Progress 09/01/23 to 08/31/24
Outputs
Target Audience:During this reporting period, Dr. Sherif reached a diverse international audience through an invited talk at the 15th International Conference on Plant Pathogenic Bacteria and 5th International Symposium on Biological Control of Bacterial Plant Diseases. The session was well attended by more than 70 participants, including several researchers and students from a number of countries. There, he discussed dsRNA applications in disease management with potential environmental implications. Further outreach activities included an invited seminar given by Dr. Sherif at the USDA Appalachian Fruit Research Station in West Virginia, which brought together researchers, scientists, staff, and students for discussion of his work. Changes/Problems:NA What opportunities for training and professional development has the project provided?This project provided extensive training and professional development opportunities, enabling a postdoctoral fellow, a lab technician, and two graduate students to develop expertise in dsRNA and its ecological interactions. Participants received rigorous training in advanced methodologies, experimental design, data acquisition, analytical procedures, and the operation of sophisticated laboratory equipment. They gained proficiency in adhering to safety protocols and regulatory standards while enhancing soft skills such as communication, teamwork, and scientific presentation. A Ph.D. student led entomology-related experiments, including constructing cages, procuring plants, coordinating with the research team, maintaining insect and mite colonies, and developing expertise in thrips and mite identification, data management, statistical analysis, and report preparation. Regular mentorship, feedback, and access to scientific resources fostered continuous learning, professional growth, and the refinement of technical and analytical skills, ensuring a well-rounded and impactful training experience. How have the results been disseminated to communities of interest?During this reporting period, Dr. Sherif reached a diverse international audience through an invited talk at the 15th International Conference on Plant Pathogenic Bacteria and 5th International Symposium on Biological Control of Bacterial Plant Diseases. The session was well attended by more than 70 participants, including several researchers and students from a number of countries. There, he discussed dsRNA applications in disease management with potential environmental implications. Further outreach activities included an invited seminar given by Dr. Sherif at the USDA Appalachian Fruit Research Station in West Virginia, which brought together researchers, scientists, staff, and students for discussion of his work. What do you plan to do during the next reporting period to accomplish the goals? Expand the intensive field studies and molecular investigations to assess the impacts of environmental factors and dsRNA formulations on microbial communities and stability in various mediums. Perform in-depth analyses of soil samples for any long-term effects of the dsRNA formulations on soil microbiota and microbial dynamics. Employ advanced bioinformatic tools and molecular techniques to analyze the collected DNA samples for shifts in microbial communities associated with the application of dsRNA and ME-dsRNA and interpret the ecological implications in detail. Improve the methods of extraction and analysis of dsRNA to ensure consistent, accurate, and reliable data collection across different samples and experimental conditions. Showcase results of Lahiri's lab in 1-2 professional and extension meetings to contribute towards knowledge of biofungicides and their impacts on beneficial arthropods. Preparing manuscripts for publication will also be prioritized focusing on the non-target impacts on insects and mites.
Impacts
What was accomplished under these goals?
