Aspergillus flavus pangenome (AflaPan) uncovers novel aflatoxin and secondary metabolite associated gene clusters.

BACKGROUND: Aspergillus flavus is an important agricultural and food safety threat due to its production of carcinogenic aflatoxins. It has high level of genetic diversity that is adapted to various environments. Recently, we reported two reference genomes of A. flavus isolates, AF13 (MAT1-2 and highly aflatoxigenic isolate) and NRRL3357 (MAT1-1 and moderate aflatoxin producer). Where, an insertion of 310 kb in AF13 included an aflatoxin producing gene bZIP transcription factor, named atfC. Observations of significant genomic variants between these isolates of contrasting phenotypes prompted an investigation into variation among other agricultural isolates of A. flavus with the goal of discovering novel genes potentially associated with aflatoxin production regulation. Present study was designed with three main objectives: (1) collection of large number of A. flavus isolates from diverse sources including maize plants and field soils; (2) whole genome sequencing of collected isolates and development of a pangenome; and (3) pangenome-wide association study (Pan-GWAS) to identify novel secondary metabolite cluster genes. RESULTS: Pangenome analysis of 346 A. flavus isolates identified a total of 17,855 unique orthologous gene clusters, with mere 41% (7,315) core genes and 59% (10,540) accessory genes indicating accumulation of high genomic diversity during domestication. 5,994 orthologous gene clusters in accessory genome not annotated in either the A. flavus AF13 or NRRL3357 reference genomes. Pan-genome wide association analysis of the genomic variations identified 391 significant associated pan-genes associated with aflatoxin production. Interestingly, most of the significantly associated pan-genes (94%; 369 associations) belonged to accessory genome indicating that genome expansion has resulted in the incorporation of new genes associated with aflatoxin and other secondary metabolites. CONCLUSION: In summary, this study provides complete pangenome framework for the species of Aspergillus flavus along with associated genes for pathogen survival and aflatoxin production. The large accessory genome indicated large genome diversity in the species A. flavus, however AflaPan is a closed pangenome represents optimum diversity of species A. flavus. Most importantly, the newly identified aflatoxin producing gene clusters will be a new source for seeking aflatoxin mitigation strategies and needs new attention in research.

Assessment of Prickly Sida as a Potential Inoculum Source for Sida Golden Mosaic Virus in Commercial Snap Bean Farms in Georgia, United States.

Sida golden mosaic virus (SiGMV), an obligate pathogen that infects snap beans (Phaseolus vulgaris), is known to infect prickly sida (Sida spinosa L.), which is a common weed in agricultural farms in Georgia. Prickly sida has also been reported as a suitable host of sweetpotato whitefly (Bemisia tabaci), the vector of SiGMV. Despite being a host for both SiGMV and its vector, the role of prickly sida as a reservoir and inoculum source for SiGMV in snap bean farms has not been evaluated. This study was conducted to document the occurrence of SiGMV-infected prickly sida plants and to assess its potential role as a source of SiGMV inoculum in snap bean farms. A survey of 17 commercial snap bean farms conducted in spring 2021 confirmed the presence of SiGMV-infected prickly sida in southern Georgia. In fall 2021 and 2022, on-farm field trials were conducted in four commercial farms where SiGMV-infected prickly sida plants were documented earlier as a part of survey in spring 2021. The spatial distribution and temporal patterns of adult whiteflies and SiGMV on snap bean were compared between macroplots (13.7 × 30.5 m) "with prickly sida" or "without prickly sida" that were at least 232 m apart from each other. We did not observe any consistent differences in counts of adult whiteflies between macroplots with or without prickly sida in the four commercial farms. SiGMV infection was detected earlier and with higher incidences in snap bean macroplots "with prickly sida" compared with macroplots "without prickly sida." An apparent disease gradient was observed in two of the four farms assessed. Higher SiGMV incidences were observed on the edges of macroplots "with prickly sida." These findings indicate prickly sida as a potential natural reservoir and a source for SiGMV spread in snap bean farms in southern Georgia.

Cotton leafroll dwarf disease: An enigmatic viral disease in cotton.

