Genetic and Pathogenic Variability among Isolates of Sporisorium reilianum Causing Sorghum Head Smut.

Sporisorium reilianum, the causal agent of sorghum (Sorghum bicolor (L.) Moench) head smut, is present in most sorghum-producing regions. This seed replacement fungal disease can reduce yield by up to 80% in severely infected fields. Management of this disease can be challenging due to the appearance of different pathotypes within the pathogenic population. In this research, the genetic variability and pathogenicity of isolates collected from five Texas Counties was conducted. Due to the lack of available space, 21 out of 32 sequenced isolates were selected and evaluated for virulence patterns on the six sorghum differentials, Tx7078, BTx635, SC170-6-17 (TAM2571), SA281 (Early Hegari), Tx414, and BTx643. The results reveal the occurrence of a new pathotype, 1A, and four previously documented US pathotypes when the 21 isolates were evaluated for virulence patterns on the differentials. The most prevalent was pathotype 5, which was recovered from Brazos, Hidalgo, Nueces, and Willacy Counties, Texas. This pathotype was followed by 1A and 6 in frequency of recovery. Pathotype 4 was identified only from isolates collected from Hidalgo County, while pathotype 1 was from Burleson County, Texas. It appeared that the previous US head smut pathotypes (2 and 3) are no longer common, and the new pathotypes, 1A, 5, and 6, are now predominant. The phylogenetic tree constructed from the single-nucleotide polymorphism (SNP) data through the neighbor-joining method showed high genetic diversity among the tested isolates. Some of the diverse clades among the tested isolates were independent of their sampled locations. Notably, HS37, HS49, and HS65 formed a clade and were classified as 1A in the virulence study, while HS 61 and HS 66, which were collected from Nueces County, were grouped and identified as pathotype 5.

Genetic mapping and prediction for novel lesion mimic in maize demonstrates quantitative effects from genetic background, environment and epistasis.

KEY MESSAGE: A novel locus was discovered on chromosome 7 associated with a lesion mimic in maize; this lesion mimic had a quantitative and heritable phenotype and was predicted better via subset genomic markers than whole genome markers across diverse environments. Lesion mimics are a phenotype of leaf micro-spotting in maize (Zea mays L.), which can be early signs of biotic or abiotic stresses. Dissecting its inheritance is helpful to understand how these loci behave across different genetic backgrounds. Here, 538 maize recombinant inbred lines (RILs) segregating for a novel lesion mimic were quantitatively phenotyped in Georgia, Texas, and Wisconsin. These RILs were derived from three bi-parental crosses using a tropical pollinator (Tx773) as the common parent crossed with three inbreds (LH195, LH82, and PB80). While this lesion mimic was heritable across three environments based on phenotypic ([Formula: see text] = 0.68) and genomic ([Formula: see text] = 0.91) data, transgressive segregation was observed. A genome-wide association study identified a single novel locus on chromosome 7 (at 70.6 Mb) also covered by a quantitative trait locus interval (69.3-71.0 Mb), explaining 11-15% of the variation, depending on the environment. One candidate gene identified in this region, Zm00001eb308070, is related to the abscisic acid pathway involving in cell death. Genomic predictions were applied to genome-wide markers (39,611 markers) contrasted with a marker subset (51 markers). Population structure explained more variation than environment in genomic prediction, but other substantial genetic background effects were additionally detected. Subset markers explained substantially less genetic variation (24.9%) for the lesion mimic than whole genome markers (55.4%) in the model, yet predicted the lesion mimic better (0.56-0.66 vs. 0.26-0.29). These results indicate this lesion mimic phenotype was less affected by environment than by epistasis and genetic background effects, which explain its transgressive segregation.

Pathogenic and Saprophytic Growth Rates of Fusarium oxysporum f. sp. vasinfectum Interact to Affect Variation in Inoculum Density and Interannual Infection Risk.

