Progress 11/19/19 to 11/18/24
Outputs
PROGRESS REPORT Objectives (from AD-416): 1. Develop biopesticides to improve control of arthropods. 1.1. Discover novel bioinsecticide active ingredients from crude plant extract screening and bioassay-directed fractionation. 1.2. Discovery of fungal compounds with activity against permethrin- resistant and susceptible mosquitoes. 2. Develop biorepellents to improve control of arthropods. 2.1. Discover novel arthropod repelling biopesticides from mosquito repelling folk remedies and plant essential oils. 2.2. Development and optimization of the chromene derivative 131-1 as a biopesticide. 3. Develop natural product synthetic analogs for management and control of arthropods. 3.1. Use synthetic methods to develop and optimize fatty acids as natural topical and spatial arthropod repellents. 3.2. 131-1 Development into a commercial mosquito repelling product. Approach (from AD-416): A bioassay-directed investigative approach will be performed on bioactive extracts in efforts to discover bioinsecticides and repellents for use against arthropod pests. General procedures for isolation of active compounds from plants and microbes will be utilized. Solvent extractions, liquid-liquid partitioning, column chromatography and thin layer chromatography will be employed as needed. Extracts, fractions and pure compounds will be tested for insecticidal and insect repellent activity in assays being carried out routinely at collaborator's laboratories. Identification of active compounds will be done using spectroscopic methods including mass spectroscopy (EI, CI, and high resolution ESI), nuclear magnetic resonance (one- and two-dimensional). Chemical modification will be performed on compound(s) identified as "highly active" to improve activity or bioavailability. This is the final year of research for this research project. Project termination was effective prior to the end of this project with the FY24 congressional budget (retroactive to Oct 1, 2023) resulting in ceasing of research related to this project in April of 2024 following final passage of the FY24 budget. Effective upon termination, the lead researcher has been reassigned to the Crop Bioprotection Research Unit in Peoria, Illinois. Other research plan researcher has been moved fully (1.0 FTE) to an NP306 project, ⿿Biobased Pesticide Discovery and Product Optimization and Enhancement from Medicinal and Aromatic Crops project number 6060-41000-015-000D. A. For objective 1, develop biopesticides to improve control of arthropods, milestones have been met and significant progress has been made. A1. In prior years, the following significant accomplishment was reported. This accomplishment relates to the discovery of microbial bio- insecticide compositions for controlling Aedes mosquito larvae, which are formulated from microbial formulations and/or metabolic products of Streptomyces orinoci, including neoantimycin. The active compound was identified as neoantimycin, and LC50 values of the crude extract of S. orinoci and neoantimycin demonstrated 1.20 and 1.93 ppm in the lab- susceptible Orlando (ORL) 1952 strain of Ae. aegypti, respectively. Notably, neoantimycin also exhibited a LC50 value of 2.03 ppm in the pyrethroid-resistant Puerto Rican strain (PR) of Ae. aegypti. We submitted an invention disclosure based on the efficacy of neoantimycin that is mentioned above. Also related to investigation of Streptomyces species, a study evaluated S. distallicus for the discovery of larvicidal compounds against Ae. aegypti for the first time. We reported the isolation and characterization of six compounds from S. distallicus, as well as their larvicidal activity against Ae. aegypti. Thus, the study discovered that S. distallicus and crude extracts thereof are promising sources of potential microbial larvicides. Invention disclosures were filed. A2. Propolis is a natural product produced by bees using resins collected from plants. The primary role of propolis for bees is to protect the beehive against invaders, but it also performs functions such as regulating hive temperature and in controlling parasites and microorganisms. Due to its botanical origin, propolis exhibits a rich diversity of metabolites. So, we evaluated the activity of the main constituents isolated from green, red, and brown propolis against Ae. aegypti mosquito larvae. From green propolis, four prenylated compounds derived from 4-hydroxycinnamic acid were isolated: artepillin C, baccharin, drupanin and 2,2-dimethyl-6-carboxyethenyl-2H-1-benzopyran. Red propolis provided five compounds: a chalcone, isoliquiritigenin, the pterocarpane medicarpin and three isoflavonoids, neovestitol, vestitol and 7-O-methylvestitol. Four diterpenes were isolated from brown Propolis: totarol, dehydroabietic acid, communic acid and isopimaric acid. Regarding larvicidal activity, artepillin C and drupanin induced mortality of 6.7 and 3.3% of larvae respectively at 100 PPM. Totarol and isopimaric acid were inactive, while communic acid and dehydroabietic acid