TAXONOMY: Cotton leafroll dwarf virus (CLRDV) is a member of the genus Polerovirus, family Solemoviridae. Geographical Distribution: CLRDV is present in most cotton-producing regions worldwide, prominently in North and South America. PHYSICAL PROPERTIES: The virion is a nonenveloped icosahedron with T = 3 icosahedral lattice symmetry that has a diameter of 26-34 nm and comprises 180 molecules of the capsid protein. The CsCl buoyant density of the virion is 1.39-1.42 g/cm3 and S20w is 115-127S. Genome: CLRDV shares genomic features with other poleroviruses; its genome consists of monopartite, single-stranded, positive-sense RNA, is approximately 5.7-5.8 kb in length, and is composed of seven open reading frames (ORFs) with an intergenic region between ORF2 and ORF3a. TRANSMISSION: CLRDV is transmitted efficiently by the cotton aphid (Aphis gossypii Glover) in a circulative and nonpropagative manner. Host: CLRDV has a limited host range. Cotton is the primary host, and it has also been detected in different weeds in and around commercial cotton fields in Georgia, USA. SYMPTOMS: Cotton plants infected early in the growth stage exhibit reddening or bronzing of foliage, maroon stems and petioles, and drooping. Plants infected in later growth stages exhibit intense green foliage with leaf rugosity, moderate to severe stunting, shortened internodes, and increased boll shedding/abortion, resulting in poor boll retention. These symptoms are variable and are probably influenced by the time of infection, plant growth stage, varieties, soil health, and geographical location. CLRDV is also often detected in symptomless plants. CONTROL: Vector management with the application of chemical insecticides is ineffective. Some host plant varieties grown in South America are resistant, but all varieties grown in the United States are susceptible. Integrated disease management strategies, including weed management and removal of volunteer stalks, could reduce the abundance of virus inoculum in the field.

Strong Resistance to Early and Late Leaf Spot in Peanut-Compatible Wild-Derived Induced Allotetraploids.

Early (ELS) and late leaf spots (LLS) are two of the most destructive diseases in peanut (Arachis hypogaea). They can cause severe plant defoliation and tremendous yield loss in the absence of fungicide applications. The high costs of fungicides, their potential for deleterious effects on the environment, the tightening of regulations, and the development of fungicide resistance call for additional management strategies to mitigate both diseases. The use of resistant cultivars is an economical way to manage these diseases, but there are limited sources of leaf spot resistance available in cultivated peanuts. Wild peanut species are excellent sources of resistance, but because of the ploidy level, they do not produce fertile progeny when crossed with peanut. To circumvent this, induced allotetraploids were developed so that resistance traits can be introgressed from wild species into peanut. Here we screened 13 induced allotetraploids (BatDur1, BatDur2, BatSten1, GregSten1, IpaCor1, IpaCor2, IpaDur1, IpaDur2, IpaDur3, IpaVillo1, MagDur1, MagSten1, and ValSten1) against ELS and LLS using a detached leaf bioassay to characterize various components of resistance and identify genotypes that can be used for breeding. Strong associations were detected between the measured components of disease resistance (r = 0.91 to 1.0; P < 0.001) and between ELS and LLS bioassays (r = 0.81 to 0.91; P < 0.001). Induced allotetraploids, particularly those derived from A. stenosperma, exhibited fewer and smaller lesions with limited sporulation and reductions in disease progression in both bioassays. Interestingly, allotetraploids derived from the B-genome peanut progenitor A. ipaënsis were almost as susceptible as cultivated genotypes. The overall results reveal several sources of foliar disease resistance that can be used in breeding programs for ELS and LLS resistance.[Formula: see text] Copyright © 2023 The Author(s). This is an open-access article distributed under the CC BY 4.0 International license.

Meta-Analysis of the Field Efficacy of Seed- and Soil-Applied Nematicides on Meloidogyne incognita and Rotylenchulus reniformis Across the U.S. Cotton Belt.