Fusarium wilt of cotton caused by the soilborne fungal pathogen Fusarium oxysporum f. sp. vasinfectum race 4 (FOV4) is a contemporary epidemic affecting cotton production in Far West Texas. The spatial distribution of soilborne FOV4 can be heterogeneous at small scales, and the factors that lead to this heterogeneity require investigation. Hypothetical causes include dissemination of spores through soils and variable saprophytic growth of the fungus. In the field, FOV4 DNA was quantified from soil during and after the cotton-growing season, and though the average amounts of DNA were not different between these time points, the variances of DNA across space were significantly different. Variability was higher when pathogenic growth of the fungus was expected owing to the presence of live cotton plants and lower when saprophytic growth was expected after cropping. In sterile-environment growth chamber experiments, the abundance of organic matter influenced the fungal vegetative growth rate and maximum amount as measured through quantitative PCR and the timing of the fungus' increasing its rate of spore production as measured through dilution plating. To investigate movement of spores in soils, spore mobility in experimental columns was quantified. Soil composition and organic matter abundance affected spore mobility, indicating that the timing of spore production relative to the availability of growth resources will affect the spatial spread of FOV4 and suggesting that soil properties affect the retention of conidia. The spatial spread of FOV4 through soil varies temporally and is affected by the shift between pathogenic and saprophytic growth modes of the fungus.

The genome of the oomycete Peronosclerospora sorghi, a cosmopolitan pathogen of maize and sorghum, is inflated with dispersed pseudogenes.

Several species in the oomycete genus Peronosclerospora cause downy mildew on maize and can result in significant yield losses in Asia. Bio-surveillance of these pathogens is a high priority to prevent epidemics on maize in the United States and consequent damage to the US economy. The unresolved taxonomy and dearth of molecular resources for Peronosclerospora spp. hinder these efforts. P. sorghi is a pathogen of sorghum and maize with a global distribution, for which limited diversity has been detected in the southern USA. We characterized the genome, transcriptome, and mitogenome of an isolate, representing the US pathotype 6. The highly homozygous genome was assembled using 10× Genomics linked reads and scaffolded using Hi-C into 13 chromosomes. The total assembled length was 303.2 Mb, larger than any other oomycete previously assembled. The mitogenome was 38 kb, similar in size to other oomycetes, although it had a unique gene order. Nearly 20,000 genes were annotated in the nuclear genome, more than described for other downy mildew causing oomycetes. The 13 chromosomes of P. sorghi were highly syntenic with the 17 chromosomes of Peronospora effusa with conserved centromeric regions and distinct chromosomal fusions. The increased assembly size and gene count of P. sorghi is due to extensive retrotransposition, resulting in putative pseudogenization. Ancestral genes had higher transcript abundance and were enriched for differential expression. This study provides foundational resources for analysis of Peronosclerospora and comparisons to other oomycete genera. Further genomic studies of global Peronosclerospora spp. will determine the suitability of the mitogenome, ancestral genes, and putative pseudogenes for marker development and taxonomic relationships.

Genetic Diversity and Classification of Colletotrichum sublineola Pathotypes Using a Standard Set of Sorghum Differentials.

Anthracnose, incited by Colletotrichum sublineola, is the most destructive foliar disease of sorghum and, under severe conditions, yield losses can exceed 80% on susceptible cultivars. The hyper-variable nature of the pathogen makes its management challenging despite the occurrence of several resistant sources. In this study, the genetic variability and pathogenicity of 140 isolates of C. sublineola, which were sequenced using restriction site-associated sequencing (RAD-Seq), resulted in 1244 quality SNPs. The genetic relationship based on the SNP data showed low to high genetic diversity based on isolates' origin. Isolates from Georgia and North Carolina were grouped into multiple clusters with some level of genetic relationships to each other. Even though some isolates from Texas formed a cluster, others clustered with isolates from Puerto Rico. The isolates from Puerto Rico showed scattered distribution, indicating the diverse nature of these isolates. A population structure and cluster analysis revealed that the genetic variation was stratified into eight populations and one admixture group. The virulence pattern of 30 sequenced isolates on 18 sorghum differential lines revealed 27 new pathotypes. SC748-5, SC112-14, and Brandes were resistant to all the tested isolates, while BTx623 was susceptible to all. Line TAM428 was susceptible to all the pathotypes, except for pathotype 26. Future use of the 18 differentials employed in this study, which contains cultivars/lines which have been used in the Americas, Asia, and Africa, could allow for better characterization of C. sublineola pathotypes at a global level, thus accelerating the development of sorghum lines with stable resistance to the anthracnose pathogen.

Asymmetrical lineage introgression and recombination in populations of Aspergillus flavus: Implications for biological control.

