Zachary Stansell

Septoria leaf spot, caused by the fungal pathogen Septoria cannabis, is a common disease of field-grown hemp (Cannabis sativa L.). The development of disease-resistant cultivars presents a promising strategy for managing this disease. To evaluate hemp germplasm for susceptibility to Septoria leaf spot, diverse hemp entries from the U.S. Department of Agriculture Agricultural Research Service Plant Genetic Resources Unit, commercially available cultivars, and Cornell breeding lines were evaluated in replicated field trials in Geneva, NY. In 2023, 81 entries were screened, and in 2024, 105 entries were screened for disease severity on a weekly basis. Relative area under disease progress curve (rAUDPC) values were calculated to quantify susceptibility across two years. Significant variations in rAUDPC were observed among entries, ranging from 0.01 to 13.60 in 2023 and from 0.10 to 21.20 in 2024. Final disease severity ranged from 0.01 to 31.30% in 2023 and from 0.00 to 36.00% in 2024. Nine entries that consistently displayed low disease severity and are likely resistant to S. cannabis were identified. Disease severity was not significantly correlated with plant sex. Several resistant entries were of Asian origin. This study establishes foundational knowledge for breeding programs targeting Septoria leaf spot resistance and emphasizes the value of diverse hemp germplasm in disease management.

Pseudoperonospora cannabina, the causal agent of downy mildew in Cannabis sativa, was first identified in New York in 2020. The pathogen causes angular brown lesions on foliar tissue and dark sporulation on the leaf underside, often leading to leaf curling, defoliation, and yield loss. To date, no sources of genetic resistance or effective chemical controls have been reported. In this study, 108 hemp entries were evaluated for resistance to downy mildew using three phenotyping methods: 1) high-throughput Blackbird robotic imaging coupled with convolutional neural network-based leaf disc analysis, 2) visual rating of the same leaf discs, and 3) detached leaf assays. Susceptibility ranged from 0 to 100%, with two entries, G 33532 and ‘Santhica 27’, exhibiting 0% severity across all three phenotyping methods. Detached leaf assays produced the lowest disease severity estimates, whereas Blackbird convolutional neural network ratings exhibited the lowest residual variance but consistently underestimated disease severity relative to visual assessments. Visual leaf disc ratings produced the highest severity estimates and had the greatest variability. These results suggest that while Blackbird convolutional neural network analysis holds promise for high-throughput phenotyping of the hemp downy mildew interaction, additional refinement is required to improve accuracy.

Powdery mildew is one of the most common diseases affecting indoor hemp (Cannabis sativa L. <0.3% tetrahydrocannabinol) cultivation. The primary causal organism is the biotrophic fungus Golovinomyces ambrosiae, whose polycyclic nature and short latent phase allow it to rapidly produce asexual conidia that spread to other plants. Control strategies that rely on the use of fungicides are limited by the efficacy of products that are registered for use on hemp, thus the breeding of resistant cultivars is critical. Two resistance genes and one susceptibility gene involved in powdery mildew resistance have been described for C. sativa. In the present work, we evaluated the susceptibility to G. ambrosiae of 70 C. sativa entries in one year and 98 in a second year, including accessions from the United States Department of Agriculture – Agricultural Research Service germplasm repository, breeding lines, and commercial cultivars. Entries represented different geographic origins, levels of improvement, and market classes. The trials were inoculated with G. ambrosiae, and disease severity was rated on a weekly basis. A wide range of disease severity was observed among and within entries. Several entries displayed no or very low levels of disease across years, suggesting that they have genetic resistance to G. ambrosiae. This screening provides a foundation for further characterization of the mechanisms of powdery mildew resistance in hemp and contributes to broaden the publicly available phenotypic data that will better serve stakeholders who rely on germplasm collections.

Hemp (Cannabis sativa L.) has historically played a vital role in agriculture across the globe. Feral and wild populations have served as genetic resources for breeding, conservation, and adaptation to changing environmental conditions. However, feral populations of Cannabis, specifically in the Midwestern United States, remain poorly understood. This study aims to characterize the abiotic tolerances of these populations, estimate suitable areas, identify regions at risk of abiotic suitability change, and highlight the utility of ecological niche models (ENMs) in germplasm conservation. The Maxent algorithm was used to construct a series of ENMs. Validation metrics and MOP (Mobility‐oriented Parity) analysis were used to assess extrapolation risk and model performance. We also projected the final projected under current and future climate scenarios (2021–2040 and 2061–2080) to assess how abiotic suitability changes with time. Climate change scenarios indicated an expansion of suitable habitat, with priority areas for germplasm collection in Indiana, Illinois, Kansas, Missouri, and Nebraska. This study demonstrates the application of ENMs for characterizing feral Cannabis populations and highlights their value in germplasm conservation and breeding efforts. Populations of feral C. sativa in the Midwest are of high interest, and future research should focus on utilizing tools to aid the collection of materials for the characterization of genetic diversity and adaptation to a changing climate.

