Browsing by Author "Nachappa, Punya, advisor"
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Item Open Access Beet curly top virus-beet leafhopper dynamics in hemp in Colorado(Colorado State University. Libraries, 2021) Chiginsky, Judith, author; Nachappa, Punya, advisor; Prenni, Jessica, committee member; Cranshaw, Whitney, committee memberHemp (Cannabis sativa L.) production within North America has dramatically increased in recent years following legislative changes in the 2014 and 2018 Farm Bills that allowed legal paths for its production. However, due to previous restrictions on this crop it has been understudied in the U.S. since its production declined, and ultimately was eliminated. Restrictive laws largely prevented any research regarding management of this crop. Among the understudied issues were those associated with disease identification and management. One newly described disease of the crop in Colorado beet curly top virus (BCTV). This viral pathogen is transmitted by an insect vector, the beet leafhopper. To better understand the diversity and prevalence of BCTV strains infecting hemp in Colorado, beet curly top virus (BCTV) was detected at high incidence (81%) in leaf samples from 12 counties in 2019. Two different strains of BCTV, BCTV-Worland and BCTV-Colorado were found present in single or mixed infection in hemp leaf samples. Phylogenetic analysis revealed BCTV sequences from hemp formed a distinct group along with BCTV-Colorado and BCTV-Worland strains. To determine other potential viral and viroid pathogens in hemp, shotgun metagenomic analysis was performed. Virome analysis revealed the presence of seven viruses and one viroid. Of these, cannabis cryptic virus, cannabis sativa mitovirus, citrus yellow vein associated, opuntia-like virus and hop latent viroid sequences that had high sequence similarity with their corresponding sequences in GenBank. In contrast, tobacco streak virus sequence was highly variable compared to sequences in GenBank suggesting a new genotype of this virus. The data presented here has important implications for the epidemiology and management of the various diseases of hemp and will lead to the development of integrated pest management strategies designed to interrupt transmission cycles and facilitate efficient crop production. Beet leafhopper abundance was monitored throughout the hemp season to understand timing of emergence and flight patterns in the north and western regions of Colorado as well as identify timing of population peaks. Virus incidence in hemp and weed species were assessed using PCR analysis. Beet curly top virus was detected earlier in western field sites of Colorado before being detected in northern survey sites. Of the 41 different weed species surveyed, the weeds that most often tested positive for BCTV, contributing to transmission prevalence were Lactuca serriola (prickly lettuce), Taraxacum officinale (dandelion), and Cichorium intybus (chicory). Life history assays were conducted using viruliferous and non-viruliferous beet leafhoppers in both sugar beet and hemp plants to understand if there were any fitness advantages or costs associated with being a carrier of the virus. Viruliferous beet leafhoppers reared on sugar beet produced more offspring than non-viruliferous treatments. There was no difference between viruliferous, and non-viruliferous beet leafhoppers reared on hemp, suggesting that these virus mediated differences in life history are induced in host plant interactions with the vector. However, beet leafhoppers were able to survive 7-day periods and successfully oviposit and develop on hemp. Understanding migration timing and patterns will result in a more thorough understanding of the pest ecology of the beet leafhopper, which will lead to targeted control strategies to incorporate into integrated pest management tactics to interrupt BCTV transmission cycles, in turn improving yield and farming efficiency.Item Open Access Characterizing host plant-virus-vector interactions of the potato virus Y and aphid pathosystem(Colorado State University. Libraries, 2023) Pitt, William Jacob, author; Nachappa, Punya, advisor; Charkowski, Amy O., committee member; MacRae, Ian V., committee member; Peairs, Frank B., committee member; Smith, Melinda D., committee memberAphid-transmitted potato virus Y (PVY) is one of the most damaging pathogens of potato worldwide. Plant virus prevalence is influenced by landscape composition, host-use patterns of vectors, and the range of capable vector species. Regarding these influences, there are important knowledge gaps that remain within the PVY-aphid pathosystem. The overall goal of this research was to better understand host-virus-vector interactions