Progress 10/01/11 to 09/30/16
Outputs
Target Audience: Maize producers, extension educators, NGOs, and scientific community Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two MS graduate students were trained. 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?
Nothing Reported
Impacts
What was accomplished under these goals?
Because we lacked funding to continue our original objectives, we refocusedour project objectives in 2014. One study completed in 2016 was to determine the impact of routine opening of the storage bags for maize consumption on fungal growth and aflatoxin contamination. Maize with moisture contents (MC) high enough to support fungal growth (15%, 16%, 18% and 20%) was stored in PICS bags, which were opened weekly and exposed to humid conditions (85% RH) for 30 min over a period of 8 weeks and 24 weeks. Monitors indicated that oxygen defused into the open bags but did not reach equilibrium with the bottom layers of grain during the 30-min exposure period. Fungal colony forming units obtained from the grain surface increased 3-fold (at 15% MC) to 10,000-fold (at 20% MC) after 8 weeks. At both 8 weeks and 24 weeks, aflatoxin was detected in at least one bag at each grain moisture, suggesting that aflatoxin contamination spread from at planted source of A. flavus-colonized grain to non-inoculated grain. The results indicate that repeatedly breaking the hermetic seal of the PICS bags will increase fungal growth and the risk of aflatoxin contamination, especially in maize stored at high moisture content. This work also further demonstrates that maize should be properly dried prior to storage in PICS bags. A second study was conducted to evaluate the effect of three months storage of maize in triple-layer hermetic (PICS) bags on the population of Aspergillus spp. and levels of aflatoxin. Maize production practices including time of harvesting, drying and storage methods were obtained with a questionnaire. Aspergillus spp. in soil and maize were isolated by serial dilution-plating and aflatoxin content was measured using Vicam. Maize was mostly stored in woven polypropylene (PP) and sisal bags within granaries and living houses. Aspergillus flavus L-strain was the most predominant isolate from soil (8.4 x102 CFU/g), on the harvested grain (4.1 x 102 CFU/g) and grain sampled after three months of storage (1.1 x 103 CFU/g). The type of storage bag significantly (P ≤ 0.05) influenced the population of members of Aspergillus section Flavi, with A. flavus (S and L strains) and A. parasiticus being 71% higher in PP bags than in PICS bags. Total aflatoxin in maize sampled at harvest and after three months storage ranged from <5 to 42.7 ppb with 55% lower aflatoxin content in PICS bags than in PP bags.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Timothy Tubbs, Dieudonne Baributsa, Charles Woloshuk. Impact of opening hermetic storage bags on grain quality, fungal growth and aflatoxin accumulation. Journal of Stored Products Research 69 (2016) 276-281
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Angeline W. Maina, John M. Wagacha, F.B. Mwaura, James W. Muthomi, Charles P. Woloshuk. 2016. Postharvest Practices of Maize Farmers in Kaiti District, Kenya and the Impact of Hermetic Storage on Populations of Aspergillus Spp. and Aflatoxin Contamination. Journal of Food Research. DOI: http://dx.doi.org/10.5539/jfr.v5n6p53
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:Maize producers, extension educators, NGOs, scientific community Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two graduate students are working on MS degrees. This project is part of their research. Two undergraduate were train is microbilogical techniques 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?Publishing research results
Impacts
What was accomplished under these goals?
Storage of maize is a significant challenge for farmers in developing countries. Fungal growth and mycotoxin accumulation are some of the leading causes of postharvest losses, which are estimated to be $4 billion annually in the Sub-Sahara. One of the largest postharvest threats is the growth of Aspergillus flavus and the accumulation of aflatoxin. As maize production in Africa continues to grow, so does the need for storage methods that will help mitigate postharvest losses. This study investigated the use of Purdue Improved Crop Storage (PICS) bags, a three-layer hermetic-storage system, for the prevention of grain rewetting in humid environments in order to mitigate fungal growth and aflatoxin accumulation. Maize (14% grain moisture) was stored in PICS and woven polypropylene bags in Lafayette, IN and Marianna, AR, two environments contrasting in temperature and relative humidity. Each bag was filled with 40 kilograms of maize and contained three satchels of maize colonized by A. flavus. Each bag contained temperature and relative humidity data loggers. After three months of summer storage samples were collected for analysis of grain insect populations, grain moisture, aflatoxin accumulation, seed germination, and fungal spread. Our initial results indicate stark differences between hermetic storage and conventional mesh bags in both locations. Most obvious was the huge insect populations in the mesh bags. In addition, moisture content of grain stored in woven bags in Indiana rose significantly as well as surface mold counts and infection rate.
Publications
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: This year's audience was the scientific community interested in stored grain technology. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Two graduate students and an ungraduate student were participanted in the research as part of the professional training. How have the results been disseminated to communities of interest? Dupnik, M.A., Woloshuk, C., Nelson, S.D., Schuster, G. And Wise, K.A. 2014. Effects of low oxygen on the growth of Aspergillus flavus and aflatoxin production. Poster, American Society of Agronomy, Southern Regional Branch meeting, February 2, Dallas, TX. Scott B. Williams, Dieudonne Baributsa, and Charles Woloshuk, Impact of Grain Moisture on Aflatoxin and Seed Germination under Hermetic Conditions. Poster. Hermetic Storage Technologies Workshop, Nairobi, Kenya April 29. 2014. What do you plan to do during the next reporting period to accomplish the goals? Our goal is to address issues related to grain storage in hot and humid environments. Over the next year we will try to answer the following questions. How well do the hermetic storage units prevent dry grain (14-15%) from rewetting during storage? How well do the hermetic storage units prevent spoilage (molding) and aflatoxin accumulation? What is the impact of repeated opening/closing of the hermetic storage units during storage?
Impacts
What was accomplished under these goals?