In 2022, virus-like symptoms were observed in a field of diverse hemp (Cannabis sativa L.) germplasm in Ontario County, New York. Less than 1% of plants exhibited stunting and curled leaves (Figure S1), consistent with tobacco streak virus (TSV) symptoms on other plants (Liu et al. 2022). Most typically, the plants were considerably reduced in overall size, with upwards, adaxial curling along the leaf margin with newer leaves appearing to be the most affected. Fifteen symptomatic plants representing nine accessions were tested for 12 viruses and viroids through Agdia Testing Services (Elkhart, IN). Of these, eight plants representing five accessions including: G 33204 21UO SD ('Cherry Wine S1'), G 33211 21UO SD ('Wife'), G 33225 22CL01 CL ('Candida #2'), G 33270 22UO SD ('Falkowski CBD Mix'), and G 33365 22UO SD ('Queen Dream'), were positive for TSV, a type of Ilarvirus in the Bromoviridae family. Presence of TSV was confirmed through enzyme-linked immunosorbent assay testing. TSV is a positive-sense, single-stranded RNA virus with a wide host range that can be transmitted by thrips, mechanical injury, seed, and pollen (Zambrana-Echevarría et al. 2021). To confirm the presence of TSV, two putatively TSV-infected samples were subjected to RNA-Seq analysis. RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Aarhus, Denmark) per manufacturer's direction. Stranded RNA libraries were prepared using the Illumina TruSeq Stranded Total RNA with Ribo-Zero Plant kit (San Diego, California, USA). Paired-end 2x150bp sequencing was performed on an Illumina NovaSeq6000 sequencer. RNA-Seq data was trimmed using the fastp program (Chen et al. 2018) with default parameters to remove adapter sequences and low-quality bases. After filtering, 49,696,041 and 56,126,804 paired-end reads were retained from 'Wife' and 'Falkowski CBD Mix' samples, respectively. Filtered RNA-seq reads were mapped to TSV genome accession GCF_000865505.1 using the bowtie2 (Langmead & Salzberg 2012) aligner with default parameters. From 'Wife' and 'Falkowski CBD Mix' samples, 153 and 139 reads mapped to the TSV reference genome. To further validate the presence of TSV reads, RNA-Seq data was analyzed using the Kraken2 pipeline (Wood et al. 2019). Using the Kraken2 virus database, reads associated with TSV (NCBI taxonomy ID: 12317) were identified. This analysis identified 172 and 151 TSV reads from 'Wife' and 'Falkowski CBD Mix,' respectively. Higher numbers of reads identified using the Kraken2 analysis is due to the more permissive k-mer matching approach implemented in Kraken2. Furthermore, we identified several other virus taxa in the samples. Of note, both samples had a high number of reads associated with Amazon lily mild mottle virus with 254,493 and 116,150 reads from 'Wife' and 'Falkowski CBD Mix,' respectively. Among other virus species belonging to Ilarviruses, Cassava Ivorian bacilliform virus and Cowpea chlorotic mottle viruses were detected from both samples. To further validate infection by TSV, samples from both ELISA-positive and ELISA-negative plants were subjected to PCR using the primers and protocol described in Zambrana-Echevarría et al. 2021. Amplification of an approximately 700 base-pair product was observed in the putatively ELISA-positive samples, but not in the ELISA-negative samples. The amplicons were further cloned into the pGEM-T Easy vector (Promega, Madison, WI, U.S.A) using the manufacturer's protocol and sequenced using M13 forward and M13 reverse primers (Functional Biosciences, Madison, WI, U.S.A). Sequencing results indicated considerable similarity to TSV genomes available in GenBank, between 88% and 99%. Raw sequence data generated from this study was deposited in NCBI under the bioproject ID PRJNA1009441. Though it cannot be ruled out that the observed symptoms were caused exclusively by TSV infection due to the high number of other viral reads, the results contribute to the literature that indicates hemp can host TSV and should be considered a potential source of TSV inoculum (Chiginsky et al. 2021). This new inoculum source could cause significant crop damage and economic loss when grown with TSV susceptible row and specialty crops.

Drought, a crucial abiotic stressor, markedly reduces the growth and yield of tomato crops (Solanum lycopersicum L.). Consequently, adopting drought-resistant cultivars and implementing breeding programs to enhance drought tolerance have emerged as enduring solutions to alleviate the adverse effects of drought in various tomato cultivation regions. In this study, 68 United States Department of Agriculture (USDA) tomato accessions were assessed in a controlled greenhouse experiment, encompassing both water deficit treatment and a control group subjected to standard watering conditions. The experiment was arranged in a randomized complete block design with three replications. The results of this study pinpointed four accessions, PI 365956, PI 584456, PI 390510, and PI 370091, as drought-tolerant accessions. Additionally, high broad-sense heritability was revealed for leaf wilting, leaf rolling, and SPAD chlorophyll content (total leaf chlorophyll). Furthermore, positive correlations were found among parameters associated with leaf wilting, leaf rolling, and SPAD chlorophyll content. The findings offer valuable insights for tomato breeding initiatives, especially those focused on enhancing drought tolerance in elite cultivars. Future studies will expand the evaluation to include a larger pool of tomato accessions and conduct a genome-wide association study to identify single nucleotide polymorphism (SNP) markers for molecular breeding in tomatoes.

Drought, a crucial abiotic stressor, markedly reduces the growth and yield of tomato crops (Solanum lycopersicum L.). Consequently, adopting drought-resistant cultivars and implementing breeding programs to enhance drought tolerance have emerged as enduring solutions to alleviate the adverse effects of drought in various tomato cultivation regions. In this study, 68 tomato accessions from the United States Department of Agriculture (USDA) were assessed in a controlled greenhouse experiment, encompassing both water deficit treatment and a control group subjected to standard watering conditions. The experiment was arranged in a randomized complete block design with three replications. The results of this study pinpointed four accessions PI 365956, PI 584456, PI 390510, and PI 370091, as drought-tolerant accessions. Additionally, high broad-sense heritability was revealed for leaf wilting, leaf rolling, and SPAD chlorophyll content (total leaf chlorophyll). Furthermore, positive correlations were found among parameters associated with leaf wilting, leaf rolling, and SPAD chlorophyll content. The findings of this study provide valuable insights relevant to tomato breeding initiatives, particularly those aimed at fortifying drought tolerance within the elite cultivars of this essential crop.