within the PVY-aphid pathosystem at multiple levels of ecological organization. There is limited information on the effect of landscape-scale crop diversity on prevalence of insect-vectored viruses. In my dissertation, I investigate how landscape composition of crops (Shannon diversity of crops, percent crop cover) affects aphid vector communities and prevalence of aphid-transmitted PVY. I conducted a two-year field study in the San Luis Valley in Colorado where I sampled aphid communities with pan traps, quantified PVY incidence in potato crops with ELISA, and determined the association with landscape variables (Shannon diversity index of crops and percent crop cover) surrounding sampling sites. Crop diversity negatively influenced aphid species richness, but positively influenced PVY incidence. The negative association of crop diversity with aphid species richness could have been due to differences in management between crops and/or increased predation/parasitization of aphids. The positive association between crop diversity and PVY is likely because PVY has a wide host range and increasing crop diversity may positively influence PVY incidence due to increased inoculum in the landscape. Additionally, there was a positive association of potato (virus host) and a negative association of barley (virus non-host) with PVY incidence. In summary, I found that crop species diversity influenced both PVY prevalence and aphid communities, and that the virus host/non-host status of crops likely modulates this effect. Aphid vectors frequently probe upon various plants within a landscape, but the host use patterns of aphid vectors has not been adequately described. It would be useful to identify plants that aphid vectors are probing upon within a landscape in order to identify 1) vector movement within a landscape and 2) potential sources of aphid-transmitted virus inoculum. I used high throughput molecular gut content analysis (GCA) to characterize plant-aphid vector associations within a major potato producing region, the San Luis Valley, CO, where aphid-transmitted potato virus Y (PVY) is a major issue for potato production. Aphids were trapped weekly with suction traps during the growing seasons of 2020 and 2021. Plant-specific DNA in 200 individual aphids representing 9 vector species of PVY (Acyrthosiphon kondoi, A. pisum, Capitophorus elaeagni, Diuraphis noxia, Hayhurstia atriplicis, Myzus persicae, Phorodon cannabis, Protaphis middletonii, and Rhopalosiphum padi) were amplified by PCR, sequenced with the PacBio sequencing platform, and sequences were identified to genus using NCBI BLASTn. I found that all species of aphid vectors probed on plant genera that are outside of their reported host ranges, with Solanum, Medicago, Populus, Brassica, and Glycine as the most frequently detected plant genera. Moreover, aphids probed on many plant genera likely to be PVY host plants. These findings provide information that is essential to develop sustainable and effective management strategies to reduce PVY spread. With the increasing acreage of hemp (Cannabis sativa L.) (Rosales: Cannabaceae) in the United States, I was interested to know whether the cannabis aphid (Phorodon cannabis Passerini) (Hemiptera: Aphididae) is a potential vector of PVY. I conducted transmission assays and used the electrical penetration graph (EPG) technique to determine whether cannabis aphids can transmit PVY to hemp (host) and potato (non-host) (Solanum tuberosum L.) (Solanales: Solanaceace). I showed, for the first time, that the cannabis aphid is an efficient vector of PVY to both hemp (96% transmission rate) and potato (91%) using cohorts of cannabis aphids. In contrast, individual aphids transmitted the virus more efficiently to hemp (63%) compared to potato (19%). During the initial 15 min of EPG recordings, aphids performed fewer intracellular punctures and spent less time performing intracellular punctures on potato compared to hemp, which may in part explain low virus transmission to potato using individual aphids. During the entire 8-hour recording, viruliferous aphids spent less time ingesting phloem compared to non-viruliferous aphids on hemp. This reduced host acceptance could potentially cause viruliferous aphids to disperse thereby increasing virus transmission. Overall, my study shows that cannabis aphid is an efficient vector of PVY and that virus infection and host plant suitability affect feeding behaviors of the cannabis aphid in ways which may increase virus transmission.Item Open Access Developing integrated pest management (IPM) strategies for hemp russet mite (Aculops cannabicola Farkas) on hemp (Cannabis sativa L.)