currently attempting to use CRISPR/Cas9 to perform gene editing on A. flavus. Our goal is to recreate the types of mutations found in nature that cause some isolates to be nontoxigenic. This would provide a means of comparing isogenic isolates that differ only in aflatoxin production. We are at preliminary stages of this gene editing work. We recently abandoned Objective 2 as originally proposed. We originally proposed to test the fitness of aflatoxigenic and non-aflatoxigenic isolates of A. flavus in maize kernels. However, we have found that we cannot detect difference in nutrient-rich substrates such as maize kernels (see Accomplishments section). However, we identified environments in which aflatoxigenic isolates are favored by selection (in the presence of insects) and where they are selected against (in natural soil). These latter results are significant for demonstrating possible selective forces that can maintain the persistent polymorphisms observed for aflatoxin production in field populations. As an alternative to the original Objective 2, we propose to conduct a population genetic analysis of A. flavus, focusing on aflatoxin production. Previous surveys hypothesized that aflatoxigenic strains are more prevalent in hotter climates. Our preliminary data do not support this hypothesis, but additional sampling is underway to test this hypothesis with larger sample sizes. What opportunities for training and professional development has the project provided?This project is funding a full-time PhD candidate. His research on this project will make up the bulk of his PhD dissertation. We are also engaging two undergraduate students on this project. One was a summer intern who concentrated on studies with insect/fungus interactions. How have the results been disseminated to communities of interest?Result for Objective 4 were published in The Proceedings of the Royal Society B in December 2017. The PD presented a poster of these results to the NIFA-AFRI project directors' meeting in Washington in December 2017. What do you plan to do during the next reporting period to accomplish the goals?The main strategy for the next reporting period is to continue from our previous results Objectives 1 and 3. Objective 4 is now completed and Objective 2 has become infeasible (as explained in the Accomplishments section). Plans for the next reporting period are listed here by objectives (hypotheses): Objective 1: We will experiment with adding aflatoxin to soil microcosms to see whether exogenous aflatoxin affects fungal fitness. We expect to see some effects if aflatoxin added suppresses resident microbial communities in the soil, which reduce the fitness of A. flavus in microcosms. We also plan to repeat some of the microcosm studies using additional aflatoxigenic and non-aflatoxigenic field isolates. Finally, we will use next-generation sequencing to determine whether the addition of aflatoxin or the presence of aflatoxigenic isolates of A. flavus in soil microcosms affect the bacterial and fungal communities in non-sterile field soils. This will be done with amplicon sequencing of 16S rRNA genes in bacteria and the ITS region of the rRNA genes in fungi. Objective 3: As mentioned in accomplishments, we recovered sclerotia of A. flavus from the field in April 2018 that were buried in soil in early February 2017. We will sort through the soil in the tea balls to recover sclerotia. Each sclerotium, or piece of sclerotium, will be scored for damage by soil invertebrates, surface sterilized and plated on culture medium to test for viability and confirmation of its identity as A. flavus. These data will allow us to test whether sclerotia of toxigenic isolates survive better than nontoxigenic under field conditions.
Impacts
What was accomplished under these goals?
Objective 1: We tested the role of aflatoxin production on the fitness of A. flavus in both synthetic media and in soil. We did not observe any differences in fitness on synthetic media. This result is consistent with other systems in which fitness effects of environmental stressors are only observable in nutrient-poor conditions like soils. Therefore, we tested the growth of aflatoxigenic and non-aflatoxigenic isolates of A. flavus under sub-optimal, optimal, and stressful temperatures (25, 37, 42 °C, respectively), in both sterile and non-sterile soil. This approach has been made possible by two techniques developed in our lab: First, we developed a modified protocol for extracting high-quality DNA from soil. Second, we developed quantitative PCR (qPCR) markers that are specific to A. flavus, even in the presence of complex soil microbial communities that include other Aspergillus species. Interaction between temperature and soil community (sterile and non-sterile) appears to differentially affect growth of aflatoxigenic and non-aflatoxigenic isolates in several ways. First we have demonstrated that while the fungus grows similarly in sterile soils at the lower two temperatures, A. flavus achieves only about one fifth the growth suboptimal temperatures as it does at optimal temperature in non-sterile soil. This suggests a suppressive effect of soil microbial communities on A. flavus that has not been described before. Secondly at optimal temperatures for growth, the production of aflatoxin resulted in decreased fitness of A. flavus when soil microbes are present, but not in sterile soils. This result is contrary to predictions that aflatoxin contributes to the competitive ability of A. flavus in complex microbial communities. However, this result is consistent with the hypothesis that aflatoxin production does incur a fitness cost under some conditions in soil. Objective 2: Similar to results reported for Objective 1, where we did not observe differences in fitness between aflatoxigenic and nonaflatoxigenic isolates growing on nutrient-rich media, we did not observe differences in fitness when A. flavus colonized maize kernels. Furthermore, attempts to compare fungal growth and fitness on surface-sterilized maize kernels has proven difficult because of high variation among isolates in their ability to colonize maize kernels, independent of their ability to produce aflatoxin. Results from these two objectives are consistent in the fact that maize kernels are nutrient-rich substrates. While there is no apparent cost to aflatoxin production in nutrient-rich environments, we did observe a fitness cost in natural soil, which is a nutrient-poor environment where microbial competitors are present. We have furthered our