Chemical Profiles of Heterodera glycines Suppressive Soils in Double Cropping Soybean Production.

We previously reported soybean fields double-cropped with winter wheat having reduced soybean cyst nematode (SCN) (Heterodera glycines) counts compared to fallow. A follow-up metagenomics study identified several fungal and bacterial taxa enriched in wheat fields, and some were reported to parasitize SCN. Knowing that phytocompounds with potential nematicidal activity are released via wheat roots and stubble, we implemented a dichloromethane-based extraction method and a gas chromatography-mass spectrometry (GCMS) system to investigate soil chemical profiles of samples collected from these fields and review the potential nematicidal activity of compounds with higher concentration in double cropping fields. 51 compounds were detected during the GCMS analysis, eight with unknown identification. Several compounds, including multiple fatty acids, had larger relative peak areas when double-cropped, compared to fallow samples. This study, along with our previously published one, provided a better understanding of the mechanisms that govern the effect of wheat on SCN populations. Rather than driven by a single mechanism, the suppression of SCN in soybean fields double-cropped with winter wheat was potentially linked to enriched microbial communities, increased populations of beneficial organisms, and higher concentrations of chemicals with potential nematicidal activity. To our knowledge, this is the first study using GCMS to characterize soil chemical profiles in soybean fields double-cropped with winter wheat regarding the suppression of SCN populations.

Fluopyram activates systemic resistance in soybean.

The soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) is a significant yield-limiting factor in soybean production in the Midwestern US. Several management practices are implemented to mitigate yield losses caused by SCN, including using SDHI (succinate dehydrogenase inhibitors) fungicides delivered as seed treatments. A set of studies was conducted to evaluate the effect of two seed-applied succinate dehydrogenase inhibitors (SDHI) compounds, fluopyram and pydiflumetofen, on SCN population densities, plant injury, and plant growth. Cyst counts in untreated control and pydiflumetofen treated plants were 3.44 and 3.59 times higher than fluopyram, respectively, while egg counts were 8.25 and 7.06 times higher in control and pydiflumetofen. Next-generation sequencing was later employed to identify transcriptomic shifts in gene expression profiles in fluopyram and pydiflumetofen -treated seedlings. RNA expression patterns of seed treatments clustered by sampling time (5 DAP vs. 10 DAP); therefore, downstream analysis was conducted by timepoint. At 5 DAP, 10,870 and 325 differentially expressed genes (DEG) were identified in fluopyram and pydiflumetofen, respectively. These same treatments generated 219 and 2 DEGs at 10 DAP. Multiple DEGs identified in soybean seedlings treated with fluopyram are linked to systemic resistance, suggesting a potential role of systemic resistance in the suppression of SCN by fluopyram, in addition to the known nematicidal activity. The non-target inhibition of soybean succinate dehydrogenase genes by fluopyram may be the origin of the phytotoxicity symptoms observed and potentially the source of the systemic resistance activation reported in the current study. This work helps to elucidate the mechanisms of suppression of SCN by fluopyram.

Ecology and diversity of culturable fungal species associated with soybean seedling diseases in the Midwestern United States.

AIMS: To isolate and characterize fungi associated with diseased soybean seedlings in Midwestern soybean production fields and to determine the influence of environmental and edaphic factors on their incidence. METHODS AND RESULTS: Seedlings were collected from fields with seedling disease history in 2012 and 2013 for fungal isolation. Environmental and edaphic data associated with each field was collected. 3036 fungal isolates were obtained and assigned to 76 species. The most abundant genera recovered were Fusarium (73%) and Trichoderma (11.2%). Other genera included Mortierella, Clonostachys, Rhizoctonia, Alternaria, Mucor, Phoma, Macrophomina and Phomopsis. Most recovered species are known soybean pathogens. However, non-pathogenic organisms were also isolated. Crop history, soil density, water source, precipitation and temperature were the main factors influencing the abundance of fungal species. CONCLUSION: Key fungal species associated with soybean seedling diseases occurring in several US production regions were characterized. This work also identified major environment and edaphic factors affecting the abundance and occurrence of these species. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification and characterization of the main pathogens associated with seedling diseases across major soybean-producing areas could help manage those pathogens, and devise more effective and sustainable practices to reduce the damage they cause.