Objective 1: Determine the biodegradability rate of naked and minicell-encapsulated dsRNAs in water and soil samples, as well as on leaf and fruit samples.A comprehensive study on the persistence of dsRNA on strawberry plants was conducted in 2024. Greenhouse and field experiments were performed to test the durability of naked and carrier-based dsRNA treatments. Applications of dsRNA at double the recommended dose were made until visible residues could be seen on the plant surfaces, simulating worst-case scenarios. Leaf and fruit samples were collected every week for a total of 30 days. A pot experiment was also run to test the persistence of dsRNA in the soil. Two different types of soils were used to represent different environmental conditions: sandy loam (10% clay, 30% soil, 60% sand) and clay (60% clay, 30% soil, 10% sand). Both Naked and minicell-encapsulated dsRNA treatments were applied, and soil samples were collected from 0.5, 1, and 3 inches of depth. In both experiments, the collected samples were processed for dsRNA extraction, which was then converted to cDNA and stored at -20°C until further analysis. This comprehensive approach allowed for a thorough evaluation of dsRNA persistence in different environmental contexts. Objective 2: Characterize bacterial and fungal communities of the soil microbiota after frequent applications of dsRNA and ME-dsRNA. The aim of this study was to describe the effects of dsRNA and minicell-encapsulated dsRNA applications on soil microbial communities. In 2023, soil samples were collected from an open field, with three replicates of 0.5-, 1-, and 2-inch depths for pre-treatment and post-treatment conditions, which were taken 30 days after application. For simplicity in the analysis, samples from different depths of the same treatment were pooled, so three replicates per treatment existed. Additional soil samples collected in 2024 were similarly processed. DNA was extracted from all the collected soil samples and will be subjected to DNA barcoding and sequencing analysis to assess changes in bacterial and fungal community composition and diversity. Objective 3. Demonstrate that RNAi-enabled biofungicide has no adverse effects on beneficial microorganisms and insects. Seven commercially available biocontrol agents-five bacterial and two fungal-were exposed to naked and minicell-encapsulated dsRNA at elevated doses in order to assess the possible effects of dsRNA-based biopesticides on beneficial microorganisms. The results indicated that the growth of all biocontrol agents was not affected by the treatment, hence posing minimal risk to these non-target organisms. To further explore the compatibility of the dsRNA-based biopesticide with other control approaches, we explored any synergistic or antagonist effects of the combination of dsRNA with different biocontrol agents against Botrytis cinerea. According to the results obtained in our experiments, these combined treatments did not notably increase or reduce disease control in relation to the individual treatments. Such evidence places dsRNA-based bio-pesticides as promising effective and sustainable components in IPM practices. The effect of an RNAi biofungicide on two predatory mite species, Amblyseius swirskii and Phytoseiulus persimilis (Acari: Phytoseiidae), and their efficacy against two economically important strawberry pests, chili thrips, Scirtothrips dorsalis, and twospotted spider mite, Tetranychus urticae, were evaluated in a greenhouse study conducted at the University of Florida, Gulf Coast Research and Education Center. Strawberry cultivar "Florida Brilliance" transplants supplied from Crown Nursery, California, were grown in pots. Plants used in the experiments had 4-5 expanded trifoliates. Laboratory colonies of S. dorsalis and T. urticae maintained in the Lahiri lab were separately used to infest the plants. Plants infested with S. dorsalis were treated with the predatory mite A. swirskii, while those infested with T. urticae were treated with P. persimilis. The populations of pests and their predators were recorded 7, 14, and 21 days after treatment with the RNAi biofungicide. Each predatory mite treatment included six replicates, and the experiment was repeated twice (Trial 1 and Trial 2). Results: An analysis of variance followed by Tukey's HSD test (α = 0.05, SAS Version 9.4) revealed the following: a) Impact on S. dorsalis and A. swirskii: In the first trial, no significant effects of the RNAi treatments were observed on either S. dorsalis or its predator A. swirskii and in the second trial, the naked RNAi biofungicide significantly reduced S. dorsalis populations (7.61 ± 1.35) as compared to the non-treated control (10.38 ± 0.8) with no adverse effects on A. swirskii. b) Effects on T. urticae and P. persimilis: In the first trial, the encapsulated RNAi treatment significantly reduced both T. urticae and its predator P. persimilis populations by day 21, and in the second trial, the encapsulated RNAi biofungicide significantly reduced T. urticae populations (38.06 ± 4.87) compared to the non-treated control (55.06 ± 6.68), while P. persimilis remained unaffected. These findings demonstrate the potential of RNAi-enabled biofungicides for pest control without negatively impacting beneficial predatory mites under specific conditions.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Zarrabian, Mohammad, and Sherif M. Sherif. "Silence is not always golden: A closer look at potential environmental and ecotoxicological impacts of large-scale dsRNA application." Science of The Total Environment (2024): 175311.