Meta-analysis was used to compare yield protection and nematode suppression provided by two seed-applied and two soil-applied nematicides against Meloidogyne incognita and Rotylenchulus reniformis on cotton across 3 years and several trial locations in the U.S. Cotton Belt. Nematicides consisted of thiodicarb- and fluopyram-treated seed, aldicarb and fluopyram applied in furrow, and combinations of the seed treatments and soil-applied fluopyram. The nematicides had no effect on nematode reproduction or root infection but had a significant impact on seed cotton yield response ([Formula: see text]), with an average increase of 176 and 197 kg/ha relative to the nontreated control in M. incognita and R. reniformis infested fields, respectively. However, because of significant variation in yield protection and nematode suppression by nematicides, five or six moderator variables (cultivar resistance [M. incognita only], nematode infestation level, nematicide treatment, application method, trial location, and growing season) were used depending on nematode species. In M. incognita-infested fields, greater yield protection was observed with nematicides applied in furrow and with seed-applied + in-furrow than with solo seed-applied nematicide applications. Most notable of these in-furrow nematicides were aldicarb and fluopyram (>131 g/ha) with or without a seed-applied nematicide compared with thiodicarb. In R. reniformis-infested fields, moderator variables provided no further explanation of the variation in yield response produced by nematicides. Furthermore, moderator variables provided little explanation of the variation in nematode suppression by nematicides in M. incognita- and R. reniformis-infested fields. The limited explanation by the moderator variables on the field efficacy of nematicides in M. incognita- and R. reniformis-infested fields demonstrates the difficulty of managing these pathogens with nonfumigant nematicides across the U.S. Cotton Belt.

Comparative genomics of host-specialized populations of Corynespora cassiicola causing target spot epidemics in the southeastern United States.

Numerous plant-pathogenic fungi secrete necrotrophic effectors (syn. host-selective toxins) that are important determinants of pathogenicity and virulence in species that have a necrotrophic lifestyle. Corynespora cassiicola is a necrotrophic fungus causing emerging target spot epidemics in the southeastern United States (US). Previous studies revealed that populations of C. cassiicola from cotton, soybean, and tomato are clonal, host specialized and genetically distinct. Additionally, cassiicolin - the necrotrophic effector identified in some C. cassiicola isolates - is an important toxin for virulence on rubber. It is encoded by seven Cas gene variants. Our goal was to conduct comparative genomic analyses to identify variation among putative necrotrophic effector genes and to determine if lack of one of the mating-types explained clonal populations in C. cassiicola causing outbreaks in the southeastern US and the apparent absence of sexual reproduction worldwide. A total of 12 C. cassiicola genomes, with four each from isolates from tomato, soybean, and cotton, were sequenced using an Illumina Next Seq platform. Each genome was assembled de novo, compared with the reference genome from rubber, and searched for known Cas, and other gene clusters with homologs of secondary metabolites. Cas2 and/or Cas6 were present in isolates from soybean in the southeastern US, whereas Cas1 and Cas2 were present in isolates from cotton in the southeastern US. In addition, several toxin genes, including the T-toxin biosynthetic genes were present in all C. cassiicola from cotton, soybean, and tomato. The mating-type locus was identified in all of the sequenced genomes, with the MAT1-1 idiomorph present in all cotton isolates and the rubber isolate, whereas the MAT1-2 idiomorph was present in all soybean isolates. We developed a PCR-based marker for mating-type in C. cassiicola. Both mating types were present in isolates from tomato. Thus, C. cassiicola has both mating-types necessary for sexual reproduction, but the absence of both mating-types within soybean and cotton populations could explain clonality in these populations. Variation in necrotrophic effectors may underlie host specialization and disease emergence of target spot on cotton, soybean, and tomato in the southeastern US.

Genome analysis of cotton leafroll dwarf virus reveals variability in the silencing suppressor protein, genotypes and genomic recombinants in the USA.

Cotton leafroll dwarf virus (CLRDV) is an emerging virus in cotton production in Georgia and several other Southeastern states in the USA. To better understand the genetic diversity of the virus population, the near complete genome sequences of six isolates from Georgia and one from Alabama were determined. The isolates sequenced were 5,866 nucleotides with seven open reading frames (ORFs). The isolates from Georgia were >94% identical with other isolates from the USA and South America. In the silencing suppressor protein (P0), at amino acid position 72, the isolates from Georgia and Alabama had a valine (V), similar to resistant-breaking 'atypical' genotypes in South America, while the Texas isolate had isoleucine (I), similar to the more aggressive 'typical' genotypes of CLRDV. At position 120, arginine (R) is unique to Georgia and China isolates, but absent in Alabama, Texas and South American isolates. Ten potential recombinant events were detected in the isolates sequenced. An increased understanding of CLRDV population structure and genetic diversity will help develop management strategies for CLRDV in the USA cotton belt.