(Colorado State University. Libraries, 2022) Hayes, Christopher, author; Nachappa, Punya, advisor; Cranshaw, Whitney, advisor; Prenni, Jessica, committee memberCannabis sativa L. is a plant that is rapidly becoming a crop of global agricultural importance. However, because of the historical peculiar regulatory status of this crop little has been developed on the pests and pest management needs of the crop. Among the more serious pests that have become established with this plant is hemp russet mite (HRM) Aculops cannabicola Farkas (Acari: Eriophyidae). In order to assess the efficacy of various IPM approaches to mitigating HRM infestations, a series of field and lab experiments were conducted including: 1) evaluation of the effects of sprays of sulfur on control of hemp russet mite, yield, and cannabinoid produduction; 2) evaluation of the efficacy of field release of the phytoseiid mites Amblyseius andersoni, A. swirskii, Neoseiulus fallacis, and N. californicus on HRM-infested hemp plants; and 3) evaluation of hot water immersion as a potential disinfestation method for HRM-infested cuttings used in propagation. The results of the sulfur sprays in field trials showed excellent ability to suppress HRM by up to 98%. Yields of treated plants improved by up to 33% and there was a further increase in the percentage of phytocannabinoids by up to 45% relative to untreated plants. Greatest effects were seen in all trials with plants receiving two applications, one during the vegetative period in July and the second at the initiation of flower production in August. Mass releases of N. fallacis and A. swirskii, but not N. californicus and A. amblysieus, did produce a significant reduction in HRM populations, but no treatments significantly affected yield or percentage of phytocannabinoids, relative to untreated plants. No reproduction was observed of any of the released mites on HRM-infested plants. Immersion treatments to disinfest cuttings included use of a water bath at temperatures of 106°F or 109°F for 10 or 15 minutes, and dips in room temperature surfactant solution of Dr. Bonner's Pure-Castile lavender soap at 1.0% and 0.1% concentrations. All treatments were able to cause significant reduction of HRM on infested hemp cuttings, although none caused complete elimination. No phytotoxicity, as evidenced by effects on subsequent rooting, were observed with any treatment. This study provides novel effective approaches to mitigating HRM at multiple stages in hemp production operations. Outcomes of this research may provide hemp producers and other stakeholders with key pest management strategies needed to produce hemp plants that are free of HRM.Item Open Access Exploring the hemp virome and assessing hemp germplasm for resistance to emerging pathogens(Colorado State University. Libraries, 2024) Hackenberg, Laine, author; Nachappa, Punya, advisor; Roberts, Robyn, committee member; Stenglein, Mark, committee memberHemp (Cannabis sativa L.), commonly grown for its seeds, fiber, and non-psychoactive cannabinoids, has been experiencing a resurgence in the United States with its recent legalization. While farmers across the nation have readily adopted this crop, resources for pest management are still lacking, particularly regarding the diversity and distribution of pathogens. As production increases and the crop diversifies, the emergence and spread of these pathogens are certain. To circumvent loss due to disease, research is needed understand the threats and to identify sustainable management options. The goal of this study is to describe the diversity and distribution of viruses/viroids infecting hemp in Colorado and to determine if there is genetic resistance to pathogens in hemp. The objectives of this study are to 1) characterize the virome of different hemp cultivars throughout the growing season across different locations and 2) screen a panel of genetically unique genotypes of hemp for resistance to emerging viruses/viroids of hemp. Throughout 2021 and 2022, the hemp virome was examined in four major hemp producing regions of Colorado. In total, nine fields were sampled, and each field was visited during three phenological stages (early vegetative, late vegetative, and mature flowering) in order to characterize the virome throughout the growing season. Leaf tissue samples were collected from two cultivars of hemp from each field site. These tissue samples were submitted for High Throughput Sequencing (HTS) and upon bioinformatic analysis, candidate virus/viroid sequences were validated. Across both years, a total of seven viruses were identified: Alfalfa mosaic virus (AMV), Beet curly top virus (BCTV), Cannabis cryptic virus (CanCV), Cannabis sativa mitovirus (CasaMV1), Grapevine line pattern virus (GLPV), Opuntia umbra-like virus (OULV), and