Last year I added the new objectives to this projects, to determine the affects of reduce oxygen atmosphere on Aspergillus flavus growth and aflatoxin production and to determine the efficacy of hermetic storage of grain to reduce losses from A. flavus and mycotoxin contamination in tropical climates. Storing maize in regions of the world without sufficient drying and storage capacity is challenging due to the potential risk of aflatoxin contamination produced by Aspergillus flavus. This study sought to determine if storage of maize in Purdue Improved Crop Storage (PICS) bags prevents mold growth and aflatoxin accumulation. PICS bags are a three-layer, hermitic bag-system that forms a barrier against the influx of oxygen and the escape of carbon dioxide. Maize conditioned at 12, 15, 18, and 21% grain moisture was inoculated with 50 g of maize kernels infected with fluorescent-marked strain of Aspergillus flavus. The grain was stored in either PICS or woven bags at 26 ?C, and percent oxygen/carbon dioxide levels, fungal growth, aflatoxin, moisture content, and kernel germination were assessed after 1 and 2 months incubation. Maize stored in woven bags was found to equilibrate with the ambient moisture environment over both storage periods, while PICS bags retained their original moisture levels. A. flavus growth and aflatoxin accumulation were not observed in maize stored in any PICS bags. No aflatoxin B1 was detected in woven bags containing low-moisture maize (12 and 15%), but detectable levels of aflatoxin were observed in high moisture maize (18 and 21%). The percentage of oxygen and carbon dioxide within PICS bags were dependent on initial grain moisture. Higher carbon dioxide levels were observed in the bags stored for 1 month than for 2 months. High initial moisture and carbon dioxide levels correlated with low kernel germination, with the 18 and 21% treatment groups having no seeds germinate. The results of the study demonstrate that storage of maize in PICS bags is a viable management tool for preventing aflatoxin accumulation in storage.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Scott B. Williams, Dieudonne Baributsa, and Charles Woloshuk. 2014. Assessing Purdue Improved Crop Storage (PICS) bags to mitigate fungal growth and aflatoxin contamination. Journal of Stored Product Research 59: 190-196.
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience: The information generated from this research was presented to the scientific community at professional meetings. Graduate and undergraduate student participated in the work, gaining laboratory and technical experience. Changes/Problems: The ozone generator and ozone monitor that I was using is not available. I added a new objective to this project that investigates the affects of reduce oxygen atmosphere on A. flavus growth and aflatoxin production. The work will help establish the response of A. flavus when maize is stored in plastic bags, especially in tropical climates. What opportunities for training and professional development has the project provided? Undergraduate student was trained on techniques on fungal molecular biology. That student has moved on to graduate school. 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? I added a new objective to this project that investigates the affects of reduce oxygen atmosphere on A. flavus growth and aflatoxin production. The work will help establish the response of A. flavus when maize is stored in plastic bags, especially in tropical climates. I will examine the effects of various moisture content on storage. We will use an A. flavus strain that expresses a green fluorescent protein to follow stread of the fungus during storage. We will also examine O2 and CO2 levels during storage as well as seed germination and aflatoxin contamination levels.
Impacts
What was accomplished under these goals?
Microarray experiments indicated that the Cat5 gene of A. flavus is up regulated during exposure to sub-lethal concentrations of ozone, and these results were verified by quantitative PCR analysis. We hypothesized that disruption of Cat5 would result in a strain more sensitive to ozone. A Cat5-deletion mutant with was generated by targeted homologous recombination with a specific disruption construct. Growth and aflatoxin were similar to the wild-type strain. When exposed to 2-4 and 8-10 ppm ozone, the mutant growth and development were also similar to the wild type. These results suggested that one of the other four catalases might compensate for lost Cat5 activity. Cat2 expression was elevated in the mutant when exposed to ozone. We are attempting to over-express Cat5 to determine if this results in resistance to ozone.
Publications
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: 1) To better understand the molecular responses to ozone, liquid cultures of Aspergillus flavus were grown for 3 days in an ozone atmosphere. Fungal growth below the surface of the medium was not affected, but aerial growth was suppressed. In contrast, cultures exposed to air produced abundant aerial mycelia and conidia. When the ozone-treated cultures were shifted to an air environment, aerial hyphae were visible after 4 h and conidia were visible after 24 h. 2) Total RNA was isolated from cultures 0, 4, 12 and 24 h after removal from ozone and from cultures grown in an air environment. The RNA was hybridized to microarrays that contain probes representing 14,163 A. flavus genes. 3) Expression profiles indicated that transcription of hydrophobins and conidiation genes were significantly reduced in the ozone-treated cultures. Among the few genes significantly up-regulated in ozone-treated cultures was CAT5, one of five putative catalase genes in A. flavus. Within 4 h after shifting the ozone-treated cultures to an air environment, CAT5 expression decreased to control levels. Furthermore, when cultures grown in air were shifted to the ozone environment, CAT5 expression increased 3-fold after 4 h. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Plant pathologists, especially those with interest in corn diseases. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Based our results, we have hypothesize that CAT5 has a role in protection against ozone.
Publications
- No publications reported this period
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: Ph.D Graduation: Marissa X. McDonough, Purdue University 2011. Presentation: Strategies to Reduce Mycotoxin Contamination in Grain and Grain-Based Food, Woloshuk, 2011 AACC International Annual Meeting Palm Springs, CA. 10/19/2011. PARTICIPANTS: Charles Woloshuk, Marissa X. McDonough, Carlos A. Campabadal, Linda J. Mason, Dirk E. Maier, Adrian Denvir TARGET AUDIENCES: Food safety and grain storage professionals. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
An experimental auger was tested to determine its efficacy with ozone to control mold, mycotoxins and insects. Mold counts, specifically Aspergillus flavus, were reduced by 96 % in a single pass through the auger. Three passes through the auger reduced the mold count by more than 2-log units. The ozone reduced aflatoxin applied to the grain, however, the reduction was not sufficient enough to be of commercial value. The results of this study provide valuable information for estimating the parameters needed for effectively treating grain in an auger under commercial settings.