Patterns of Seed-to-Seedling Transmission of Xanthomonas citri pv. malvacearum, the Causal Agent of Cotton Bacterial Blight.

Cotton bacterial blight (CBB), caused by Xanthomonas citri pv. malvacearum, was a major disease of cotton in the United States in the early part of the twentieth century. The reemergence of CBB revealed many gaps in our understanding of this important disease. In this study, we employed a wild-type (WT) field isolate of X. citri pv. malvacearum from Georgia (U.S.A.) to generate a nonpathogenic hrcV mutant lacking a functional type-III secretion system (T3SS-). We tagged the WT and T3SS- strains with an auto-bioluminescent Tn7 reporter and compared colonization patterns of CBB-susceptible and CBB-resistant cotton seedlings using macroscopic image analysis and bacterial load enumeration. WT and T3SS- X. citri pv. malvacearum strains colonized cotton cotyledons of CBB-resistant and CBB-susceptible cotton cultivars. However, X. citri pv. malvacearum populations were significantly higher in CBB-susceptible seedlings inoculated with the WT strain. Additionally, WT and T3SS- X. citri pv. malvacearum strains systemically colonized true leaves, although at different rates. Finally, we observed that seed-to-seedling transmission of X. citri pv. malvacearum may involve systemic spread through the vascular tissue of cotton plants. These findings yield novel insights into potential X. citri pv. malvacearum reservoirs for CBB outbreaks.

Effect of In-Furrow Application of Fluopyram on Leaf Spot Diseases of Peanut.

In peanut (Arachis hypogaea) production, in-furrow applications of the premix combination of the succinate-dehydrogenase-inhibitor (SDHI) fungicide and nematicide fluopyram and the insecticide imidacloprid are used primarily for management of nematode pests and for preventing feeding damage on foliage caused by tobacco thrips (Frankliniella fusca). Fluopyram is also active against many fungal pathogens. However, the effect of in-furrow applications of fluopyram on early leaf spot (Passalora arachidicola) or late leaf spot (Nothopassalora personata) has not been characterized. The purpose of this study was to determine the effects of in-furrow applications of fluopyram + imidacloprid or fluopyram alone on leaf spot epidemics. Field experiments were conducted in Tifton, GA in 2015, 2016, and 2018 to 2020. In all experiments, in-furrow applications of fluopyram + imidacloprid provided extended suppression of early leaf spot and late leaf spot epidemics compared with the nontreated control. In 2020, there was no difference between the effects of fluopyram + imidacloprid and fluopyram alone on leaf spot epidemics. Results indicated that fluopyram could complement early-season leaf spot management programs. Use of in-furrow applications of fluopyram should be considered as an SDHI fungicide application for resistance management purposes.

Assessment of Quinone Outside Inhibitor Sensitivity and Frogeye Leaf Spot Race of Cercospora sojina in Georgia Soybean.

