During the 2019 growing season, cotton (Gossypium hirsutum L.) plants in North Carolina were observed to have virus-like symptoms including leaf rugosity, leaf curling, and shortened upper internodes, consistent with Cotton Leafroll Dwarf Disease (CLRDD) associated with cotton leafroll dwarf virus (CLRDV, family: Luteoviridae, genus: Polerovirus) (Avelar et al. 2020). Sentinel plots planted on June 17, 2019 at the Sandhills Research Station in Moore County, NC exhibited CLRDD symptoms and disease incidence was estimated at 75% on a 0.1-ha field. Cotton aphids (Aphis gossypii Glover), the reported vector of CLRDV (Michelotto and Busoli 2007; Heilsnis et al. 2020; McLaughlin et al. 2020), were detected on plants throughout the growing season. Samples (n = 24) were collected from sentinel plots on September 20, 2019 to test for CLRDV through RT-PCR. Each sample represented five symptomatic plants collected from a single plot. Total RNA was extracted from the petiole tissue of each sample using Qiagen RNeasy Plant Mini kit (Qiagen, Germantown, MD), following the manufacturer’s recommendations. The cDNA was synthesized using SuperScript IV first-strand synthesis system (ThermoFisher Scientific, Waltham, MA) and amplified with CLRDV-specific PCR primers CLRDV3675F/Pol3982R (Sharman et al. 2015) targeting a 310 bp genome segment of ORF3-5. Seven CLRDV positive samples were further amplified with two additional primer sets specifically designed to detect CLRDV: AL674F/AL1407R (Avelar et al. 2019), targeting a 733 bp portion of the ORF0-ORF1, and CLPOF/CLPOR (Cascardo et al. 2015), amplifying a 880 bp fragment spanning the ORF0. Nucleotide BLAST searches showed that the best matches for all sequences in this study were derived from CLRDV with a range of pairwise identity of 99.2¬¬ - 100% for all genome segments. From symptomatic samples (n = 14 ), the isolated virus was confirmed as CLRDV in several cotton varieties, including Deltapine 1646 B2XF (GenBank Accessions MN958131 (ORF3-5), MN958147 (ORF0-ORF1), MN958138 (ORF0), MN958133 (ORF3-5), MN958145 (ORF0-ORF1), MN958140 (ORF0)), Phytogen 480 W3FE (MN958134 (ORF3-5), MN958144 (ORF0-ORF1), MN958141 (ORF0), Stoneville 5471 GLTP (GenBank Accessions MN958135 (ORF3-5), MN958143 (ORF0-ORF1), MN958142 (ORF0)), and Nextgen 5711 B3XF (GenBank Accessions MN958130 (ORF3-5), MN958148 (ORF0-ORF1), MN958137 (ORF0), MN958132 (ORF3-5), MN958146 (ORF0-ORF1), MN958139 (ORF0), and MN958136 (ORF3-5). CLRDD is a newly named disease of cotton in the United States that was first reported in Alabama (Avelar et al. 2019), Georgia (Tabassum et. al. 2019), Mississippi (Aboughanem-Sabanadzovic et. al. 2019), and Texas (Alabi et al. 2019). While the virus has been reported with variable impacts, losses can be extensive in some fields that are affected (Avelar et al. 2019). North Carolina produced over one million bales of cotton in 2019 and given reported losses among fields with high disease incidence, CLRDV has the potential to significantly reduce cotton yield and quality for the state if it becomes widespread.

ResumoO trabalho foi desenvolvido em Dourados-MS, com o objetivo de estudar o efeito de pontas de pulverização e horários de aplicação de fungicida no controle químico da ferrugem asiática da soja (Phakopsora pachyrhizi Sidow). Avaliaram-se quatro pontas de pulverização: jato plano defletor, jato plano defletor duplo, jato plano de faixa ampliada, jato plano duplo. As aplicações ocorreram nos horários de 14h e 17h 30min com pulverizador costal à pressão constante. O fungicida utilizado foi uma formulação comercial de epoxiconazol e piraclostrobina (0,5 L ha -1 ). Foram coletados folíolos nos terços superior, médio e inferior para avaliação do número de lesões, urédias, e área foliar lesionada. Menor número de urédias no terço inferior foi observado em plantas que receberam aplicação de fungicida às 14h via ponta jato plano defletor duplo em relação às demais pontas. A aplicação de fungicida, independente do tipo de ponta, não resultou em alterações nos níveis de controle da ferrugem asiática nos terços superior e médio. Menor desfolha e maior massa de mil grãos foram detectadas quando a aplicação foi realizada com a ponta jato plano defletor duplo em relação à jato plano defletor. A produtividade não foi afetada pelo uso de diferentes pontas e horários de aplicação do fungicida. Palavras-chave: Glycine max, Phakopsora pachyrhizi, controle de doença, tecnologia de aplicação AbstractSpray nozzles were evaluated at two different spraying times (14h and 17h 30m), in Mato Grosso do Sul, Brazil regarding Asian soybean rust control (Phakopsora pachyrhizi Sidow). Four spray nozzles were evaluated: deflector plain tips, double deflector plain tips, extended range flat spray tips and double plain tips. A commercial formulation of epoxiconazole and pyraclostrobin (0,5 L ha -1 ) was sprayed in the plots. Soybean leaves were collected at the top, middle and lower canopy, for assessment of the number of lesions and uredinias. In addition, leaf area affected by P. pachyrhizi was estimated. Plots sprayed with double deflector plain tips had lower uredinia counts in the lower canopy compared to the other tips tested. Sprays done early in the afternoon with double deflector plain nozzles had lower uredinia counts compared to the other treatments. Smaller defoliation and greater a-thousand grain mass were detected in plots sprayed with fungicide using a double deflector plain tip compared to the deflector plain nozzle. Soybean yield was not affected by nozzles and time of fungicide spray.