Aspergillus flavus is an agriculturally important fungus that causes ear rot of maize and produces aflatoxins, of which B1 is the most carcinogenic naturally-produced compound. In the US, the management of aflatoxins includes the deployment of biological control agents that comprise two nonaflatoxigenic A. flavus strains, either Afla-Guard (member of lineage IB) or AF36 (lineage IC). We used genotyping-by-sequencing to examine the influence of both biocontrol agents on native populations of A. flavus in cornfields in Texas, North Carolina, Arkansas, and Indiana. This study examined up to 27,529 single-nucleotide polymorphisms (SNPs) in a total of 815 A. flavus isolates, and 353 genome-wide haplotypes sampled before biocontrol application, three months after biocontrol application, and up to three years after initial application. Here, we report that the two distinct A. flavus evolutionary lineages IB and IC differ significantly in their frequency distributions across states. We provide evidence of increased unidirectional gene flow from lineage IB into IC, inferred to be due to the applied Afla-Guard biocontrol strain. Genetic exchange and recombination of biocontrol strains with native strains was detected in as little as three months after biocontrol application and up to one and three years later. There was limited inter-lineage migration in the untreated fields. These findings suggest that biocontrol products that include strains from lineage IB offer the greatest potential for sustained reductions in aflatoxin levels over several years. This knowledge has important implications for developing new biocontrol strategies.

Phenomic data-facilitated rust and senescence prediction in maize using machine learning algorithms.

Current methods in measuring maize (Zea mays L.) southern rust (Puccinia polyspora Underw.) and subsequent crop senescence require expert observation and are resource-intensive and prone to subjectivity. In this study, unoccupied aerial system (UAS) field-based high-throughput phenotyping (HTP) was employed to collect high-resolution aerial imagery of elite maize hybrids planted in the 2020 and 2021 growing seasons, with 13 UAS flights obtained from 2020 and 17 from 2021. In total, 36 vegetation indices (VIs) were extracted from mosaicked aerial images that served as temporal phenomic predictors for southern rust scored in the field and senescence as scored using UAS-acquired mosaic images. Temporal best linear unbiased predictors (TBLUPs) were calculated using a nested model that treated hybrid performance as nested within flights in terms of rust and senescence. All eight machine learning regressions tested (ridge, lasso, elastic net, random forest, support vector machine with radial and linear kernels, partial least squares, and k-nearest neighbors) outperformed a general linear model with both higher prediction accuracies (92-98%) and lower root mean squared error (RMSE) for rust and senescence scores (linear model RMSE ranged from 65.8 to 2396.5 across all traits, machine learning regressions RMSE ranged from 0.3 to 17.0). UAS-acquired VIs enabled the discovery of novel early quantitative phenotypic indicators of maize senescence and southern rust before being detectable by expert annotation and revealed positive correlations between grain filling time and yield (0.22 and 0.44 in 2020 and 2021), with practical implications for precision agricultural practices.

DNA-Based Quantification of Fusarium oxysporum f. sp. vasinfectum in Environmental Soils to Describe Spatial Variation in Inoculum Density.

Fusarium wilt of cotton, caused by the soilborne fungal pathogen Fusarium oxysporum f. sp. vasinfectum (FOV), occurs in regions of the United States where cotton (Gossypium spp.) is grown. Race 4 of this pathogen (FOV4) is especially aggressive, and does not require the co-occurrence of the root knot nematode (Meloidogyne incognita) to infect cotton. Its sudden appearance in far-west Texas in 2016 after many years of being restricted to California is of great concern, as is the threat of its continued spread through the cotton-producing regions of the United States. The aim of this research was to analyze the spatial variability of FOV4 inoculum density in the location where FOV4 is locally emerging, using quantitative and droplet digital PCR methods. Soil samples collected from a field with known FOV4 incidence in Fabens, Texas, were analyzed. Appreciable variation in inoculum density was found to occur at spatial scales smaller than the size of plots involved in cultivar trial research, and was spatially autocorrelated (Moran's I, Z = 17.73, P < 0.0001). These findings indicate that, for cultivar trials, accounting for the spatial distribution of inoculum, either by directly quantifying it or through the use of densely distributed calibration checks, is important to the interpretation of results.

Cotton Leafroll Dwarf Virus US Genomes Comprise Divergent Subpopulations and Harbor Extensive Variability.