understanding of A. flavus' saprophytic ability by showing that almost no growth occurs in field soils that are not amended with a nutrient source, in this case, with small quantities of ground maize. Together these results suggest that aflatoxin production has no effect on fitness when A. flavus colonizes maize kernels--the "pathogenic" phase we tested--because they are nutrient-rich, whereas we did detect a fitness cost of aflatoxin production in the saprophytic phase in nutrient-poor soil. Objective 3: We buried sclerotia in a North Carolina field in February 2017 and recovered them in April 2018, 14 months later. Sclerotia were harvested from 13 aflatoxigenic and 13 nonaflatoxigenic field isolates sampled from North Carolina. Sclerotia were buried in metal tea balls that had a mesh small enough to contain sclerotia, but would also allow small invertebrates to gain access to sclerotia inside. Although we originally proposed conducting these experiments under greenhouse conditions, a preliminary study proved that it was not feasible to maintain some soil organisms (e.g., mites or insects). These small invertebrates are potentially of great importance for the survival of sclerotia, and, as demonstrated for Objective 4, aflatoxigenic isolates have increased fitness that in the presence of some insects. Sclerotia recovered from the field will be assessed for damage and viability to test whether aflatoxin production is associated with survival in dormancy. Objective 4: This objective has been completed. We demonstrated that the addition of aflatoxin to culture medium dramatically increased the fitness of A. flavus in presence of Drosophila melanogaster larvae. We observed a 26-fold increase in fungal biomass when aflatoxin was added. No differences in fitness were observed due to added aflatoxin when larvae were not present. We observed small but significant differences in fitness between aflatoxigenic and non-aflatoxigenic isolates when larvae were present. Greater fitness of aflatoxigenic isolates was consistent with the effects of added aflatoxin. In addition to demonstrating a fitness advantage of aflatoxin production to the fungus, we also showed that aflatoxin production was induced both by insect feeding and by physical wounding of fungal mycelium that simulated feeding. This last finding raises the possibility that aflatoxin production represents an induced resistance mechanism against insects. Altogether, our results suggest that aflatoxin may benefit the fungus in the presence of insects, but may be costly when insects are absent and resources are scarce. Results of this objective were published in The Proceedings of the Royal Society B.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Drott, M.T., Lazzaro, B.P., Brown, D.L., Carbone, I., Milgroom, M.G. 2017. Balancing selection for aflatoxin in Aspergillus flavus is maintained through interference competition with, and fungivory by insects. Proc. R. Soc. B 284(1869):20172408. DOI: 10.1098/rspb.2017.2408
|
Progress 02/15/16 to 02/14/17
Outputs
Target Audience:The PD attended the NIFA project directors' meeting in Washington DC in June 2016 and presented a poster on the progress to date. No other outreach was done for this project. Changes/Problems:The original proposal was based on comparing toxigenic and nontoxigenic fungal isolates from sexual crosses made in the laboratory to control for the effects on fitness of background genotypes. We have changed this approach after observing small numbers of isolates available and large variances in fitness among these sexual progeny. We are now working with field isolates, in addition to sexual progeny, because these may better represent the types of nontoxigenic strains found in nature. In addition, we are also exploring the possibility of using CRISPR/Cas9 to perform gene editing on A. flavus. Our goal would be to try to recreate the types of mutations found in nature that cause some isolates to be nontoxigenic. This would provide a means of comparing (isogenic) isolates that differ only in aflatoxin production. We are at preliminary stages of this gene editing work. What opportunities for training and professional development has the project provided?This project is funding a full-time PhD candidate. His research on this project will make up the bulk of his PhD dissertation. We are also engaging two undergraduate students on this project. One was a summer intern who concentrated on studies with insect/fungus interactions. 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 main strategy for the next reporting period is to continue from our preliminary results for all objectives. Plans are listed here by objectives (hypotheses): Objectives 1 and 2: Because we are not seeing any obvious differences in fitness between toxigenic and nontoxigenic isolates, we will experiment with adding aflatoxin to soil and maize kernels microcosms to see whether exogenous aflatoxin affects fungal fitness. We expect to see some effects if aflatoxin added suppresses resident microbial communities in the soil, which reduce the fitness of A. flavus in microcosms. We have no specific prediction for maize kernels. An additional plan is to repeat some of the microcosm studies using field isolates that either toxigenic or nontoxigenic, instead of relying solely on differences between sexual progeny from laboratory crosses. Objective 3: The next activity for this objective is to recover the sclerotia from the field that were buried in soil in early February 2017. Although most of this activity will spill over into the following reporting period, we will sort through the soil in the tea balls to recover sclerotia. Each sclerotium, or piece of sclerotium, will be scored for damage by soil invertebrates, surface sterilized and plated on culture medium to test for viability and confirmation of its identity as A. flavus. These data will allow us to test whether sclerotia of toxigenic isolates survive better than nontoxigenic under field conditions. Objective 4: Preliminary results from these experiments with Drosophila and A. flavus have encouraged us to repeat some experiments and increase our sample sizes/replications. However, instead of adding aflatoxin to the culture medium, we will explore only the effects of aflatoxin produced by the isolates under study. As in Objectives 1 and 2, we will also expand this study to include field isolates, not just progeny from laboratory crosses. In addition, we will conduct parallel experiments with maize weevils in maize-kernel microcosms.