Frogeye leaf spot (FLS), caused by the fungal pathogen Cercospora sojina K. Hara, is a foliar disease of soybean (Glycine max L. [Merr.]) responsible for yield reductions throughout the major soybean-producing regions of the world. In the United States, management of FLS relies heavily on the use of resistant cultivars and in-season fungicide applications, specifically within the class of quinone outside inhibitors (QoIs), which has resulted in the development of fungicide resistance in many states. In 2018 and 2019, 80 isolates of C. sojina were collected from six counties in Georgia and screened for QoI fungicide resistance using molecular and in vitro assays, with resistant isolates being confirmed from three counties. Additionally, 50 isolates, including a "baseline isolate" with no prior fungicide exposure, were used to determine the percent reduction of mycelial growth to two fungicides, azoxystrobin and pyraclostrobin, at six concentrations: 0.0001, 0.001, 0.01, 0.1, 1, and 10 µg ml-1. Mycelial growth observed for resistant isolates varied significantly from both sensitive isolates and baseline isolate for azoxystrobin concentrations of 10, 1, 0.1, and 0.01 µg ml-1 and for pyraclostrobin concentrations of 10, 1, 0.1, 0.01, and 0.001 µg ml-1. Moreover, 40 isolates were used to evaluate pathogen race on six soybean differential cultivars by assessing susceptible or resistant reactions. Isolate reactions suggested 12 races of C. sojina present in Georgia, 4 of which have not been previously described. Species richness indicators (rarefaction and abundance-based coverage estimators) indicated that within-county C. sojina race numbers were undersampled in this study, suggesting the potential for the presence of either additional undescribed races or known but unaccounted for races in Georgia. However, no isolates were pathogenic on 'Davis', a differential cultivar carrying the Rcs3 resistance allele, suggesting that the gene is still an effective source of resistance in Georgia.

Draft Genome Sequences of One Aspergillus parasiticus Isolate and Nine Aspergillus flavus Isolates with Varying Stress Tolerance and Aflatoxin Production.

Aspergillus flavus and Aspergillus parasiticus produce carcinogenic aflatoxins during crop infection, with extensive variations in production among isolates, ranging from atoxigenic to highly toxigenic. Here, we report draft genome sequences of one A. parasiticus isolate and nine A. flavus isolates from field environments for use in comparative, functional, and phylogenetic studies.

Two New Aspergillus flavus Reference Genomes Reveal a Large Insertion Potentially Contributing to Isolate Stress Tolerance and Aflatoxin Production.

Efforts in genome sequencing in the Aspergillus genus have led to the development of quality reference genomes for several important species including A. nidulans, A. fumigatus, and A. oryzae However, less progress has been made for A. flavus As part of the effort of the USDA-ARS Annual Aflatoxin Workshop Fungal Genome Project, the isolate NRRL3357 was sequenced and resulted in a scaffold-level genome released in 2005. Our goal has been biologically driven, focusing on two areas: isolate variation in aflatoxin production and drought stress exacerbating aflatoxin production by A. flavus Therefore, we developed two reference pseudomolecule genome assemblies derived from chromosome arms for two isolates: AF13, a MAT1-2, highly stress tolerant, and highly aflatoxigenic isolate; and NRRL3357, a MAT1-1, less stress tolerant, and moderate aflatoxin producer in comparison to AF13. Here, we report these two reference-grade assemblies for these isolates through a combination of PacBio long-read sequencing and optical mapping, and coupled them with comparative, functional, and phylogenetic analyses. This analysis resulted in the identification of 153 and 45 unique genes in AF13 and NRRL3357, respectively. We also confirmed the presence of a unique 310 Kb insertion in AF13 containing 60 genes. Analysis of this insertion revealed the presence of a bZIP transcription factor, named atfC, which may contribute to isolate pathogenicity and stress tolerance. Phylogenomic analyses comparing these and other available assemblies also suggest that the species complex of A. flavus is polyphyletic.

Genetic Diversity and Population Structure of Races of Fusarium oxysporum Causing Cotton Wilt.

To better understand the evolution of virulence we are interested in identifying the genetic basis of this trait in pathogenic fungi and in developing tools for the rapid characterization of variation in virulence among populations associated with epidemics. Fusarium oxysporum f. sp. vasinfectum (FOV) is a haploid fungus that causes devastating outbreaks of Fusarium wilt of cotton wherever it is grown. In the United States, six nominal races and eleven genotypes of FOV have been characterized based on the translation elongation factor (EF-1α) gene and intergenic spacer region (IGS), but it is unclear how race or genotype based on these regions relates to population structure or virulence. We used genotyping-by-sequencing to identify SNPs and determine genetic diversity and population structure among 86 diverse FOV isolates. Six individuals of Fusarium oxysporum closely related to FOV were genotyped and included in some analyses. Between 193 and 354 SNPs were identified and included in the analyses depending on the pipeline and filtering criteria used. Phylogenetic trees, minimum spanning networks (MSNs), principal components analysis (PCA), and discriminant analysis of principal components (DAPC) demonstrated that races and genotypes of FOV are generally not structured by EF-1α genotype, nor are they monophyletic groups with the exception of race 4 isolates, which are distinct. Furthermore, DAPC identified between 11 and 14 genetically distinct clusters of FOV, whereas only eight EF-1α genotypes were represented among isolates; suggesting that FOV, especially isolates within the widely distributed and common race 1 genotype, is more genetically diverse than currently recognized.