Reduction of Pythium Damping-Off in Soybean by Biocontrol Seed Treatment.

Pythium spp. is one of the major groups of pathogens that cause seedling diseases on soybean, leading to both preemergence and postemergence damping-off and root rot. More than 100 species have been identified within this genus, with Pythium irregulare, P. sylvaticum, P. ultimum var ultimum, and P. torulosum being particularly important for soybean production given their aggressiveness, prevalence, and abundance in production fields. This study investigated the antagonistic activity of potential biological control agents (BCAs) native to the U.S. Midwest against Pythium spp. First, in vitro screening identified BCAs that inhibit P. ultimum var. ultimum growth. Scanning electron microscopy demonstrated evidence of mycoparasitism of all potential biocontrol isolates against P. ultimum var. ultimum and P. torulosum, with the formation of appressorium-like structures, short hyphal branches around host hyphae, hook-shaped structures, coiling, and parallel growth of the mycoparasite along the host hyphae. Based on these promising results, selected BCAs were tested under field conditions against six different Pythium spp. Trichoderma afroharzianum 26 used alone and a mix of T. hamatum 16 + T. afroharzianum 19 used as seed treatments protected soybean seedlings from Pythium spp. infection, as BCA-treated plots had on average 15 to 20% greater plant stand and vigor than control plots. Our results also indicate that some of these potential BCAs could be added with a fungicide seed treatment with minimum inhibition occurring, depending on the fungicide active ingredient. This research highlights the need to develop tools incorporating biological control as a facet of soybean seedling disease management programs. The harnessing of native BCAs could be integrated with other management strategies to provide efficient control of seedling diseases.

Impact of Wheat on Soybean Cyst Nematode Population Density in Double-Cropping Soybean Production.

Double-cropping is defined as producing more than one crop on the same parcel of land in a single growing season. It is reported to have many benefits when incorporated in cropping systems, including improving soil health. In some double-cropping systems, soybean is planted following winter wheat. The soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) is a major soybean pathogen, and several reports suggest suppressive effects of wheat on SCN populations. Field trials were conducted from 2017 to 2018 to investigate the effect of wheat on SCN populations in double-cropping soybean. Nine fields with three levels of initial SCN populations (low, moderate, and high) were selected in Illinois. Wheat was planted in strips alternating with strips-maintained weed-free and under fallow over winter and early spring. Soybean was planted in all strips after wheat harvest. SCN egg densities were acquired at four time points: wheat establishment, post-wheat/pre-soybean, mid-soybean (R1 growth stage or beginning of flowering), and post-soybean harvest. Wheat strips reduced SCN egg densities compared with fallow strips at the R1 stage (-31.8%) and after soybean harvest (-32.7%). Double-cropping soybean with wheat has the potential to suppress SCN field populations and is a system with the potential to provide additional farm income. This study is meant to be a first step toward a better understanding of the mechanisms that govern the suppression of SCN by wheat.

Microbial Communities Associated With Long-Term Tillage and Fertility Treatments in a Corn-Soybean Cropping System.