Publications
- McDonough, M.X., Campabadal, C.A., Mason, L.J., Maier, D.E., Denvir, A., Woloshuk, C. P. 2011. Ozone application in a modified screw conveyor to treat grain for insect pests, fungal contaminants, and mycotoxins. J. Stored Prod. Res. 47:249-254.
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: 1) Research was conducted on gene transcription in Aspergillus flavus when grown on kernels of the four developmental stages, blister (R2), milk (R3), dough (R4), and dent (R5). Five days after inoculation, total RNA was isolated from kernels and hybridized to Affymetrix Gene Chip arrays containing probes representing 12,834 A. flavus genes. Statistical comparisons of the expression profile data revealed significant differences that included unique sets of up-regulated genes in each kernel stage and six patterns of expression over the four stages. 2) One graduate student was mentored and received a Master's degree. One undergraduate was mentored. 1) A poster "Effect of Maize Kernels Maturation on Transcriptional Activity in Aspergillus flavus" describing this work was present at the annual meeting of the American Phytopathological Society August 9, Charlotte, NC. 2) Seminar presented "Gene Expression Profile and Response to Maize Kernels by Aspergillus flavus" at Purdue University March 28. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Plant pathologists working on corn pathogens PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Among the genes expressed in colonized dent kernels were a phytase gene and six putative genes involved in zinc acquisition. Disruption of the phytase gene phy1 resulted in reduced growth on medium containing phytate as the sole source of phosphate. Furthermore, growth of the mutant (Δphy1) was 20 % of the wild-type strain when wound-inoculated into maize ears. In contrast, no difference was detected in the amount of aflatoxin produced relative to fungal growth, indicating that phy1 does not affect aflatoxin production. The study revealed the genome-wide effects of immature maize kernels on A. flavus and suggest that phytase has a role in pathogenesis. A manuscript has been submitted for publication.
Publications
- No publications reported this period
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: The genome of Fusarium verticillioides has at least 147 putative genes that encode proteins with significant similarity to sugar transporters, none of which has been biochemically characterized. FST1 in was identified by comparative microarray analysis of RNA isolated from colonized maize germ and degermed (endosperm) tissues. Of the 50 microarray probes specific for putative sugar transporter genes, FST1 was one of six genes that were highly expressed in the colonized endosperm tissues compared to germ. We hypothesized that FST1 functions as an environmental sensor that is required for maximum FB1 production. Our current objectives are to determine if FST1 in involve in pathogenicity, the conditions for activate transcription of FST1, the cellular location of FST1, and the biochemical activity of FST1. We anticipate that this information will help lead to a management solution of fumonisin contamination of food and feed Results from theis work were presented at the annual meeting of the American Phytopathological Society. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We learned that FST1 has a potential role in pathogenicity on maize. We learned that FST1 does not function as a hexose transporter. Expression of FST1 was shown to be specifically up-regulated in the endosperm tissue, the tissue where fumonsin is produced. The results have given us a better understanding of how the pathogen must perceive and respond to different kernel environments during pathogenesis.
Publications
- Woloshuk, C. P. 2009. Aspects of habitat important to Fusarium verticillioides during pathogenesis of maize kernels. Annual Meeting of the American-Phytopathology-Society. Phytopathology 99:S152.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: Fumonisins comprise a class of carcinogenic mycotoxins produced by Fusarium verticillioides during colonization of maize kernels. We characterized the role of the fungal gene ZFR1 during colonization of maize kernels. The ZFR1 deletion strain grew approximately 2.5 fold less than wild type on endosperm tissue and a variety of other carbon sources including glucose and amylopectin. However, the ZFR1-mutant strain displayed higher α-amylase activity and expression of genes involved in starch saccharification than wild type, thus indicating that the reduced growth of the ZFR1-mutant strain was not due to inhibition of amylolytic enzymes. In the wild-type strain, expression of six genes encoding putative sugar transporters was significantly greater on endosperm tissue relative to germ tissue, and expression of at least 3 of the 6 genes was negatively affected by disruption of ZFR1. Intriguingly, disruption of FST1 had no effect on growth, kernel colonization, or kernel pH but decreased FB1 production by approximately 82% on maize kernels. Fumonisin B1 biosynthesis is repressed in cultures containing ammonium as the nitrogen source and when grown on blister kernels, the earliest stages of kernel development. In this study AREA, a regulator of nitrogen metabolism, was disrupted in Fusarium verticilliodes. The mutant (areA) grew poorly on mature maize kernels, but grew similar to wild type (WT) with the addition of ammonium phosphate. Fumonisin was not produced by areA under any condition or by the WT with added ammonium phosphate. Constitutive expression of AREA (strain AREA-CE) rescued the growth and fumonisin defects in areA. Growth of WT, areA, and AREA-CE on blister-stage kernels was similar. After seven days of growth, none of the strains produced fumonisin and the pH of the kernel tissues was 8.0. Addition of amylopectin to the blister kernels resulted in a pH near 6.6 and fumonisin production by WT and AREA-CE. PARTICIPANTS: Burt Bluhm and Hun Kim, graduate students and Robert Butchko, USDA ARS TARGET AUDIENCES: Research scientist, maize breeders and grain industry. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Based on our findings, we hypothesize that ZFR1 controls fumonsin biosynthesis by regulating genes involved in the perception or uptake of carbohydrates. Also our results support the hypothesis that fumonisin biosynthesis is regulated by the gene AREA. The failure to produce fumonisin in blister kernels is due to high pH conditions generated because of an unfavorable carbon/nitrogen environment.