Cotton leafroll dwarf virus (CLRDV) was first reported in the United States (US) in 2017 from cotton plants in Alabama (AL) and has become widespread in cotton-growing states of the southern US. To investigate the genomic variability among CLRDV isolates in the US, complete genomes of the virus were obtained from infected cotton plants displaying mild to severe symptoms from AL, Florida, and Texas. Eight CLRDV genomes were determined, ranging in size from 5865 to 5867 bp, and shared highest nucleotide identity with other CLRDV isolates in the US, at 95.9-98.7%. Open reading frame (ORF) 0, encoding the P0 silencing suppressor, was the most variable gene, sharing 88.5-99.6% and 81.2-89.3% amino acid similarity with CLRDV isolates reported in cotton growing states in the US and in Argentina and Brazil in South America, respectively. Based on Bayesian analysis, the complete CLRDV genomes from cotton in the US formed a monophyletic group comprising three relatively divergent sister clades, whereas CLRDV genotypes from South America clustered as closely related sister-groups, separate from US isolates, patterns reminiscent of phylogeographical structuring. The CLRDV isolates exhibited a complex pattern of recombination, with most breakpoints evident in ORFs 2 and 3, and ORF5. Despite extensive nucleotide diversity among all available CLRDV genomes, purifying selection (dN/dS < 1) was implicated as the primary selective force acting on viral protein evolution.

First report of squash vein yellowing virus naturally infecting butternut squash (Cucurbita moschata) in Texas.

Virus diseases are major constraints to the production of cucurbits in the Texas Lower Rio Grande Valley. In September 2020, a ~8.1 ha butternut squash (Cucurbita moschata) field in Hidalgo County, Texas, was observed with virus-like symptoms of vein yellowing, leaf curl, mosaic, and foliar chlorosis. The proportion of plants with virus-like symptoms in this field was estimated at 30% and seven samples (symptomatic = 5; non-symptomatic = 2) were collected randomly for virus diagnosis. Initially, equimolar mixtures of total nucleic acid extracts (Dellaporta et. al. 1983) from two symptomatic samples from this field and extracts from 12 additional symptomatic samples from six other fields across south and central Texas was used to generate one composite sample for diagnosis by high throughput sequencing (HTS). The TruSeq Stranded Total RNA with Ribo-Zero Plant Kit (Illumina) was used to construct cDNA library from the composite sample, which was then sequenced on the Illumina NextSeq 500 platform. More than 26 million single-end HTS reads (75 nt each) were obtained and their bioinformatic analyses (Al Rwahnih et al. 2018) revealed several virus-like contigs belonging to different species (data not shown). Among them, 6 contigs that ranged in length from 429 to 3,834 nt shared 96 to 100% identities with isolates of squash vein yellowing virus (SqVYV), genus Ipomovirus, family Potyviridae. To confirm the HTS results, total nucleic acid extracts from the cucurbit samples from all seven fields (n = 46) were used for cDNA synthesis with random hexamers and the PrimeScript 1st strand cDNA Synthesis Kit (Takara Bio). A 1-µL aliquot of cDNA was used in 12.5-µL PCR reaction volumes with PrimeSTAR GXL DNA Polymerase (Takara Bio) and two pairs of SqVYV-specific primers designed based on the HTS derived contigs. The primer pairs SqYVV-v4762: 5'-CTGGATTCTGCTGGAAGATCA & SqYVV-c5512: 5'-CCACCATTAAGGCCATCAAAC and SqYVV-v8478: 5'-TTTCTGGGCAAACAAACATGG & SqYVV-c9715: 5'-TTCAGCGACGTCAAGTGAG targeted ~0.75 kb and ~1.2 kb fragments of the cylindrical inclusion (CI) and the complete coat protein (CP) gene sequences of SqVYV, respectively. The expected DNA band sizes were obtained only from the five symptomatic butternut squash samples from the Hidalgo Co. field. Two amplicons per primer pair from two samples were cloned into pJET1.2/Blunt vector (Life Technologies) and bidirectionally Sanger sequenced, generating 753 nt partial CI specific sequences (MW584341-342) and 1,238 nt that encompassed the complete CP (MW584343-344) of SqVYV. In pairwise comparisons, the partial CI sequences shared 100% nt/aa identity with each other and 98-99% nt/aa identity with corresponding sequences of SqVYV isolate IL (KT721735). The CP cistron of TX isolates shared 100% nt/aa identity with each other and 90-98% nt (97-100% aa) identities with corresponding sequences of several SqVYV isolates in GenBank, with isolates IL (KT721735) and Florida (EU259611) being at the high and low spectrum of nt/aa identity values, respectively. This is the first report of SqVYV in Texas, naturally occurring in butternut squash. SqVYV was first discovered in Florida (Adkins et al. 2007) and subsequently reported from few other states in the U.S. (Adkins et al. 2013; Egel and Adkins 2007; Batuman et al. 2015), Puerto Rico (Acevedo et al. 2013), and locations around the world. The finding shows an expansion of the geographical range of SqVYV and adds to the repertoire of cucurbit-infecting viruses in Texas. Further studies are needed to determine the prevalence of SqVYV in Texas cucurbit fields and an assessment of their genetic diversity.