Tillage and fertilization are common practices used to enhance soil fertility and increase yield. Changes in soil edaphic properties associated with different tillage and fertility regimes have been widely examined, yet, the microbially mediated pathways and ecological niches involved in enhancing soil fertility are poorly understood. The effects of long-term conventional tillage and no-till in parallel with three fertility treatments (No fertilization, N-only, and NPK) on soil microbial communities were investigated in a long-term field study that was established in the 1970's. Here, we used high-throughput sequencing of bacterial, fungal and oomycetes markers, followed by community-level functional and ecological assembly to discern principles governing tillage and fertility practices' influence on associated soil microbiomes. Both tillage and fertilizer significantly altered microbial community structure, but the tillage effect was more prominent than the fertilizer effect. Tillage significantly affected bacteria, fungi, fusaria, and oomycete beta-diversity, whereas fertilizer only affected bacteria and fungi beta-diversity. In our study different tillage and fertilizer regimes favored specific networks of metabolic pathways and distinct ecological guilds. No-till selected for beneficial microbes that translocate nutrients and resources and protect the host against pathogens. Notably, ecological guilds featuring arbuscular mycorrhizae, mycoparasites, and nematophagous fungi were favored in no-till soils, while fungal saprotrophs and plant pathogens dominated in tilled soils. Conventional till and fertilizer management shifted the communities toward fast growing competitors. Copiotrophic bacteria and fusarium species were favored under conventional tillage and in the presence of fertilizers. The analysis of the metagenomes revealed a higher abundance of predicted pathways associated with energy metabolism, translation, metabolism of cofactors and vitamins, glycan biosynthesis and nucleotide metabolism in no-till. Furthermore, no specific pathways were found to be enriched under the investigated fertilization regimes. Understanding how tillage and fertilizer management shift microbial diversity, structure and ecological niches, such as presented here, can assist with designing farming systems that can maintain high crop yield, while reducing soil erosion and nutrient losses.

Use of Dual RNA-seq for Systems Biology Analysis of Zea mays and Aspergillus flavus Interaction.

The interaction between Aspergillus flavus and Zea mays is complex, and the identification of plant genes and pathways conferring resistance to the fungus has been challenging. Therefore, the authors undertook a systems biology approach involving dual RNA-seq to determine the simultaneous response from the host and the pathogen. What was dramatically highlighted in the analysis is the uniformity in the development patterns of gene expression of the host and the pathogen during infection. This led to the development of a "stage of infection index" that was subsequently used to categorize the samples before down-stream system biology analysis. Additionally, we were able to ascertain that key maize genes in pathways such as the jasmonate, ethylene and ROS pathways, were up-regulated in the study. The stage of infection index used for the transcriptomic analysis revealed that A. flavus produces a relatively limited number of transcripts during the early stages (0 to 12 h) of infection. At later stages, in A. flavus, transcripts and pathways involved in endosomal transport, aflatoxin production, and carbohydrate metabolism were up-regulated. Multiple WRKY genes targeting the activation of the resistance pathways (i.e., jasmonate, phenylpropanoid, and ethylene) were detected using causal inference analysis. This analysis also revealed, for the first time, the activation of Z. mays resistance genes influencing the expression of specific A. flavus genes. Our results show that A. flavus seems to be reacting to a hostile environment resulting from the activation of resistance pathways in Z. mays. This study revealed the dynamic nature of the interaction between the two organisms.

Trichoderma Isolates Inhibit Fusarium virguliforme Growth, Reduce Root Rot, and Induce Defense-Related Genes on Soybean Seedlings.

Sudden death syndrome (SDS) caused by Fusarium virguliforme is among the most important diseases affecting soybean in the United States. The use of biological control agents (BCAs) such as Trichoderma spp. can be a valuable resource to suppress F. virguliforme populations. Therefore, this research focused on screening possible BCAs against F. virguliforme and evaluating mycoparasitism and the induction of systemic resistance as mechanisms underlying the antagonistic activity of selected BCAs against F. virguliforme. In total, 47 potential BCAs, including 41 Trichoderma isolates and 6 Mortierella isolates, were screened in a dual-plate assay. The most effective isolates belonged to the Trichoderma harzianum species and were able to inhibit F. virguliforme radial growth by up to 92%. Selected Trichoderma isolates were tested in the greenhouse and in a microplot study. They reduced root rot caused by F. virguliforme when the plants were coinoculated with the pathogen and the BCA. The tested BCA's ability to reduce F. virguliforme growth may be related to several mechanisms of action, including mycoparasitism and induction of defense-related genes in plants, as revealed by monitoring the expression of defense-related genes in soybean. Our results highlight the potential of native Trichoderma isolates to inhibit F. virguliforme growth and reduce SDS severity, providing the basis for future implementation of biological control in soybean production. More efforts are needed to implement the use of these approaches in production fields, and to deepen the current knowledge on the biology of these highly antagonistic isolates.