Adding Epidemiologically Important Meteorological Data to Peanut Rx, the Risk Assessment Framework for Spotted Wilt of Peanut.

Management of disease affecting peanut in the southeastern United States has benefited from extensive field research identifying disease-associated risk factors since the 1990s. An assessment of risk factors associated with tomato spotted wilt (TSW), caused by tomato spotted wilt virus and spread exclusively by thrips, is available to growers through Peanut Rx, a tool developed to inform peanut management decisions. Peanut Rx provides an assessment of relative TSW risk as an index. The assessment provides information about the relative degree to which a field characterized by a specified suite of practices is at risk of crop loss caused by TSW. Loss results when infection occurs, and infection rates are determined, in part, by factors outside a grower's control, primarily the abundance of dispersing, viruliferous thrips. In this study, we incorporated meteorological variables useful for predicting thrips dispersal, increasing the robustness of the Peanut Rx framework in relation to variation in the weather. We used data from field experiments and a large grower survey to estimate the relationships between weather and TSW risk mediated by thrips vectors, and developed an addition to Peanut Rx that proved informative and easy to implement. The expected temporal occurrence of major thrips flights, as a function of heat and precipitation, was translated into the existing risk-point system of Peanut Rx. Results from the grower survey further demonstrated the validity of Peanut Rx for guiding growers' decisions to minimize risk of TSW.

Relating Peanut Rx Risk Factors to Epidemics of Early and Late Leaf Spot of Peanut.

Previous research has demonstrated the efficacy of prescription fungicide programs, based upon Peanut Rx, to reduce combined effects of early leaf spot (ELS), caused by Passalora arachidicola (Cercospora arachidicola), and late leaf spot (LLS), caused by Nothopassalora personata (syn. Cercosporidium personatum), but the potential of Peanut Rx to predict each disease has never been formally evaluated. From 2010 to 2016, non-fungicide-treated peanut plots in Georgia and Florida were sampled to monitor the development of ELS and LLS. This resulted in 168 cases (unique combinations of Peanut Rx risk factors) with associated total leaf spot risk points ranging from 40 to 100. Defoliation ranged from 13.9 to 100%, and increased significantly with increasing total risk points (conditional R2 = 0.56; P < 0.001). Leaf spot onset (time in days after planting [DAP] when either leaf spot reached 1% lesion incidence), ELS onset, and LLS onset ranged from 29 to 140, 29 to 142, and 50 to 143 DAP, respectively, and decreased significantly with increasing risk points. Standardized AUDPC of ELS was significantly affected by risk points (conditional R2 = 0.53, P < 0.001), but not for LLS. After removing redundant Peanut Rx factors, planting date, rotation, historical leaf spot prevalence, cultivar, and field history were used as fixed effects in mixed effect regression models to evaluate their contribution to leaf spot, ELS or LLS prediction. Results from mixed effects regression confirmed that the selected Peanut Rx risk factors contributed to the variability of at least one measurement of development of combined or separate epidemics of ELS and LLS, but not all factors affected ELS and LLS equally. Historical leaf spot prevalence, a new potential preplant risk factor, was a consistent predictor of the dominant disease(s) observed in the field. Results presented here demonstrate that Peanut Rx is a very effective tool for predicting leaf spot onset regardless of which leaf spot is predominant, but also suggest that associated risk does not reflect the same development for each disease. These data will be useful for refining thresholds for differentiating high, moderate, and low risk fields, and reevaluating the timing of fungicide applications in reduced input programs with respect to disease onset.

Carbohydrate, glutathione, and polyamine metabolism are central to Aspergillus flavus oxidative stress responses over time.