Tomato bushy stunt virus (TBSV). All viruses identified had >97% nucleotide identity to the nearest GenBank accession. Between individual cultivars isolated from the same field, both similar and unique viromes were observed. Viral diversity and incidence increased as the growing season progressed for both years. The three viruses that were most commonly found across all regions were CasaMV1, GLPV, and BCTV. Dominating the virome in viral load were CasaMV1 and GLPV. Given the prevalence of BCTV in the virome, in addition to its prevalence in hemp across the western United States, 13 genotypes of hemp were screened for resistance to this pathogen. These genotypes of hemp are genetically diverse, which will aide in the discovery of candidate genes involved with resistance. BCTV is the causal agent of curly top disease which can have drastic symptomology in hemp plants, causing malformed growth, stunted plants, and crop loss up to 100%. Varying BCTV copy number was observed across the hemp genotypes. Additionally, percent disease index (PDI) was analyzed to determine the frequency of infection of individual genotypes. Two of the genotypes were observed to have a lower PDI than the others, 4587 and 4710. Hop latent viroid (HLVd) has been emerging as a threat to the cannabis industry. It has been described across North America but is believed to be worldwide due to its global distribution in hops. HLVd has been documented to cause drastic reduction in cannabinoid content in mature inflorescences and therefore has the potential for substantial economic losses. Although not identified within the 2021 or 2022 virome, HLVd was determined to be an important threat facing hemp production therefore it was included in the screening. Similarly to BCTV, a panel of 14 genetically unique genotypes of hemp were analyzed for resistance to HLVd. Resistance was identified in a single genotype, 517, which had a lower frequency of infection than the others. However, no varying viroidal loads were observed between genotypes. Throughout this study, viruses associated with hemp were described as well as the identification of genetic resistance to emerging pathogens. This work will help to further integrated pest management strategies and promote sustainable agriculture.Item Open Access Studies on the Odonata and Trichoptera of high-elevation lakes of northern Colorado and southern Wyoming(Colorado State University. Libraries, 2021) Al Mousa, Moh'd Anwar, author; Nachappa, Punya, advisor; Fairchild, Mathew, advisor; Boone, Randall, committee memberFreshwater biodiversity loss is a major concern, and global warming is already causing a significant role in species extinctions. The main goal of this research was to provide a baseline for specific aquatic insect species distributions at high-elevation lentic habitats in Northcentral Colorado and Southern Wyoming. I provided occurrence records of the Hudsonian Emerald dragonfly (Somatochlora hudsonica, HED) in Northcentral Colorado and Southern Wyoming. The HED is the only Colorado dragonfly listed as threatened by the US Forest Service. It was ranked as critically imperiled in Colorado and vulnerable in Wyoming. I used Maxent (Maximum entropy), a machine learning program that uses species presence data and environmental variables to predict the potentially suitable habitat for species. Maxent was used to plot a map of the potentially suitable habitats of HED. Temperature seasonality, mean temperature of wettest quarter, precipitation of warmest quarter, precipitation of driest quarter, and precipitation seasonality were the key environmental factors for predicting the occurrence of HED in appropriate high-elevation lakes of Northcentral Colorado and Southern Wyoming with an accumulated contribution of 91%. Results of this study provided baseline data for the US Forest Service to assist to evaluate the conservation status of HED and potentially initiate protection plans in two national forests (The Arapaho & Roosevelt National Forest and the Medicine Bow & Routt National Forest) in Colorado and Wyoming. I report adult caddisflies from 136 montane and alpine lentic habitats, primarily lakes, of seven northern Colorado counties for the first time. My objective was to provide species records of adult and larval caddisflies from high-altitude lentic habitats that are not generally well sampled. These lakes may be potentially impacted by current and future global climate change scenarios. Field collection of adults and rearing of larvae were included with available unpublished and published records, resulting in 541 confirmed records of caddisfly species. Forty-nine species, representing 24% of all known Colorado caddisflies are documented. Seven families and 