The Pathogenesis-Related Maize Seed (PRms) Gene Plays a Role in Resistance to Aspergillus flavus Infection and Aflatoxin Contamination.

Aspergillus flavus is an opportunistic plant pathogen that colonizes and produces the toxic and carcinogenic secondary metabolites, aflatoxins, in oil-rich crops such as maize (Zea mays ssp. mays L.). Pathogenesis-related (PR) proteins serve as an important defense mechanism against invading pathogens by conferring systemic acquired resistance in plants. Among these, production of the PR maize seed protein, ZmPRms (AC205274.3_FG001), has been speculated to be involved in resistance to infection by A. flavus and other pathogens. To better understand the relative contribution of ZmPRms to A. flavus resistance and aflatoxin production, a seed-specific RNA interference (RNAi)-based gene silencing approach was used to develop transgenic maize lines expressing hairpin RNAs to target ZmPRms. Downregulation of ZmPRms in transgenic kernels resulted in a ∼250-350% increase in A. flavus infection accompanied by a ∼4.5-7.5-fold higher accumulation of aflatoxins than control plants. Gene co-expression network analysis of RNA-seq data during the A. flavus-maize interaction identified ZmPRms as a network hub possibly responsible for regulating several downstream candidate genes associated with disease resistance and other biochemical functions. Expression analysis of these candidate genes in the ZmPRms-RNAi lines demonstrated downregulation (vs. control) of a majority of these ZmPRms-regulated genes during A. flavus infection. These results are consistent with a key role of ZmPRms in resistance to A. flavus infection and aflatoxin accumulation in maize kernels.

FvSTR1, a striatin orthologue in Fusarium virguliforme, is required for asexual development and virulence.

The soil-borne fungus Fusarium virguliforme causes sudden death syndrome (SDS), one of the most devastating diseases of soybean in North and South America. Despite the importance of SDS, a clear understanding of the fungal pathogenicity factors that affect the development of this disease is still lacking. We have identified FvSTR1, a F. virguliforme gene, which encodes a protein similar to a family of striatin proteins previously reported to regulate signalling pathways, cell differentiation, conidiation, sexual development, and virulence in filamentous fungi. Striatins are multi-domain proteins that serve as scaffolding units in the striatin-interacting phosphatase and kinase (STRIPAK) complex in fungi and animals. To address the function of a striatin homologue in F. virguliforme, FvSTR1 was disrupted and functionally characterized using a gene knock out strategy. The resulting Fvstr1 mutants were largely impaired in conidiation and pigmentation, and displayed defective conidia and conidiophore morphology compared to the wild-type and ectopic transformants. Greenhouse virulence assays revealed that the disruption of FvSTR1 resulted in complete loss of virulence in F. virguliforme. Microtome studies using fluorescence microscopy showed that the Fvstr1 mutants were defective in their ability to colonize the vascular system. The Fvstr1 mutants also showed a reduced transcript level of genes involved in asexual reproduction and in the production of secondary metabolites. These results suggest that FvSTR1 has a critical role in asexual development and virulence in F. virguliforme.

FvSNF1, the sucrose non-fermenting protein kinase gene of Fusarium virguliforme, is required for cell-wall-degrading enzymes expression and sudden death syndrome development in soybean.