Impacts
What was accomplished under these goals?
Accomplishments testing Hypothesis 1: 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 testing hypotheses 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. Our hypothesis is that microbial populations in the soil suppress A. flavus growth. No differences were observed, however, between strains that produce aflatoxin and those that are nontoxigenic. Therefore, we are continuing these experiments with soil microcosms by adding known quantities of aflatoxin and determining whether it affects fungal fitness. These experiments are still in progress. Accomplishments testing Hypothesis 2: This objective is closely linked to testing Hypothesis 1 (see above). Lack of differences between toxigenic and nontoxigenic strains of Aspergillus flavus in maize kernel microcosms mirrors that observed in soil. We have optimized methods for working with maize kernel microcosms by growing A. flavus on different sizes of fragments of maize, from whole kernels alone to small particle sizes in soil. We currently do not have enough data to make any conclusions. So far, we consistently fail to see any differences between toxigenic and nontoxigenic isolates of A. flavus in soil or on kernels. Accomplishments testing Hypothesis 3: Preliminary results on the effects of aflatoxin production on the survival of sclerotia were obtained from a growth chamber study in which sclerotia were buried in field soil kept in bins for approximately one year. This pilot study allowed us to optimize methods of producing and harvesting sclerotia on maize kernels (not on agar medium); for burying and recovering sclerotia from "tea balls"; and scoring the damage of recovered sclerotia. We recovered large percentages of sclerotia after one year (80-90% recovery). Nearly 100% of the sclerotia recovered were viable when surface sterilized and incubated on a nutrient medium, even when they had experienced extensive damage from soil invertebrates. Although we did not see large differences in survival or damage of sclerotia between toxigenic and nontoxigenic isolates we did optimize methods for this study. Because of the limitations of growth chamber conditions being unrepresentative of conditions in nature, we are now repeating this experiment under field conditions in North Carolina. Sclerotia of 12 toxigenic and 12 nontoxigenic field isolates (originally isolated in NC) were buried in the field in early February 2017. We will recover these one year later to determine their damage and survival. Accomplishments testing Hypothesis 4: For this objective we have been working with the interactions of A. flavus and insects in two systems. We are using fruit flies (Drosophila melanogaster) competing for artificial culture medium with A. flavus as a model system. We are also performing experiments on maize weevils in whole maize kernels and on processed maize so that we can visualize weevil behavior better. In both systems, we have found that A. flavus produces significantly more aflatoxin when insects are present than when they are not present. This preliminary result suggests that aflatoxin may have a role in defending nutritional substrates--culture medium or maize kernels--from competition with insects. These experiments are being repeated to confirm these results We have also tested the effects of adding aflatoxin to the culture medium on the fitness of flies and fungus. In dose-response experiments, Drosophila survival decreased markedly as aflatoxin concentration increased. We also studied the fitness of A. flavus as a function of aflatoxin concentration. The fitness of A. flavus (measured in terms of fungal biomass by quantitative PCR) increases as aflatoxin concentration increases, but only in the presence of Drosophila. When Drosophila is not present, aflatoxin added to the medium has no effect on fungal fitness. This result is important in demonstrating that aflatoxin benefits the fungus (increases fitness) under some conditions and not others, in this case, in the presence of Drosophila but not in their absence. This differential effect is the type of response that can explain balancing selection and the high prevalence of isolates in nature that do not produce aflatoxin.
Publications
|
|