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Progress 09/01/22 to 08/31/23
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided multifaceted opportunities for training and professional development, allowing two postdoctoral fellows, and a lab technician to delve deeply into the nuanced scientific principles of dsRNA and its multifarious interactions within ecological systems. The participants were afforded rigorous training in advanced methodologies, ensuring precision and reliability in experimental design, data acquisition, and analytical procedures. Technological prowess was emphasized, promoting proficiency in the utilization and maintenance of sophisticated laboratory equipment and analytical tools. A strong focus was placed on meticulous adherence to safety protocols and regulatory compliances, fostering a secure and compliant research ambiance. Soft skills such as effective communication and collaborative teamwork were honed, enriching the research environment and facilitating seamless conveyance of scientific findings. Continuous learning was encouraged through access to a plethora of scientific resources and regular insightful sessions, fostering an atmosphere of perpetual knowledge enrichment and professional growth. The mentorship provided was invaluable, offering consistent support, guidance, and insights, thereby contributing to the creation of a nurturing and conducive learning environment. Regular assessments and constructive feedback were integral to ensuring the continuous elevation of skills and refinement of acquired knowledge. A PhD candidate under Dr. Lahiri's guidance spearheaded the entomological experiments. Their responsibilities ranged from constructing cages, sourcing plants from nurseries, to liaising with the research team on the nuances of biofungicide application. They also managed diverse insect and mite colonies, oversaw data, coordinated logistics, and provided training on thrips and mite identification. Finally, they took on the tasks of statistical analysis and report drafting How have the results been disseminated to communities of interest?Dr. Sherif's group has delivered presentations on dsRNA and its applications in disease management at two international conferences: the American Society for Horticultural Science in Orlando, USA and the International Congress of Plant Pathology in Lyon, France. Since the entomological experiments are still underway, the results haven't been shared with a broader professional audience yet. What do you plan to do during the next reporting period to accomplish the goals?1. Intensive Field and Molecular Studies: Intensify field studies and advanced molecular analysis to analyze the impacts of environmental variables and dsRNA formulations on microbial communities and dsRNA stability in varied mediums. Utilize advanced analytical techniques like RT-qPCR and HPLC to enhance precision in quantifying dsRNA and understanding its stability and biodegradability in diverse environmental conditions. 2. Advanced Analytical and Bioinformatic Assessments: Undertake detailed molecular and bioinformatic analyses of the collected DNA samples, leveraging advanced tools and methodologies, to discern shifts in microbial communities due to dsRNA and ME-dsRNA applications and interpret the ecological impacts meticulously. 3. Comprehensive Examination of Water and Soil Samples: Extend the examination of dsRNA and ME-dsRNA stability in diverse water sources, correlating the findings with the water's pH and mineral composition, alongside conducting advanced analysis on soil samples to uncover the longitudinal impacts of dsRNA formulations on soil microbiota. 4. Optimization of Methodologies: Refine dsRNA extraction protocols and analytical methodologies to ensure reliability, accuracy, and consistency in data acquisition and interpretation across different samples and experimental conditions. 5. Synthesis, Interpretation, and Dissemination of Findings: Consolidate, interpret, and synthesize the findings from various experiments to draw cohesive and robust conclusions about the behavior, stability, and impacts of dsRNA in varied environmental settings, and disseminate the insights to enrich the scientific community's understanding of microbial community dynamics and interactions. 6. Repeat the experiments for Objective 3: The Lahiri lab will conduct the experiment again in the greenhouse to validate the findings from the first trial concerning the two predatory mites and pest species. They plan to share the results at one or two professional gatherings, including extension meetings, to enhance understanding of biofungicides and their effects on beneficial arthropods
Impacts
What was accomplished under these goals?