Fusarium virguliforme is a soil-borne pathogenic fungus that causes sudden death syndrome (SDS) in soybean. Its pathogenicity is believed to require the activity of cell-wall-degrading enzymes (CWDEs). The sucrose non-fermenting protein kinase 1 gene (SNF1) is a key component of the glucose de-repression pathway in yeast, and a regulator of gene expression for CWDEs in some plant pathogenic fungi. To elucidate the functional role of the SNF1 homolog in F. virguliforme, FvSNF1 was disrupted using a split-marker strategy. Disruption of FvSNF1 in F. virguliforme abolishes galactose utilization and causes poor growth on xylose, arabinose and sucrose. However, the resulting Fvsnf1 mutant grew similar to wild-type and ectopic transformants on glucose, fructose, maltose, or pectin as the main source of carbon. The Fvsnf1 mutant displayed no expression of the gene-encoding galactose oxidase (GAO), a secretory enzyme that catalyzes oxidation of D-galactose. It also exhibited a significant reduction in the expression of several CWDE-coding genes in contrast to the wild-type strain. Greenhouse pathogenicity assays revealed that the Fvsnf1 mutant was severely impaired in its ability to cause SDS on challenged soybean plants. Microscopy and microtome studies on infected roots showed that the Fvsnf1 mutant was defective in colonizing vascular tissue of infected plants. Cross and longitudinal sections of infected roots stained with fluorescein-labeled wheat germ agglutinin and Congo red showed that the Fvsnf1 mutant failed to colonize the xylem vessels and phloem tissue at later stages of infection. Quantification of the fungal biomass in inoculated roots further confirmed a reduced colonization of roots by the Fvsnf1 mutant when compared to the wild type. These findings suggest that FvSNF1 regulates the expression of CWDEs in F. virguliforme, thus affecting the virulence of the fungus on soybean.

Targeting Aflatoxin Biosynthetic Genes.

Chemical detoxification and physical destruction of aflatoxins in foods and feed commodities are mostly unattainable in a way that preserves the edibility of the food. Therefore, preventing mycotoxins in general and aflatoxins in particular from entering the food chain is a better approach. This requires early detection of the aflatoxin-causing organisms. Detection and quantification of aflatoxin-producing fungi has always been a challenge, especially within species of Aspergillus and Penicillium. Culture-based methods require a high level of expertise and a list of sophisticated equipment. Furthermore, even for a trained taxonomist, species that are identical in morphology, physiology, and nutritional aspects can be challenging to classify. Fungal taxonomy has changed over the past few decades; more species are being reclassified, and new species are being described due to advances in sequencing and genome assembly. These developments make the use of PCR-based approaches practical, rapid, and more reliable for the identification of fungi to the species level. This chapter presents a variety of protocols to detect and quantify aflatoxin-producing fungi using mycotoxin biosynthesis pathway genes.

A Network Approach of Gene Co-expression in the Zea mays/Aspergillus flavus Pathosystem to Map Host/Pathogen Interaction Pathways.

A gene co-expression network (GEN) was generated using a dual RNA-seq study with the fungal pathogen Aspergillus flavus and its plant host Zea mays during the initial 3 days of infection. The analysis deciphered novel pathways and mapped genes of interest in both organisms during the infection. This network revealed a high degree of connectivity in many of the previously recognized pathways in Z. mays such as jasmonic acid, ethylene, and reactive oxygen species (ROS). For the pathogen A. flavus, a link between aflatoxin production and vesicular transport was identified within the network. There was significant interspecies correlation of expression between Z. mays and A. flavus for a subset of 104 Z. mays, and 1942 A. flavus genes. This resulted in an interspecies subnetwork enriched in multiple Z. mays genes involved in the production of ROS. In addition to the ROS from Z. mays, there was enrichment in the vesicular transport pathways and the aflatoxin pathway for A. flavus. Included in these genes, a key aflatoxin cluster regulator, AflS, was found to be co-regulated with multiple Z. mays ROS producing genes within the network, suggesting AflS may be monitoring host ROS levels. The entire GEN for both host and pathogen, and the subset of interspecies correlations, is presented as a tool for hypothesis generation and discovery for events in the early stages of fungal infection of Z. mays by A. flavus.