Publications
- Kim, H., and Woloshuk, C. P. 2008. Role of AREA, a regulator of nitrogen metabolism, during colonization of maize kernels and fumonisin biosynthesis in Fusarium verticillioides. Fungal Genet. Biol. 45:957-953.
- Bluhm, B. H., Kim, H., Butchko, R. A. E., and Woloshuk, C. P. 2008. Involvement of ZFR1 of Fusarium verticillioides in kernel colonization and the regulation of FST1, a putative sugar transporter gene required for fumonisin biosynthesis on maize kernels. Molecular Plant Pathology. 9:203-211.
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: This study has successfully characterized the complete structure of the afl-1 mutation in a strain of Aspergillus flavus, which remained unresolved for a number of years. With a large deletion and duplication, this strain is unique with a readily scorable phenotype; diploids between a wild-type strain and the strain (649) fail to produce aflatoxin or transcripts of the aflatoxin pathway genes.. We used new approaches to better understand the unique phenotype of this strain, namely the whole genome sequence of A. flavus and expression of the regulatory gene AflR. To better understand the role of metabolic pathways in A. flavus during colonization of corn, we have used a whole genome gene chip to monitor expression of A. flavus during growth on various kernel environments. We examine expression during growth on germ, endosperm, blister stage, milk stage, dough stage, dent stage, and mature stage. We are also interested in determining the response of the fungus to active oxygen
species such as ozone and hydrogen peroxide.
TARGET AUDIENCES: plant pathologists
Impacts
The dominant phenotype is rescued in diploids between a wild-type strain and a transformant of the mutant containing an ectopic copy of aflR, the transcriptional regulator of the aflatoxin biosynthetic gene cluster. Further characterization of the mutant showed that it is missing 317 kb of chromosome III, including the known genes for aflatoxin biosynthesis. In addition, 939 kb of chromosome II is present as a duplication on chromosome III in the region previously containing the aflatoxin gene cluster. Gene expression analysis revealed the silencing effect is specific to the aflatoxin gene cluster. The results showed that silencing was restricted to the aflatoxin gene cluster in 649 x wild type diploids. The results of this study provide a better understanding of the molecular genetics surrounding the novel phenotype of a secondary metabolite pathway in an asexual fungus. We are in the early stages of data analyses from the gene chip array experiment. Preliminary
results indicated that kernel environment has a major impact on metabolic activity. There are striking difference in gene expression between gene vs endosperm tissues. Similarly, large differences in gene expression were measured between blister stage and dent stage kernels, which reflect the vastly difference composition of the two kernel types. The fungus response to active oxygen species includes the expression of many peroxisomal genes.
Publications
- Smith, C. A., Woloshuk, C. P., Robertson, D., and Payne, G. A. 2007. Silencing of the aflatoxin gene cluster in a diploid strain of Aspergillus flavus is suppressed by ectopic aflR expression. Genetics 176:2077-2086.
- Ileleji, K. E., Maier, D. E. and Woloshuk, C. P. 2007. Evaluation of different temperature management strategies for suppression of Sitophilus zeamais (Motschulsky) in stored maize. J. Stored Prod. Res. 43:480-488.
- Kim, Y.-H., Woloshuk, C. P., Cho, E. H., Bae, J. M., Song, Y.-S. and Huh, G.-H. 2007. Cloning and functional expression of the gene encoding an inhibitor against Aspergillus flavus alpha-amylase, a novel seed lectin from Lablab purpureus (Dolichos lablab). Plant Cell Reports 26:395-405.
- Tsai, W.-T., Mason, L. J., and Woloshuk, C. P. 2007. Effect of three stored-grain fungi on the development of Typhaea stercorea. J. Stored Prod. Res. 43:129-133.
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Progress 10/01/05 to 09/30/06
Outputs
We found that F. verticillioides grew equally well on all stages of the developing kernel. Fumonisin B1 (FB1) was not produced in the most immature kernels. We have hypothesized that this lack of fumonisin production results from either nitrogen repression due to high levels of free amino acids or to a lack of an inducer such as starch. Microarray analysis of FUM gene expression indicated between 16 to 64-fold increase in gene expression in endosperm versus germ tissue. Disruption of the ?-amylase gene of F. verticillioides results in low levels of FB1 biosynthesis on endosperm fractions. Analyses of FB1 production in maize kernel mutants and in defined liquid media indicate that amylopectin, a component of starch, induces FB1 biosynthesis. To better understand the results we obtain with the immature corn kernels, we disrupted AreA in F. verticillioides. Homologues of AreA in other fungi are known to be global regulators of nitrogen metabolism, but also effect the
transcription of many other genes, including genes involved in secondary metabolism. The AreA-disruption mutant can grow on media containing ammonium salts or amino acids, but not on media containing nitrate or protein. The mutant also grows poorly on mature corn kernels. However the mutant does grow well on immature (blister) kernels. Disruption of Zfr1 in F. verticillioides results in a loss of FB1 production on colonized corn kernels. Our original hypothesis was that Zfr1 is a regulatory of fumonisin gene expression. Although we have not disproved this hypothesis, further analysis of the Zfr1-disruption mutant indicated that the strain might be impaired in sugar uptake. We found that the mutant grows poorly on kernel endosperm but growth on germ tissue is much greater than the wild-type fungus. Amylase production is not impaired in the mutant. By microarray analysis of RNA isolated from the wild-type strain grown on germ and endosperm tissues we found that expression of two of the
21 sugar transporter genes are up in the endosperm grown fungus.
Impacts
Our results provide a better understanding of how the metabolites in a developing corn kernel influences growth and fumonisin production in F. verticillioides when it colonizes each kernel stage. We have also determined discovered the fumonisin-inducing activity of amylopectin. This work suggests that starch chemical structure and metabolism may be a target for control strategies.
Publications
- Burton Bluhm and Charles P. Woloshuk 2005. Amylopectin induces fumonisin B1 production by Fusarium verticillioides during colonization of maize kernels. Molecular Plant-Microbial Interactions 12:1333-1339.