First report of Cucurbit chlorotic yellows virus infecting cantaloupe (Cucumis melo L.) in Texas.

Texas is a major producer of cucurbits such as cantaloupe (Cucumis melo L.), but outbreaks of virus-like diseases often adversely affect yields. Little is known about the identity of the causal or associated viruses. During studies conducted in fall 2020 to explore the virome of cucurbit fields in Texas, a commercial cantaloupe field (~4.1 ha) in Cameron County was observed with virus-like symptoms of interveinal chlorotic mottle and foliar chlorosis and disease incidence was estimated at 100%. Virus-like symptoms including mosaic and leaf curl were also observed in six additional fields across five south and central Texas counties of Atascosa, Hidalgo, Fort Bend, Frio, and Wharton. Forty-six plants, which included 32 symptomatic and 14 non-symptomatic, were sampled from these fields for virus diagnosis and each sample was subjected to total nucleic acid extraction according to Dellaporta et. al. (1983). Initially, equal amounts of nucleic acids from 14 symptomatic plants (two/field) were pooled into one composite sample for preliminary diagnosis by high throughput sequencing (HTS). The cDNA library obtained from the composite sample with a TruSeq Stranded Total RNA with Ribo-Zero Plant Kit (Illumina) was sequenced on the Illumina NextSeq 500 platform, generating ~26.3 M single-end HTS reads (75 nucleotides [nt] each). Analyses of the reads according to Al Rwahnih et al. (2018) revealed several virus-like contigs; among them 23 contigs (206 to 741 nt) shared 98 to 100% nt identities to isolates of cucurbit chlorotic yellows virus (CCYV), genus Crinivirus, family Closteroviridae. Three pairs of CCYV-specific primers were designed from the HTS contigs with primers CCYV-v1330: 5'-AGTCCCTTACCCTGAGATGAA/CCYV-c2369: 5'-CGGAGCATTCGACAACTGAATA targeting ~1 kb fragment of the ORF1a (RNA1), primers CCYV-v4881: 5'-ATAAGGCGGCGACCTAATC/CCYV-c5736: 5'-GATCACTTGACCATCTCCTTCT targeting a ~0.9 kb fragment encompassing the coat protein (CP) cistron of CCYV (RNA2), and primers CCYV-v6362: 5'-CACCTCTTCCAGAACCAGTTAAA/CCYV-c7423: 5'-TGGGAACAACTTATTTCTCCTAGC targeting ~1 kb spanning partial minor coat protein (CPm) and p26 sequences (RNA2). Total nucleic acid extracts of each of the 46 samples from the seven fields were tested by two-step reverse transcription polymerase chain reaction using all three CCYV-specific primer pairs and they yielded amplicons of expected sizes from all five symptomatic cantaloupe samples from the Cameron County field and one additional symptomatic butternut squash sample from a field in Hidalgo County. The DNA bands from three randomly chosen cantaloupe samples were cloned and sequenced as previously described (Oke et al. 2020). In pairwise comparisons, the obtained 1,040 nt ORF1a (MW584332-334), 753 nt complete CP (MW584335-337), and 1,062 nt CPm/p26 (MW584338-340) gene specific sequences from the three samples shared 100% nt identity with each other, and 99-100% nt identities with corresponding RNA1 (AB523788) and RNA2 (AB523788) sequences of the exemplar isolate of CCYV. This is the first report of CCYV in Texas, thus expanding the current geographical range of the virus in the U.S. that includes California (Wintermantel et al. 2019) and Georgia (Kavalappara et al. 2021). The abundance of whiteflies of the Bemisia tabaci species complex in south Texas and other major U.S. cucurbit production areas presents additional challenges to virus disease management.

First report of watermelon crinkle leaf-associated virus 1 (WCLaV-1) and WCLaV-2 infecting watermelon (Citrullus lanatus) in the United States.