Multilaboratory Comparison of Quantitative PCR Assays for Detection and Quantification of Fusarium virguliforme from Soybean Roots and Soil.

The ability to accurately detect and quantify Fusarium virguliforme, the cause of sudden death syndrome (SDS) in soybean, in samples such as plant root tissue and soil is extremely valuable for accurate disease diagnoses and to address research questions. Numerous quantitative real-time polymerase chain reaction (qPCR) assays have been developed for this pathogen but their sensitivity and specificity for F. virguliforme have not been compared. In this study, six qPCR assays were compared in five independent laboratories using the same set of DNA samples from fungi, plants, and soil. Multicopy gene-based assays targeting the ribosomal DNA intergenic spacer (IGS) or the mitochondrial small subunit (mtSSU) showed relatively high sensitivity (limit of detection [LOD] = 0.05 to 5 pg) compared with a single-copy gene (FvTox1)-based assay (LOD = 5 to 50 pg). Specificity varied greatly among assays, with the FvTox1 assay ranking the highest (100%) and two IGS assays being slightly less specific (95 to 96%). Another IGS assay targeting four SDS-causing fusaria showed lower specificity (70%), while the two mtSSU assays were lowest (41 and 47%). An IGS-based assay showed consistently highest sensitivity (LOD = 0.05 pg) and specificity and inclusivity above 94% and, thus, is suggested as the most useful qPCR assay for F. virguliforme diagnosis and quantification. However, specificity was also above 94% in two other assays and their selection for diagnostics and research will depend on objectives, samples, and materials used. These results will facilitate both fundamental and disease management research pertinent to SDS.

A predicted protein interactome identifies conserved global networks and disease resistance subnetworks in maize.

Interactomes are genome-wide roadmaps of protein-protein interactions. They have been produced for humans, yeast, the fruit fly, and Arabidopsis thaliana and have become invaluable tools for generating and testing hypotheses. A predicted interactome for Zea mays (PiZeaM) is presented here as an aid to the research community for this valuable crop species. PiZeaM was built using a proven method of interologs (interacting orthologs) that were identified using both one-to-one and many-to-many orthology between genomes of maize and reference species. Where both maize orthologs occurred for an experimentally determined interaction in the reference species, we predicted a likely interaction in maize. A total of 49,026 unique interactions for 6004 maize proteins were predicted. These interactions are enriched for processes that are evolutionarily conserved, but include many otherwise poorly annotated proteins in maize. The predicted maize interactions were further analyzed by comparing annotation of interacting proteins, including different layers of ontology. A map of pairwise gene co-expression was also generated and compared to predicted interactions. Two global subnetworks were constructed for highly conserved interactions. These subnetworks showed clear clustering of proteins by function. Another subnetwork was created for disease response using a bait and prey strategy to capture interacting partners for proteins that respond to other organisms. Closer examination of this subnetwork revealed the connectivity between biotic and abiotic hormone stress pathways. We believe PiZeaM will provide a useful tool for the prediction of protein function and analysis of pathways for Z. mays researchers and is presented in this paper as a reference tool for the exploration of protein interactions in maize.

The genome sequence of the fungal pathogen Fusarium virguliforme that causes sudden death syndrome in soybean.