- Bluhm, B. H., and Woloshuk. 2006. Fck1, a C-type cyclin-dependent kinase, interacts with Fcc1 to regulate development and secondary metabolism in Fusarium verticillioides Fungal Genet. Biol. 43:146-154.
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Progress 10/01/04 to 09/30/05
Outputs
Fusarium verticillioides, a fungal pathogen of maize, produces fumonisin mycotoxins that adversely affect human and animal health. Basic questions remain unanswered regarding the interactions between the host plant and the fungus that lead to the accumulation of fumonisins in maize kernels. In this study, we evaluated the role of kernel endosperm composition in regulating fumonisin B1 (FB1) biosynthesis. We found that kernels lacking starch due to physiological immaturity did not accumulate FB1. Quantitative PCR analysis indicated that kernel development also affected the expression of fungal genes involved in FB1 biosynthesis, starch metabolism, and nitrogen regulation. A mutant strain of F. verticillioides with a disrupted ?-amylase gene was impaired in its ability to produce FB1 on starchy kernels, and both the wild-type and mutant strains produced significantly less FB1 on a high-amylose kernel mutant of maize. When grown on a defined medium with amylose as the
sole carbon source, the wild-type strain produced only trace amounts of FB1, but it produced large amounts of FB1 when grown on amylopectin or dextrin, a product of amylopectin hydrolysis. We conclude that amylopectin induces FB1 production in F. verticillioides.
Impacts
This study provides new insight regarding the interaction between the fungus and maize kernel during pathogenesis and highlights important areas that need further study.
Publications
- No publications reported this period
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Progress 10/01/03 to 09/29/04
Outputs
Fumonisins are a group of mycotoxins produced by Fusarium verticillioides that contaminate corn and cause leukoencephalomalacia in equine, pulmonary edema in swine, and promote cancer in mice. A candidate regulatory gene, ZFR1, was identified in a cDNA library enriched for transcripts expressed by F. verticillioides during fumonisin B1 (FB1) biosynthesis. ZFR1 deletion mutants exhibited normal growth and development on maize kernels, but fumonisin production was reduced to less than 10% of that of the wild-type strain. ZFR1 encodes a putative protein of 705 amino acids with sequence similarity to the Zn(II)2Cys6 binuclear cluster family that are regulators of both primary and secondary metabolism in fungi. Expression of ZFR1 in colonized germ and degermed corn kernel tissues correlated with FB1 levels. Overexpression of ZFR1 in zfr1 mutants restored FB1 production to wild-type levels. The results of this study indicate that ZFR1 is a positive regulator of FB1
biosynthesis in F. verticillioides.
Impacts
The research contributes to our knowledge on genetic factors involved in the biosynthesis of fumonisin. This knowledge will assist in developing methods to prevent mycotoxin contamination in food.
Publications
- Flaherty, J. E., and Woloshuk, C. P. 2004. Regulation of fumonisin biosynthesis in Fusarium verticilliodes by a zinc binuclear cluster-type gene, ZFR1. Appl. Environ. Microbiol. 70:2653-2659.
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Progress 10/01/02 to 09/30/03
Outputs
Fumonisins are a group of mycotoxins that contaminate maize and cause leukoencephalomalacia in equine, pulmonary edema in swine, and promote cancer in mice. Fumonisin biosynthesis in Fusarium verticillioides is repressed by nitrogen and alkaline pH. We cloned a PACC-like gene (PAC1) from F. verticillioides. PACC genes encode the major transcriptional regulators of several pH-responsive pathways in other filamentous fungi. In northern blot analyses, a PAC1 probe hybridized to a 2.2-kb transcript present in F. verticillioides grown at alkaline pH. A mutant of F. verticillioides with a disrupted PAC1 gene had severely impaired growth at alkaline pH. The mutant produced more fumonisin than the wild type when grown on maize kernels and in a synthetic medium buffered at acidic pH (4.5). The mutant, but not the wild type, also produced fumonisin B1 when mycelia were resuspended in medium buffered at alkaline pH (8.4). Transcription of FUM1, a gene involved in fumonisin
biosynthesis, was correlated with fumonisin production. We conclude that PAC1 is required for growth at alkaline pH and that Pac1 may have a role as a repressor of fumonisin biosynthesis under alkaline conditions.
Impacts
The research contributes to our knowledge on genetic factors involved in the biosynthesis of fumonisin and towards understanding how the environment of a maize kernel influences mycotoxin production. This knowledge will assist in developing methods to prevent mycotoxin contamination in food.
Publications
- Flaherty, J. E., Pirttila, A. M., Bluhm, B. H., and Woloshuk, C. P. 2003. Role of PAC1, a pH regulatory gene from Fusarium verticillioides, in growth and fumonisin biosynthesis. Appl. Environ. Microbiol. 69:5222-5227.
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Progress 10/01/01 to 09/30/02
Outputs
We have hypothesized that expression of AILP in maize kernels may slow colonization by A. flavus and reduce aflatoxin contamination. To test this hypothesis, we are cloning the gene (LAI) encoding AILP, and we will express Lai in E. coli and maize. Southern analysis indicated that Lai in L. purpureus is a multigenic family containing at least nine genes. We have cloned the upstream and downstream regions of the LAI genes by genomic walker PCR. From the DNA sequence analysis of these regions we designed PCR primers to obtain all Lai genes. Of these, six genes encoded proteins containing the peptide sequences obtained from the native protein. The six putative LAI genes were cloned into the pET28C vector for expression in E. coli. All six proteins accumulated as inclusion bodies. Solubilization of the AILP proteins was achieved with non-detergent sulfobetains as solubilizing agents. We have also developed antibodies in rabbit to one of the recombinant proteins. Western
analysis indicated that the antibodies recognized all six of the soluble, renatured AILP proteins. Additionally we cloned one of the LAI genes into the binary vector pBI121 and transformed the construct into Arabidopsis. Currently we are testing the AILP proteins from E. coli for a-amylase inhibitor activity and screening Arabidopsis transformants for AILP expression. Our goal is to identify a LAI gene that encodes an active a-amylase inhibitor prior to transforming the gene into maize.