Watermelon (Citrullus lanatus) and other cucurbits are cultivated globally, and Texas ranks among its top 5 producers in the U.S. In July 2020, plants with virus-like disease symptoms consisting of mild leaf crinkling and yellow mosaic patterns were observed in a 174-ha watermelon field in Burleson Co., TX; disease incidence was visually estimated at 5%. Total nucleic acids were extracted from leaf tissues of 5 randomly sampled plants (Dellaporta 1983) and their equimolar amounts were made into a composite sample that was used for cDNA library construction with TruSeq Stranded Total RNA with Ribo-Zero Plant Kit (Illumina). The cDNA library was sequenced on the Illumina NextSeq 500 platform, generating ~37M single-end reads (each 75 nt), which were analyzed as per Al Rwahnih et al. (2018). Of these, 58,200 and 27,500 reads mapped to the genomes of watermelon crinkle leaf-associated virus 1 (WCLaV-1) and WCLaV-2 (Xin et al. 2017), respectively, along with 4 other virus-specific reads (data not shown). The near complete RNA1-RNA3 segments of WCLaV-1 (354-652X) and WCLaV-2 (144-258X) were generated from the mapped reads and they shared ≥96% nt identities with published RNA segments of both viruses. The results were verified by RT-PCR using newly designed primers WCLaV-1vRP: 5'-GGTGAGTTAGTGTGTCTGAAGG/WCLaV-1cRP: 5'-GAGGTTGCCTGAGGTGATAAG to target 881 bp of the RNA1-encoded RNA-dependent RNA polymerase (RdRP), WCLaV-1vMP: 5'-GAAGGTTTGCTCCCTTGAAATG/WCLaV-1cMP: 5'-GACTGTGGCTGAAGAGTCTATG target 538 bp of the RNA2-encoded movement protein (MP), and WCLaV-1vNP: 5'-CGAATAGACTCTGGAGGGTAGA/WCLaV-1cMP: 5'-GAAAGCAAGAAAGCTGGCTAAA target 786 bp of the RNA3-encoded nucleoprotein (NP). Similarly, the WWCLaV-2-specific primers WCLaV-2vRP: 5'-GTCTCACATTCCTGCACTAACT/WCLaV-2cRP: 5'-ATCGGTCCTGGGTTATTTGTATC target 968 bp of the RdRP, WCLaV-2vMP: 5'-GACTTCAGAACCTCAACATCCA/WCLaV-2cMP: 5'-CAAGGGAGAGTGCTGACAAA target 562 bp of the MP, and WCLaV-2vNP: 5'-ATTCCCAGTGAGAGCAACAA/WCLaV-2cMP: 5'-GAGGTGGAGGTAGGAAAGAAAG target 449 bp of the NP. Fresh cDNA synthesized from the 5 samples with PrimeScript First Strand cDNA synthesis kit (Takara Bio) were tested by PCR with all 6 primer pairs using the PrimeSTAR GXL DNA Polymerase kit (Takara Bio). Three of the 5 samples were positive for both viruses and one sample was positive for each virus. The obtained products from 4 samples were cloned individually into pJET1.2/Blunt vector (Thermo Scientific, USA), followed by bidirectional Sanger-sequencing of the plasmids with the GenElute Five-Minute Plasmid Miniprep kit (Sigma-Aldrich). In pairwise comparisons, the partial RNA1-RNA3 sequences of WCLaV-1 (GenBank accession nos. MW559074-82) shared 100% nt/aa identities with each other and with corresponding sequences of WCLaV-1 isolate KF-1 from China (KY781184-86). The partial RNA1-RNA3 sequences of WCLaV-2 (MW559083-91) shared 97-100% nt/96-100% aa identities with each other and with corresponding sequences of WCLaV-2 isolate KF-15 from China (KY781187-89). This is the first report of WCLaV-1 and WCLaV-2 in Texas and the first record of both viruses in the U.S. and elsewhere outside of China. Both negative-sense, single-stranded RNA viruses represent a novel taxon in the family Phenuiviridae (order Bunyavirales) (Xin et al. 2017). While aspects of the biology of both viruses are yet to be elucidated, our results expand their geographical range. The detection primers developed here will be useful for screening cucurbits germplasm to avert their spread.

Yield, Insect-Derived Ear Injury, and Aflatoxin Among Developmental and Commercial Maize Hybrids Adapted to the North American Subtropics.