UNLABELLED: Fusarium virguliforme causes sudden death syndrome (SDS) of soybean, a disease of serious concern throughout most of the soybean producing regions of the world. Despite the global importance, little is known about the pathogenesis mechanisms of F. virguliforme. Thus, we applied Next-Generation DNA Sequencing to reveal the draft F. virguliforme genome sequence and identified putative pathogenicity genes to facilitate discovering the mechanisms used by the pathogen to cause this disease. METHODOLOGY/PRINCIPAL FINDINGS: We have generated the draft genome sequence of F. virguliforme by conducting whole-genome shotgun sequencing on a 454 GS-FLX Titanium sequencer. Initially, single-end reads of a 400-bp shotgun library were assembled using the PCAP program. Paired end sequences from 3 and 20 Kb DNA fragments and approximately 100 Kb inserts of 1,400 BAC clones were used to generate the assembled genome. The assembled genome sequence was 51 Mb. The N50 scaffold number was 11 with an N50 Scaffold length of 1,263 Kb. The AUGUSTUS gene prediction program predicted 14,845 putative genes, which were annotated with Pfam and GO databases. Gene distributions were uniform in all but one of the major scaffolds. Phylogenic analyses revealed that F. virguliforme was closely related to the pea pathogen, Nectria haematococca. Of the 14,845 F. virguliforme genes, 11,043 were conserved among five Fusarium species: F. virguliforme, F. graminearum, F. verticillioides, F. oxysporum and N. haematococca; and 1,332 F. virguliforme-specific genes, which may include pathogenicity genes. Additionally, searches for candidate F. virguliforme pathogenicity genes using gene sequences of the pathogen-host interaction database identified 358 genes. CONCLUSIONS: The F. virguliforme genome sequence and putative pathogenicity genes presented here will facilitate identification of pathogenicity mechanisms involved in SDS development. Together, these resources will expedite our efforts towards discovering pathogenicity mechanisms in F. virguliforme. This will ultimately lead to improvement of SDS resistance in soybean.

Mating-type distribution and genetic diversity of Cercospora sojina populations on soybean from Arkansas: evidence for potential sexual reproduction.

Cercospora sojina causes frogeye leaf spot of soybean, which can cause serious economic losses in the United States. In this study, 132 C. sojina isolates were collected from six fields (from two counties, Cross and Crawford) in Arkansas. To determine mating type, a multiplex polymerase chain reaction assay was developed with primers specific for C. sojina. Of the 132 isolates, 68 isolates had the MAT1-1-1 idiomorph and 64 isolates had the MAT1-2 idiomorph; no isolates possessed both idiomorphs. Both mating types were present in a variety of spatial scales, including separate lesions on individual leaves. Clone-corrected data from eight microsatellites indicated that mating-type loci were present in approximately equal proportions in all populations analyzed, which suggests that Arkansas populations of C. sojina are undergoing cryptic sexual reproduction. All six populations evaluated had high genotypic diversity of 26 to 79%. In addition, among strains isolated from a single leaf, multiple and distinct haplotypes were associated with both mating types, supporting the hypothesis that sexual reproduction occurs within the populations. Most populations showed significant gametic disequilibrium but levels of disequilibrium were relatively low, particularly in populations from Crawford County. A low differentiation index (GST) was observed for all simple-sequence repeat markers across all populations. Furthermore, the value of G statistics between populations suggests that significant genetic exchange exists among the populations. Taken together, these results demonstrate that C. sojina populations from Arkansas are genetically diverse and most likely undergoing sexual reproduction.

Identification of genes differentially expressed during aflatoxin biosynthesis in Aspergillus flavus and Aspergillus parasiticus.

A complex regulatory network governs the biosynthesis of aflatoxin. While several genes involved in aflatoxin production are known, their action alone cannot account for its regulation. Arrays of clones from an Aspergillus flavus cDNA library and glass slide microarrays of ESTs were screened to identify additional genes. An initial screen of the cDNA clone arrays lead to the identification of 753 unique ESTs. Many showed sequence similarity to known metabolic and regulatory genes; however, no function could be ascribed to over 50% of the ESTs. Gene expression analysis of Aspergillus parasiticus grown under conditions conducive and non-conductive for aflatoxin production was evaluated using glass slide microarrays containing the 753 ESTs. Twenty-four genes were more highly expressed during aflatoxin biosynthesis and 18 genes were more highly expressed prior to aflatoxin biosynthesis. No predicted function could be ascribed to 18 of the 24 genes whose elevated expression was associated with aflatoxin biosynthesis.