BACKGROUND: The primary and secondary metabolites of fungi are critical for adaptation to environmental stresses, host pathogenicity, competition with other microbes, and reproductive fitness. Drought-derived reactive oxygen species (ROS) have been shown to stimulate aflatoxin production and regulate in Aspergillus flavus, and may function in signaling with host plants. Here, we have performed global, untargeted metabolomics to better understand the role of aflatoxin production in oxidative stress responses, and also explore isolate-specific oxidative stress responses over time. RESULTS: Two field isolates of A. flavus, AF13 and NRRL3357, possessing high and moderate aflatoxin production, respectively, were cultured in medium with and without supplementation with 15 mM H2O2, and mycelia were collected following 4 and 7 days in culture for global metabolomics. Overall, 389 compounds were described in the analysis which encompassed 9 biological super-pathways and 47 sub-pathways. These metabolites were examined for differential accumulation. Significant differences were observed in both isolates in response to oxidative stress and when comparing sampling time points. CONCLUSIONS: The moderately high aflatoxin-producing isolate, NRRL3357, showed extensive stimulation of antioxidant mechanisms and pathways including polyamines metabolism, glutathione metabolism, TCA cycle, and lipid metabolism while the highly aflatoxigenic isolate, AF13, showed a less vigorous response to stress. Carbohydrate pathway levels also imply that carbohydrate repression and starvation may influence metabolite accumulation at the later timepoint. Higher conidial oxidative stress tolerance and antioxidant capacity in AF13 compared to NRRL3357, inferred from their metabolomic profiles and growth curves over time, may be connected to aflatoxin production capability and aflatoxin-related antioxidant accumulation. The coincidence of several of the detected metabolites in H2O2-stressed A. flavus and drought-stressed hosts also suggests their potential role in the interaction between these organisms and their use as markers/targets to enhance host resistance through biomarker selection or genetic engineering.

Biocontrol Strains Differentially Shift the Genetic Structure of Indigenous Soil Populations of Aspergillus flavus.

Biocontrol using non-aflatoxigenic strains of Aspergillus flavus has the greatest potential to mitigate aflatoxin contamination in agricultural produce. However, factors that influence the efficacy of biocontrol agents in reducing aflatoxin accumulation under field conditions are not well-understood. Shifts in the genetic structure of indigenous soil populations of A. flavus following application of biocontrol products Afla-Guard and AF36 were investigated to determine how these changes can influence the efficacy of biocontrol strains in reducing aflatoxin contamination. Soil samples were collected from maize fields in Alabama, Georgia, and North Carolina in 2012 and 2013 to determine changes in the population genetic structure of A. flavus in the soil following application of the biocontrol strains. A. flavus L was the most dominant species of Aspergillus section Flavi with a frequency ranging from 61 to 100%, followed by Aspergillus parasiticus that had a frequency of <35%. The frequency of A. flavus L increased, while that of A. parasiticus decreased after application of biocontrol strains. A total of 112 multilocus haplotypes (MLHs) were inferred from 1,282 isolates of A. flavus L using multilocus sequence typing of the trpC, mfs, and AF17 loci. A. flavus individuals belonging to the Afla-Guard MLH in the IB lineage were the most dominant before and after application of biocontrol strains, while individuals of the AF36 MLH in the IC lineage were either recovered in very low frequencies or not recovered at harvest. There were no significant (P > 0.05) differences in the frequency of individuals with MAT1-1 and MAT1-2 for clone-corrected MLH data, an indication of a recombining population resulting from sexual reproduction. Population mean mutation rates were not different across temporal and spatial scales indicating that mutation alone is not a driving force in observed multilocus sequence diversity. Clustering based on principal component analysis identified two distinct evolutionary lineages (IB and IC) across all three states. Additionally, patristic distance analysis revealed phylogenetic incongruency among single locus phylogenies which suggests ongoing genetic exchange and recombination. Levels of aflatoxin accumulation were very low except in North Carolina in 2012, where aflatoxin levels were significantly (P < 0.05) lower in grain from treated compared to untreated plots. Phylogenetic analysis showed that Afla-Guard was more effective than AF36 in shifting the indigenous soil populations of A. flavus toward the non-toxigenic or low aflatoxin producing IB lineage. These results suggest that Afla-Guard, which matches the genetic and ecological structure of indigenous soil populations of A. flavus in Alabama, Georgia, and North Carolina, is likely to be more effective in reducing aflatoxin accumulation and will also persist longer in the soil than AF36 in the southeastern United States.