The development of maize (Zea mays L.) hybrids that are adapted to subtropical areas of North America should consider yield potential under heat and moisture stress, and reduced susceptibility to insect herbivory and disease. To aid in this process, maize hybrids (43 developmental and seven non-Bt commercial hybrids) were evaluated for severity of ear injury to Helicoverpa zea (Boddie) and Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), susceptibility to Aspergillus flavus (Link) (Deuteromycetes: Moniliales), and yield. In subtropical Corpus Christi and College Station, TX, field experiments conducted over three years revealed significant differences among maize hybrids with the rank of the selected measurements differing across the two locations. When the location by maize hybrid interaction was not significant, variation across the main factors of maize hybrid genetics (in all cases) and location (in some cases) was detected. In 2014, a significant location by maize hybrid interaction in yield but not aflatoxin and ear injury were likely associated with differences in weather between locations. In Corpus Christi in 2015, a location by maize hybrid interaction was detected for ear injury only. Overall, experimental maize hybrids, containing the inbred line Tx777, displayed partial resistance to insect derived ear injury in both locations, and some hybrid testcrosses exhibited low rates of aflatoxin accumulation while maintaining relatively high yields. Tx777 was selected from populations originating in Bolivia and adapted to subtropical climates. The most promising hybrid testcrosses had lower ear injury and aflatoxin accumulation, and good yield under varying heat and moisture stress at the two subtropical maize growing areas in this study.

Transmission of Cotton Seed and Boll Rotting Bacteria by the Verde Plant Bug (Hemiptera: Miridae).

Field experiments and supporting laboratory work were conducted to characterize the ability of the verde plant bug, Creontiades signatus (Distant), a boll-feeding sucking bug, to transmit a cotton seed and boll rot bacterial pathogen, Serratia marcescens (Bizio) (Enterobacteriales: Enterobacteriaceae). Serratia marcescens was originally isolated from bolls infested with verde plant bug in south Texas, and a Rifampicin resistant S. marcescens strain was used in transmission and retention experiments. Serratia-exposed and nonexposed adult verde plant bugs from a laboratory colony were placed individually on 5-, 6-, 7-, and 8-d-old bolls (postanthesis). The bacterial acquisition process did not apparently affect insect vigor based on similar average boll injury ratings observed across both exposed and nonexposed bugs. Cotton bolls caged with Serratia-exposed verde plant bugs had significantly greater presence of S. marcescens and cotton boll rot symptoms than bolls caged without bugs (no-insect controls) or nonexposed bugs. Transmission of the disease agent by verde plant bug was confirmed across all boll ages assayed. Incidence of diseased locules on 5- and 6-d-old bolls was the same or greater than on 7- and 8-d-old bolls. Verde plant bug was able to harbor the disease agent from 24- to 96-h postinfection, and transmission efficiency rates ranged from 54 to 62% during initial transmission and retention (transmission across two bolls fed upon consecutively) studies. Along with photographic evidence, the experimental data supported that boll damage associated with verde plant bug infestations was magnified when insects transmitted the cotton pathogen S. marcescens as demonstrated in this 2-yr field experiment.

Association of Insect-Derived Ear Injury With Yield and Aflatoxin of Maize Hybrids Varying in Bt Transgenes.

Environmental factors have been associated with the production of aflatoxin in maize, Zea mays L., and it is inconclusive whether transgenic, Bacillus thuringiensis (Bt), maize has an impact on aflatoxin accumulation. Maize hybrids differing in transgenes were planted in two locations from 2014 to 2017. Yield, aflatoxin, and ear injury caused by corn earworm, Helicoverpa zea (Boddie), and fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), were measured across three groups of hybrids differing in transgenes including near-isogenic hybrids, and water-stressed conditions. The hybrid groups consisted of non-Bt hybrids with no Bt transgenes, a second group with one or more Cry-Bt transgenes, and the third group with vegetative insecticidal Bt protein and Cry-Bt transgenes (Cry/Vip-Bt). Across the six data sets derived from 11 experiments, the Cry-Bt and Cry/Vip-Bt hybrids had less ear injury and aflatoxin on average than non-Bt hybrids. The effects of ear injury on yield and aflatoxin were more prominent and consistent in Corpus Christi, TX, where hybrids experienced more water-limited conditions than in College Station, TX. The trend of increased aflatoxin among hybrids with increased ear injury was further resolved when looking at Cry-Bt and Cry/Vip-Bt isogenic hybrids in Corpus Christi. The results supported that the maize hybrids with the inclusion of Cry-Bt and Cry/Vip-Bt transgenes warrant further investigation in an integrated approach to insect and aflatoxin management in sub-tropical rain-fed maize production regions. Research outcomes may be improved by focusing on areas prone to water-stress and by using hybrids with similar genetic backgrounds.

GWAS analysis of sorghum association panel lines identifies SNPs associated with disease response to Texas isolates of Colletotrichum sublineola.