24 genera are represented. The Limnephilidae comprised 76% of the 49 recorded species. The other six families were usually represented by only one to four species. Distribution maps are presented for the six families and the most common limnephilid species. Montane and alpine lakes are vulnerable ecosystems likely to be impacted by climate change. Comprehensive faunal surveys are key to understanding long–term biodiversity changes and establishing conservation needs and priorities. In addition, species lists of taxa are important to monitor future faunal biodiversity changes.Item Embargo Sweet surprise: the search for genes conferring beet curly top virus resistance(Colorado State University. Libraries, 2023) Withycombe, Jordan, author; Nachappa, Punya, advisor; Nalam, Vamsi, committee member; Nishimura, Marc, committee member; Dorn, Kevin, committee memberSugar beets (Beta vulgaris L.) are grown across the western United States and suffer economic loss annually to curly top disease. Curly top disease is caused by the beet curly top virus (BCTV) and is spread by the only known insect vector the beet leafhopper, Circulifer tenellus Baker (BLH). Current management strategies for BCTV include chemical control using neonicotinoid seed treatments and foliar insecticidal sprays, as well as the use of BCTV-resistant sugar beet varieties. However, the underlying genetic mechanism surrounding resistance in sugar beet is unknown. The overarching goal of this study was to identify the mechanism of resistance in sugar beet to BCTV and identify potential genes conferring resistance. The objectives for this study were: 1) classify the nature of BCTV resistance in a resistant (EL10) and susceptible (FC709-2) genotype of sugar beet using host suitability and host preference insect assays, as well as assess viral load within each genotype and 2) characterize the transcriptional response to BCTV infection using RNA-sequencing. To classify the nature of BCTV resistance in each genotype of sugar beet, host suitability and preference assays were conducted using virus infected and uninfected BLH. In host suitability assays, the percentage of surviving BLH adults and the number of nymphs produced when reared on a single plant of either genotype was determined over a 3-week period. There was no difference in adult survival, or the number of nymphs produced on either genotype for the virus infected or uninfected leafhoppers. Host preference assays were used to assess settling behavior of BLH over time when given a choice between the two genotypes. It was concluded that virus infected leafhoppers had a clear choice to settle on the susceptible genotype at all timepoints after 4 hours, while uninfected leafhoppers did not make as strong of a settling choice. Average viral load for each genotype across three timepoints was estimated using qPCR. The results showed that the average viral load increased in each genotype over time, yet there was no difference in the average viral load between the genotypes at any individual timepoint. The global transcriptional response to BCTV infection over time for a resistant and susceptible genotype of sugar beet was conducted using RNA-sequencing technology. Mock-inoculated and BCTV-inoculated plants from each genotype were sampled on day 1, 7 or 14 post inoculation resulting in the preparation of 36 mRNA sequencing libraries. Comparison between mock-inoculated and BCTV-inoculated plants of each genotype and timepoint were conducted separately to generate six list of differentially expressed transcripts (DETs). Each transcript was annotated with a description and further classified for its role in the plant biological, cellular or molecular processes. The results showed that both genotypes of sugar beet had a dynamic response to BCTV infection over time, although there was minimal overlap between the responses to one another. EL10, the resistant genotype, had DETs associated with phytohormone production including jasmonic acid and abscisic acid, along with proteins linked to stress reduction and the downregulation of plant primary metabolic processes. In contrast FC709-2, the susceptible genotype, was found to produce opposing phytohormones like salicylic acid and auxins, as well as the production of volatile organic compounds and an increase of primary plant metabolic processes. These opposing responses shed light on the differences in the transcriptional response of a resistant and susceptible genotype of sugar beet. Understanding and classifying the mechanisms of resistance or susceptibility to BCTV infection in sugar beet is beneficial to researchers and plant breeders as it provides a basis for further exploration of the host plant-virus-vector interactions.