Impacts
Research on the regulation of aflatoxin biosynthesis will reveal vulnerable points in the pathway that can be targets for control by bioengineering techniques. The completion of the proposed objectives will yield important information about the role of an enzyme, a-amylase, in aflatoxin production and identify novel amylase inhibitors that are specific towards the A. flavus a-amylase. The information will impact agriculture by furthering our understanding of aflatoxin contamination of grains and by determining if inhibiting a-amylase is a feasible method for controlling aflatoxin production.
Publications
- Fakhoury, A. M. and Woloshuk, C. P. 2001. Inhibition of growth of Aspergillus flavus and fungal a-amylases by a lectin-like protein from Lablab purpureus. Mol. Plant Microbiol. Interact. 18:955-961.
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Progress 10/01/00 to 09/30/01
Outputs
Aspergillus flavus is a fungal pathogen of maize causing an important ear rot disease when plants are exposed to drought and heat stress. Associated with the disease is production of aflatoxins, which are a series of structurally related mycotoxins known to be carcinogenic. Previous research has suggested that the alpha-amylase of A. flavus promotes aflatoxin production in the endosperm of infected maize kernels. We reported on the isolation and characterization of a 36-kDa alpha-amylase inhibitor from Lablab purpureus (AILP). AILP inhibited the alpha-amylases from several fungi but had little effect on those from animal and plant sources. The protein inhibited conidial germination and hyphal growth of A. flavus. Amino acid sequence indicated that AILP is similar to lectin members of a lectin-arcelin-alpha-amylase inhibitor family described in common bean and shown to be a component of plant resistance to insect pests. AILP also agglutinated papain-treated red blood
cells from human and rabbit. These data indicate that AILP represents a novel variant in the lectin-arcelin-alpha-amylase inhibitor family of proteins having both lectin-like and alpha-amylase inhibitory activity.
Impacts
Research on the regulation of aflatoxin biosynthesis will reveal vulnerable points in the pathway that can be targets for control by bioengineering techniques. The completion of the proposed objectives will yield important information about the role of an enzyme, alpha-amylase, in aflatoxin production and identify novel amylase inhibitors that are specific towards the A. flavus alpha-amylase. The information will impact agriculture by furthering our understanding of aflatoxin contamination of grains and by determining if inhibiting a-amylase is a feasible method for controlling aflatoxin production.
Publications
- Fakhoury, A. M. and Woloshuk, C. P. 2001. Inhibition of growth of Aspergillus flavus and fungal alpha-amylases by a lectin-like protein from Lablab purpureus. Molecular Plant-Microbe Interactions. 18:955-961.
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Progress 10/01/99 to 09/30/00
Outputs
Hydrolysis products of corn endosperm starch induce aflatoxin biosynthesis by Aspergillus flavus. When the a-amylase gene of A. flavus is disrupted, growth of the fungus and its ability to produce aflatoxin in the endosperm of colonized corn kernels is inhibited. We have characterized an a-amylase inhibitors from the legume Lablab purpureus that is 50 times more potent than a 14 kDa trypsin/a-amylase inhibitor found in maize. Peptide sequences of the L. purpureus protein revealed high similarity to an a-amylase inhibitor from Phaseolus maculatus and to a phytohemagglutinin from Phaseolus acutifolius. The protein inhibits the a-amylases from Magnaporthe grisea, Helminthosporium victorae and A. flavus, but not those from corn, porcine pancreas and human saliva. The protein also inhibits conidial germination and hyphal growth of A. flavus.
Impacts
Research on the regulation of aflatoxin biosynthesis will reveal vulnerable points in the pathway that can be targets for control by bioengineering techniques. The completion of the proposed objectives will yield important information about the role of an enzyme, a-amylase, in aflatoxin production and identify novel amylase inhibitors that are specific towards the A. flavus a-amylase. The information will impact agriculture by furthering our understanding of aflatoxin contamination of grains and by determining if inhibiting a-amylase is a feasible method for controlling aflatoxin production.
Publications
- Fakhoury, A. M. and Woloshuk, C. P. 1999. Amy1, the a-amylase gene of Aspergillus flavus: Involvement in aflatoxin biosynthesis in maize kernels. Phytopathology 89: 908-914.
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Progress 10/01/98 to 09/30/99
Outputs
Aspergillus flavus is the causal agent of an ear and kernel rot in maize. In this study we characterized an a-amylase-deficient mutant and assessed its ability to infect and produce aflatoxin in wounded maize kernels. The a-amylase gene Amy1 was isolated from A. flavus and its DNA sequence was determined to be nearly identical to Amy3 of A. oryzae. When Amy1 was disrupted in an aflatoxigenic strain of A. flavus, the mutant failed to produce extracellular a-amylase and grew 45% the rate of the wild-type strain on starch medium. The mutant produced aflatoxin in medium containing glucose but not in a medium containing starch. The a-amylase-deficient mutant produced aflatoxin in maize kernels with wounded embryos and the mutant occasionally produced aflatoxin only in embryos of kernels with wounded endosperm. The mutant strain also failed to produce aflatoxin when inoculated onto degermed kernels. In contrast, the wild-type strain produced aflatoxin in both the endosperm
and embryo.
Impacts
These results suggest that a-amylase facilitates aflatoxin production and growth of A. flavus from a wound in the endosperm to the embryo. A 14 kDa trypsin inhibitor associated with resistance to A. flavus and aflatoxin in maize also inhibited the a-amylase from A. flavus indicating that it is a bifunctional inhibitor. The inhibitor may have a role in resistance, limiting the growth of the fungus in the endosperm tissue by inhibiting the degradation of starch.