KEY MESSAGE: SNPs identify prospective genes related to response to Colletotrichum sublineola (anthracnose) in the sorghum association panel lines. Sorghum association panel (SAP) lines were scored over several years for response to Colletotrichum sublineola, the causal agent of the disease anthracnose. Known resistant and susceptible lines were included each year to verify successful inoculation. Over 79,000 single-nucleotide polymorphic (SNP) loci from a publicly available genotype by sequencing dataset available for the SAP lines were used with TASSEL association mapping software to identify chromosomal locations associated with differences in disease response. When the top-scoring SNPs were mapped to the published sorghum genome, in each case, the nearest annotated gene has precedence for a role in host defense.

TAL effector driven induction of a SWEET gene confers susceptibility to bacterial blight of cotton.

Transcription activator-like (TAL) effectors from Xanthomonas citri subsp. malvacearum (Xcm) are essential for bacterial blight of cotton (BBC). Here, by combining transcriptome profiling with TAL effector-binding element (EBE) prediction, we show that GhSWEET10, encoding a functional sucrose transporter, is induced by Avrb6, a TAL effector determining Xcm pathogenicity. Activation of GhSWEET10 by designer TAL effectors (dTALEs) restores virulence of Xcm avrb6 deletion strains, whereas silencing of GhSWEET10 compromises cotton susceptibility to infections. A BBC-resistant line carrying an unknown recessive b6 gene bears the same EBE as the susceptible line, but Avrb6-mediated induction of GhSWEET10 is reduced, suggesting a unique mechanism underlying b6-mediated resistance. We show via an extensive survey of GhSWEET transcriptional responsiveness to different Xcm field isolates that additional GhSWEETs may also be involved in BBC. These findings advance our understanding of the disease and resistance in cotton and may facilitate the development cotton with improved resistance to BBC.

Genome wide association study for drought, aflatoxin resistance, and important agronomic traits of maize hybrids in the sub-tropics.

The primary maize (Zea mays L.) production areas are in temperate regions throughout the world and this is where most maize breeding is focused. Important but lower yielding maize growing regions such as the sub-tropics experience unique challenges, the greatest of which are drought stress and aflatoxin contamination. Here we used a diversity panel consisting of 346 maize inbred lines originating in temperate, sub-tropical and tropical areas testcrossed to stiff-stalk line Tx714 to investigate these traits. Testcross hybrids were evaluated under irrigated and non-irrigated trials for yield, plant height, ear height, days to anthesis, days to silking and other agronomic traits. Irrigated trials were also inoculated with Aspergillus flavus and evaluated for aflatoxin content. Diverse maize testcrosses out-yielded commercial checks in most trials, which indicated the potential for genetic diversity to improve sub-tropical breeding programs. To identify genomic regions associated with yield, aflatoxin resistance and other important agronomic traits, a genome wide association analysis was performed. Using 60,000 SNPs, this study found 10 quantitative trait variants for grain yield, plant and ear height, and flowering time after stringent multiple test corrections, and after fitting different models. Three of these variants explained 5-10% of the variation in grain yield under both water conditions. Multiple identified SNPs co-localized with previously reported QTL, which narrows the possible location of causal polymorphisms. Novel significant SNPs were also identified. This study demonstrated the potential to use genome wide association studies to identify major variants of quantitative and complex traits such as yield under drought that are still segregating between elite inbred lines.

Characterization of genetic diversity and linkage disequilibrium of ZmLOX4 and ZmLOX5 loci in maize.

Maize (Zea mays L.) lipoxygenases (ZmLOXs) are well recognized as important players in plant defense against pathogens, especially in cross kingdom lipid communication with pathogenic fungi. This study is among the first to investigate genetic diversity at important gene paralogs ZmLOX4 and ZmLOX5. Sequencing of these genes in 400 diverse maize lines showed little genetic diversity and low linkage disequilibrium in the two genes. Importantly, we identified one inbred line in which ZmLOX5 has a disrupted open reading frame, a line missing ZmLOX5, and five lines with a duplication of ZmLOX5. Tajima's D test suggests that both ZmLOX4 and ZmLOX5 have been under neutral selection. Further investigation of haplotype data revealed that within the ZmLOX family members only ZmLOX12, a monocot specific ZmLOX, showed strong linkage disequilibrium that extends further than expected in maize. Linkage disequilibrium patterns at these loci of interest are crucial for future candidate gene association mapping studies. ZmLOX4 and ZmLOX5 mutations and copy number variants are under further investigation for crop improvement.