Objective 1: Determine the biodegradability rate of naked and minicell-encapsulated dsRNAs in water and soil samples, as well as on leaf and fruit samples The greenhouse experiment was embarked upon to assess the rate of biodegradability of both naked and minicell-encapsulated dsRNAs on the strawberry fruit and foliage, and to investigate the influence of soil texture on the penetration of minicell carrier and naked-dsRNA. In this exploration, 54 pots were allocated, each filled with one of two distinct soil types: sandy loam (composed of 10% clay, 30% soil, and 60% sand) and clay (constituted by 60% clay, 30% soil, and 10% sand), in triplicate. Young strawberry plants were then cultivated and housed within the greenhouse until reaching optimal condition for treatment application. To further scrutinize the degradability rate under field conditions, an analogous study was executed and concluded for the initial year. Within this experiment, the concentration for both naked and minicell dsRNA was predetermined as the worst-case scenario, precisely double the suitable dose, with application continued until residue was evident on the leaves. For each distinct treatment, specimens of both fruit and leaves were meticulously collected in quadruplicate over a 30-day period, at seven-day intervals post-inoculation. Additionally, to assess the biodegradability rate of dsRNA within the open-field soil matrix, concluding the experiment, soil samples of varied depths (0.5, 1, 3 inches) were secured, with three biological replicates each, and were stored separately. All procured samples were subsequently preserved at -80°C pending downstream analysis. This meticulous approach aimed to render comprehensive insights into the behavior and stability of dsRNAs, contributing pivotal data to the scientific community's understanding of its interaction with plant and soil ecosystems, under varied environmental conditions. Objective 2: Characterize bacterial and fungal communities of the soil microbiota after frequent applications of dsRNA and ME-dsRNA To decipher the impact of various applied formulations of dsRNA--specifically, naked and Minicell dsRNA--on the microbial communities within soil, encompassing both bacterial and fungal populations, soil samples from the initial year of the open-field experiment were meticulously gathered from three distinct soil strata (0.5, 1, and 3 inches), and were collectively categorized as bulk samples. Consequently, a sum of nine bulk soil samples, embodying three unique treatments--water, dsRNA, and ME-dsRNA--alongside three biological replicates, were amassed and prepared for the subsequent extraction of total DNA. This prepared DNA is designated for downstream analyses including barcoding and sequencing to generate profound insights into the microbial dynamics and interactions precipitated by differing dsRNA formulations within the soil ecosystem. This analytical endeavour aims to elucidate the multidimensional influences of dsRNA formulations on soil microbiota, yielding critical insights into their ecological interactions and impacts, thereby contributing to the nuanced understanding of microbial community structures and functions within applied environmental settings. Objective 3. Demonstrate that RNAi-enabled biofungicide has no adverse effects on beneficial microorganisms and insects. To assess the effect of the RNAi-enabled biofungicide on two species of predatory mites, Amblyseius swirskii Athias-Henriot and Phytoseiulus persimilis Athias-Henriot (Acari: Phytoseiidae), against two economically significant arthropod pests of strawberry, Fragaria × ananassa Duchesne (Rosales: Rosaceae), a greenhouse study was initiated at the University of Florida's Gulf Coast Research and Education Center. Strawberry transplants were sourced from Crown Nursery in California and potted. The cultivar "Florida Brilliance" was chosen for the caged plant experiments, and plants with 4-5 expanded trifoliates were utilized. Laboratory colonies of strawberry pests, namely chilli thrips Scirtothrips dorsalis Hood (Thysanoptera: Thripidae) and twospotted spider mite Tetranychus urticae Koch (Acari: Tetranychidae) from the Lahiri lab, were used to infest these plants separately. Plants infested with S. dorsalis were treated with the predatory mite A. swirskii, while those infested with T. urticae were treated with P. persimilis. The numbers of S. dorsalis, T. urticae, A. swirskii, and P. persimilis on plants were counted on days 7, 14, and 21 post biofungicide treatment. Both predatory mite treatments were replicated six times. After exposing the two predatory mites to the RNAi-enabled biofungicide, the subsequent results were noted post an analysis of variance followed by Tukey's HSD (separation of means test) (α = 0.05, SAS Version 9.4, SAS Institute, Cary, NC): a) The RNAi fungicide treatments did not show any detrimental effects on either S. dorsalis or its predator, the A. swirskii mite; and b) the populations of both P. persimilis and T. urticae were adversely affected by the encapsulated RNAi treatment in comparison to the non-treated control plants. By the 21st day post-treatment, a significant decline was observed in the numbers of both the predatory mite and the pest.
Publications
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