Publications
- Fakhoury, A. M. and Woloshuk, C. P. 1999. Amy1, the a-amylase gene of Aspergillus flavus: Involvement in aflatoxin biosynthesis in maize kernels. Phytopathology 89: 908-914.
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Progress 10/01/97 to 09/30/98
Outputs
.Aflatoxins are secondary metabolites produced by Aspergillus flavus and A. parasiticus. Genes involved in the biosynthesis of aflatoxins are clustered in the fungal genome. While most of the DNA sequence is known, the function of several genes in the cluster remains to be elucidated. We transformed a mutant of A. flavus lacking the entire aflatoxin gene cluster with two overlapping cosmids (5E6 and 8B9) and recovered transformants that accumulated the aflatoxin pathway intermediate averufin (AVR). The addition of a third cosmid (13B9) restored aflatoxin biosynthesis, suggesting that the gene, avf1, involved in the conversion of AVR to versiconal acetate (VA) is located on cosmid 13B9. Analysis of 13B9 located avf1 to a 7 kb DNA fragment of the cosmid 13B9. From nucleotide sequencing, we have identified a candidate gene that appeared to encode a P450 monooxygenase. Transformants of strain 649 containing this putative gene and cosmid 8B9 were fed averufin. When
analyzed for conversion products by thin-layer chromatography, we detected an metabolite with a Rf-value and color similar to versiconal hemiacetal acetate. However, the percent conversion was low. Several attempts to identify the metabolite by mass spectrometry failed to give any definitive information. The A. parasiticus strain ATCC 24551 that was isolated by Donkersloot in 1972 also accumulates AVR because of an apparent block in the conversion of AVR to VA. This mutation was complemented with cosmid 13B9. Transformants containing cosmid 13B9 produced wild-type levels of aflatoxin. These data suggest that strain ATCC 24551 contains a mutation in avf1.
Impacts
(N/A)
Publications
- Prieto, R., and Woloshuk, C. P. 1997. Ord1, an oxidoreductase gene responsible for the conversion of O-methylsterigmatocystin to aflatoxin in Aspergillus flavus. Appl. Environ. Microbiol. 63:1661-1666.
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Progress 10/01/96 to 09/30/97
Outputs
Using a GUS-reporter assay we have detected an aflatoxin inducing activity in ground maize kernels colonized by Aspergillus flavus. Evidence suggest that alpha amylase produced by the fungus has a role in aflatoxin production in diseased maize kernels. We have hypothesized that the action of the fungal alpha amylase on kernel starch results in the production of fermentable sugars leading to an induction of aflatoxin biosynthesis. To test this hypothesis, we disrupted the alpha amylase gene Amy1 of the aflatoxigenic strain 86-10D by site-directed mutagenesis. A transformant (T-150) having an Amy1 disruption was identified by PCR methodology, and the mutation was confirmed by Southern hybridization. The rate of growth of T-150 and two control strains was identical on potato dextrose agar (PDA) medium. On starch medium supplemented with 10 mM uracil and arginine, T-150 grew at 70% the rate of the control strains. T-150 grew on a medium containing 10 mM arginine and
uracil as the sole carbon source (without starch), suggesting that arginine and uracil were responsible for the observed growth on starch medium. Iodine staining of the cultures grown on starch resulted in clear zones around the colonies of the control strains. In contrast, no clearing was detected around the T-150 colonies indicating that this mutant does not produce an extracellular alpha amylase. Also, extracellular alpha amylase activity was not detected when T-150 was grown on ground corn. After 8 days of incubation, T-150 appeared to grow in a viscous, starchy matrix with numerous starch granules. At the same time, the control strain grew more extensively, and the corn matrix was relatively clear with very few starch granules. These data confirm that the Amy1 gene was disrupted in T-150. T-150 failed to produce aflatoxin when grown on rice kernels, ground corn kernels, coconut agar medium and PMSG (peptone mineral salt and glucose) medium. To determine whether the disruption of
Amy1 is responsible for the non-aflatoxin phenotype, we have crossed T-150 with strain 118 (tan, arg2, Tox+). We have obtained heterokaryons and putative diploids. The segregation of Amy1 and aflatoxin in the progeny from this parasexual cross will be determined. We will also determine if progeny that are arginine prototrophs with a disrupted Amy1 can grow on starch. The progeny will not require the addition of arginine to the culture medium.
Impacts
(N/A)
Publications
- Brown, R. L., Cleveland, T. E., Payne, G. A., Woloshuk, C. P., and White, D. G. 1997. Growth of an Aspergillus flavus transformant expressing Escherichia coli beta-glucuronidase in maize kernels resistant to aflatoxin production. J. Food Protection 60:84-87.
- Woloshuk, C. P., Cavaletto, J. R., and Cleveland, T. E. 1997. Inducers of aflatoxin biosynthesis from colonized maize kernels are generated by an amylase activity from Aspergillus flavus. Phytopathology 87:164-169.
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Progress 10/01/95 to 09/30/96
Outputs
Aflatoxin biosynthesis was induced by compounds in filtrates (EF) obtained from cultures consisting of ground maize kernels colonized by Aspergillus flavus. The inducing-activity increased to a maximum at 4 days of incubation and then decreased. Amylase activity was detected in the EF, suggesting that the inducers are products of starch degradation (glucose, maltose and maltotriose). Analysis of the enzyme by isoelectric focusing electrophoresis indicated a single a-amylase with a pI of 4.3. No maltase or amyloglucosidase was detected in EF. HPLC analysis of EF indicated the presence of glucose, maltose, and maltotriose in near equal molar concentrations (about 15 mM). With a beta-glucuronidase (GUS) reporter assay consisting of A. flavus transformed with an aflatoxin gene promoter-GUS reporter gene fusion to monitor induction of aflatoxin biosynthesis, the minimum concentration of glucose, maltose or maltotriose that induced measurable GUS activity was determined to
be 1 mM. These results support the hypothesis that the best inducers of aflatoxin biosynthesis are carbon sources readily metabolized via glycolysis. They also suggest that a-amylase produced by A. flavus has a role in the induction of aflatoxin biosynthesis in infected maize kernels.