Complete Genome Segment Sequences of Tomato Chlorotic Spot Virus from Peanut in Haiti.

Tomato chlorotic spot virus (TCSV) is emerging as a significant constraint to vegetable and legume crops in the Americas. The complete genome sequence of a TCSV isolate naturally infecting peanut (Arachis hypogea) in Haiti was determined in the effort to build epidemiological knowledge of the virus.

Fusarium wilt of cotton may commonly result from the interaction of Fusarium oxysporum f. sp. vasinfectum with Belonolaimus longicaudatus.

The interaction between Fusarium oxysporum f. sp. vasinfectum (Fov) and Meloidogyne incognita (root-knot nematode) resulting in Fusarium wilt (FW) of cotton is well-known. Although Belonolaimus longicaudatus (sting nematode) can also interact with Fov and cause FW, it has long been believed that virtually all of the FW in Georgia is caused by the interaction of Fov with M. incognita. In recent years, FW has been reported more frequently in Georgia, which suggests that something affecting the disease complex may have changed. In 2015 and 2016, a survey of 27 Georgia cotton fields in 10 counties was conducted. At least 10 soil and stem samples per field were collected from individual plants showing symptoms of FW to quantify plant-parasitic nematode levels and identify Fov races. Fov race 1 was identified in all samples in 2015, but one sample also had the LA110 genotype and another sample also had the LA108 genotype. In 2016, all Fov races and genotypes found in 2015 were present, however, MDS-12 and LA127/140 also were found. Meloidogyne incognita was present in 18% of fields in 2015 and 40% in 2016, whereas B. longicaudatus was present in all fields in 2015 and 75% of fields in 2016. Regardless of whether they occurred separately or together, M. incognita and B. longicaudatus were present, respectively, in 18% and 55% of individual samples in 2015 and 40% and 51% in 2016. However, M. incognita without B. longicaudatus was found in 7% of samples in 2015 and 34% in 2016, whereas B. longicaudatus without M. incognita was found in 45% of samples in 2015 and 44% in 2016. We conclude that Fov race 1 continues to be the dominant race in Georgia and many instances of FW in Georgia may be due to Fov interacting with B. longicaudatus and not M. incognita as previously believed.The interaction between Fusarium oxysporum f. sp. vasinfectum (Fov) and Meloidogyne incognita (root-knot nematode) resulting in Fusarium wilt (FW) of cotton is well-known. Although Belonolaimus longicaudatus (sting nematode) can also interact with Fov and cause FW, it has long been believed that virtually all of the FW in Georgia is caused by the interaction of Fov with M. incognita. In recent years, FW has been reported more frequently in Georgia, which suggests that something affecting the disease complex may have changed. In 2015 and 2016, a survey of 27 Georgia cotton fields in 10 counties was conducted. At least 10 soil and stem samples per field were collected from individual plants showing symptoms of FW to quantify plant-parasitic nematode levels and identify Fov races. Fov race 1 was identified in all samples in 2015, but one sample also had the LA110 genotype and another sample also had the LA108 genotype. In 2016, all Fov races and genotypes found in 2015 were present, however, MDS­12 and LA127/140 also were found. Meloidogyne incognita was present in 18% of fields in 2015 and 40% in 2016, whereas B. longicaudatus was present in all fields in 2015 and 75% of fields in 2016. Regardless of whether they occurred separately or together, M. incognita and B. longicaudatus were present, respectively, in 18% and 55% of individual samples in 2015 and 40% and 51% in 2016. However, M. incognita without B. longicaudatus was found in 7% of samples in 2015 and 34% in 2016, whereas B. longicaudatus without M. incognita was found in 45% of samples in 2015 and 44% in 2016. We conclude that Fov race 1 continues to be the dominant race in Georgia and many instances of FW in Georgia may be due to Fov interacting with B. longicaudatus and not M. incognita as previously believed.