A multi-state and interdisciplinary team was formed to address the Extension and research needs of CLRDV, an emerging cotton disease with high potential impact for U.S. cotton production. In 2017, CLRDV was identified in AL and Auburn University immediately formed an interdisciplinary working group composed of plant breeders, plant pathologists, entomologists, and agronomists. Since then, scientists from ten other states have joined the CLRDV group. Thus, allowing research to be coordinated efficiently and best deploy limited resources to attend the stakeholder’s needs. The CLRDV group produces and shares new and relevant information with the scientific community and cotton producers alike.

Worldwide, Ascochyta blight is caused by a complex of host-specific fungal pathogens, including Ascochyta pisi, Didymella pinodes, and Didymella pinodella. The application of foliar fungicides is often necessary for disease management, but a better understanding of pathogen prevalence, aggressiveness, and fungicide sensitivity is needed to optimize control. Leaf and stem samples were obtained from 56 field pea production fields in 14 counties in North Dakota from 2017 to 2020 and isolates were collected from lesions characteristic of Ascochyta blight. Based on fungal characteristics and sequencing the ITS1-5.8S-ITS2 region, 73% of isolates were confirmed to be D. pinodes (n = 177) and 27% were A. pisi (n = 65). Across pathogens, aggressiveness was similar among some isolates in greenhouse assays. The in vitro pyraclostrobin sensitivity of all D. pinodes isolates collected from 2017 to 2020 was lower than that of the three baseline isolates. Sensitivity of 91% of A. pisi isolates collected in 2019 and 2020 was lower than the sensitivity of two known sensitive isolates. Resistance factors (Rf) from mean EC50 values of pyraclostrobin baseline/known sensitive isolates to isolates collected from 2017 to 2020 ranged from 2 to 1,429 for D. pinodes and 1 to 209 for A. pisi. In vitro prothioconazole sensitivity of 91% of D. pinodes isolates collected from 2017 to 2020 was lower than the sensitivity of the baseline isolates and 98% of A. pisi isolates collected from 2019 to 2020 was lower than the sensitivity of the known sensitive isolates. Prothioconazole Rf ranged from 1 to 338 for D. pinodes and 1 to 127 for A. pisi. Based on in vitro results, 92% of D. pinodes and 98% of A. pisi isolates collected displayed reduced-sensitivity/resistance to both fungicides when compared to baseline/known sensitive isolates. Disease control under greenhouse conditions of both pathogens provided by both fungicides was significantly lower in isolates determined to be reduced-sensitive or resistant in in vitro assays when compared to sensitive. Results reported here reinforce growers desperate need of alternative fungicides and/or management tools to fight Ascochyta blight in North Dakota and neighboring regions.

Significance and Impact of the Study: This study describes MoNSTR-seq (Mutation analysis via Next-generation DNA Sequencing of T-DNA Regions), an adaptation of restriction site-associated DNA sequencing (RAD-seq) to identify the position of transfer-DNA (T-DNA) insertions in the genome of Phomopsis longicolla, an important pathogen of soybean. The technique enables high-throughput characterization of mutants generated via Agrobacterium tumefaciens-mediated transformation (ATMT), thus accelerating gene discovery via forward genetics. This technique represents a significant advancement over existing approaches to characterize T-DNA insertions in fungal genomes. With minor modifications, this technique could be easily adapted to taxonomically diverse fungal pathogens and additional mutagenesis cassettes. AbstractPhomopsis longicolla (Hobbs) causes Phomopsis seed decay and stem lesions in soybean (Glycine max). In this study, a novel, high-throughput adaptation of RAD-seq termed MoNSTR-seq (Mutation analysis via Next-generation DNA Sequencing of T-DNA Regions) was developed to determine the genomic location of T-DNA insertions in P. longicolla mutants. Insertional mutants were created via Agrobacterium tumefaciens-mediated transformation, and one mutant, strain PL343, was further investigated due to impaired stem lesion formation. Mutation analysis via Next-generation DNA Sequencing of T-DNA Regions, in which DNA libraries are created with two distinct restriction enzymes and customized adapters to simultaneously enrich both T-DNA insertion borders, was developed to characterize the genomic lesion in strain PL343. MoNSTR-seq successfully identified a T-DNA insertion in the predicted promoter region of a gene encoding a cellobiose dehydrogenase (CDH1), and the position of the T-DNA insertion in strain PL343 was confirmed by Sanger sequencing. Thus, MoNSTR-seq represents an effective tool for molecular genetics in P. longicolla, and is readily adaptable for use in diverse fungal species.