Impacts
(N/A)
Publications
- Woloshuk, C. P., Cavaletto, J. R., and Cleveland, T. E. 1997. Inducers of aflatoxin biosynthesis from colonized maize kernels are generated by an amylase activity from Aspergillus flavus Phytopathology (in press)
- Brown, R. L., Cleveland, T. E., Payne, G. A., Woloshuk, C. P., and White*, D. G.1996. Growth of an Aspergillus flavus transformant expressing Escherichia coli glucuronidase in maize kernels resistant to aflatoxin production. J. Food Prot.
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Progress 10/01/94 to 09/30/95
Outputs
Aflatoxins are toxic and carcinogenic secondary metabolites produced by the fungi Aspergillus flavus and A. parasiticus. The objective of this research was to develop a method to study the induction and regulation of aflatoxin biosynthesis by examining the expression of one aflatoxin pathway gene, ver1. The promoter region of ver1 was fused to the B-glucuronidase (GUS) gene (uidA) from E. coli to form the reporter construct, GAP13. A. flavus strain 656-2 was transformed with this construct. Transformants harboring GAP13 displayed GUS expression only when aflatoxin was detected in culture. Further, the transcription of the uidA gene driven by the ver1 promoter followed the same profile as for the ver1 genes. The results show that the GAP13 construct may be useful as a genetic tool to study the induction of aflatoxin in situ and to identify substances that affect the expression of genes involved in aflatoxin biosynthesis. Using the GUS-expressing transformant in a
bioassay, an aflatoxin-inducing activity was detected in extracts (EF) from ground maize kernels colonized with the aflatoxigenic strain of Aspergillus flavus. The inducing component was water soluble and was not inactivated by autoclaving. The inducing activity passed through ultrafiltration membranes with 100- and 10-kDa exclusion. Eighty kernal samples collected from diseased ears from the 1994 Ear Rot and Mycotoxin Survey were plated on to a medium selective for Fusarium isolation. Out of the 56 counties that were represented in the.
Impacts
(N/A)
Publications
- Flaherty, J.E., M.A. Weaver, G.A. Payne, and C.P. Woloshuk. 1995 A B-glucuronidase reporter gene construct for monitoring aflatoxin biosynthesis in Aspergillus flavus. Appl. Environ. Microbiol. 61:2482-2486.
- Brown, R.L., T.E. Cleveland, G.A. Payne, C.P. Woloshuk, K.W. Campbell, and D.G. White. 1995. Determination of resistance to aflatoxin production in maize kernels and of fungal colonization using an Aspergillus flavus transformant expressing
- Foutz, K.R., C.P. Woloshuk, and G.A. Payne. 1995. Cloning and assignment of linkage group loci to a karyotype map of the filamentous fungus Aspergillus flavus. Mycologia (in press, November issue).
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Progress 10/01/93 to 09/30/94
Outputs
I crossed strain 649 with strain 86 (afl-1(superscript 1+)) to obtain diploids. These diploids did not produce aflatoxin indicating that the afl-1 mutation is a dominate allele. Transcript levels of four aflatoxin genes in strain 649 and in two aflatoxigenic strains during aflatoxin biosynthesis indicated that no transcripts in strain 649 and expression of all four genes in the aflatoxigenic strains. In two diploids, no expression of the structural genes was detected; however, there was expression of the regulatory gene. These data suggest that inhibition of aflatoxin biosynthesis associated with the afl-1 is not totally by suppression of aflR. An assay procedure was developed for detecting substances that induce aflatoxin biosynthesis. This procedure identified a water extractable activity from maize kernels. When A. flavus NRRL 3357 was first grown on the ground kernels, higher inducing activity was recovered. These data suggest that fungal growth can facilitate the
release of the inducing activity from the kernel tissues. It was also determined that the majority of the inducing activity was heat stable. I have collected 45 isolates of Fusarium moniliforme from infected corn kernels from from 32 Indiana counties. This fungus is responsible for Fusarium fumonisin contamination in corn. Mating type testing has been completed on 13 of the isolates and results indicate that both mating types A- and A+ are present in the state. Most of the isolates are also capable of producing fumonisin in culture.
Impacts
(N/A)
Publications
- No publications reported this period.
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Progress 10/01/92 to 09/30/93
Outputs
Toward the goal of isolating genes in Aspergillus flavus mapped to linkage groupVII, I have produced heterokaryons and diploids from six haploid strains having linkage group VII mutations. The heterokaryons and diploids were produced by parasexual mating. The data thus far attained indicate that the mutations in these strains are non allelic and recessive. A method was developed for identifying substances that induce or inhibit aflatoxin biosynthesis. The promoter regions of two aflatoxin biosynthetic pathway genes (nor and ver) from Aspergillus flavus were cloned into the GUS-reporter gene construct GAP4 to generate the promoter constructs, GAP12 and GAP13; respectively. These constructs were transformed into the A. flavus strain 656-2. Aflatoxin production and GUS activity were determined in the transformants after shifting the cultures from a medium that does not support aflatoxin production to a medium that does support its production. In transformants harboring the
promoter-GUS constructs, the GUS activity paralleled aflatoxin production in culture. Based on these data, the transformants are good candidates for studying the induction of aflatoxin in situ and for indentifying substances that affect the expression of genes involved in aflatoxin biosynthesis.
Impacts
(N/A)
Publications
- No publications reported this period.
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