induced mortality of 6.7 and 3.3% respectively at 100 PPM. In contrast, the red propolis compounds demonstrated significantly higher activity, with all of them being active. Isoliquiritigenin, neovestitol, and methylvestitol caused the mortality of 93.3% of the larvae at a concentration of 100 PPM, while medicarpin induced mortality of 96.7% of the larvae at the same concentration. The molecular docking analysis of the compounds against the target model AaAChE1 of Ae. aegypti was also reported. A3. Piper cubeba L.F., commonly known as cubeb pepper or tailed pepper, is a plant species belonging to the Piperaceae family, native to Southeast Asia. P. cubeba has been historically utilized as a culinary spice and traditional medicinal ingredient. We evaluated the larvicidal activities against Ae. aegypti of lignans isolated from P. cubeba. From the crude 85% ethanol/water extract of P. cubeba four major compounds were isolated and identified as dihydroclusin, cubebin, clusin and yatein. Regarding larvicidal activity, dihydroclusin demonstrated activity, causing the mortality of 96.7% of the larvae at a concentration of 100 PPM, while cubebin induced mortality of 30% of the larvae at the same concentration. In contrast, clusin and yatein were inactive. Dihydroclusin exhibited notably high larvicidal activity, indicating that dihydroclusin is a promising candidate for mosquito control. Through molecular docking analysis, the proposed mechanism of action was related on the UDP-N-acetylglucosamine pyrophosphorylase protein interaction, crucial for chitin synthesis. B. For objective 2, develop biorepellents to improve control of arthropods., milestones have been met and significant progress has been made. B1. In prior years, the following significant repellent accomplishment was reported. Pineapple weed (Matricaria discoidea) has documented use as an insect repellent by various indigenous peoples of North America. This investigation was conducted to evaluate this practice and systematically identify chemical constituents responsible for any insect repelling effect by utilizing a mosquito (Aedes aegypti) biting deterrent assay approach. Essential oil from dried M. discoidea was the most bioactive crude extract generated and was as effective as DEET. The most active compounds were determined to be alpha-terpineol, spathulenol, and neryl isovalerate. Large cage in vitro evaluation of pure compounds isolated from M. discoidea indicated alpha-terpineol to be the most active compound providing complete protection similar to DEET. This is the first report on the mosquito repellency of neryl isovalerate and scientific evidence reported here validates the traditional use of M. discoidea as a biting-insect deterrent. B2. In prior years, the following significant repellent accomplishment was also reported. Phlebotomus papatasi (Scopoli, 1786), is a major vector for Leishmania major in the Middle East that significantly impacted US Military operations and personnel, demonstrating the need for additional sand fly control and repellent options. We report measurement of spatial repellency and avoidance to essential oils of Lippia graveolens (Mexican oregano), Pimenta dioica (allspice), Amyris balsamifera (amyris), Nepeta cateria (catnip), Mentha piperita (peppermint), Melaleuca alternifolia (tea tree), the 9-12 carbon saturated fatty acids (nonanoic acid, decanoic acid, undecanoic acid, and dodecanoic acid), and the synthetic repellents DEET and IR3535 to adult female P. papatasi sand flies in a static air olfactometer. Materials tested at 1% application exhibited varying levels of activity but were not significantly different compared to DEET and IR3535 in mean repellency and avoidance to female sand flies except for nonanoic acid. Some materials produced sand fly mortality, particularly nonanoic and undecanoic acids. Different trends were noted in mean repellency over increasing exposure time: 1) Pimenta dioica oil, tea tree oil, decanoic acid, undecanoic acid, DEET and IR3535 exhibited increasing mean repellency over time; 2) catnip oil, amyris oil, peppermint oil and dodecanoic acid exhibited relatively constant mean repellency over time; and 3) Lippia graveolens oil and nonanoic acid exhibited a general decrease in mean repellent activity over time. Essential oils of catnip, amyris, and peppermint were effective spatial repellents at reduced concentrations compared to DEET. Additional research is needed to elucidate the modes of action and potential synergism repellents and essential oil components responsible for observed activity of the oils. C. For objective 3, develop natural product synthetic analogs for management and control of arthropods, milestones have been partially met and significant progress has been made in previous years to meet this objectives milestone. C1. In prior years, the following synthetic optimization of a repellent was reported. Esters of C-11:0 and C-12:0 fatty acids were synthesized last fiscal year in an effort to both improve the mosquito repellency and improve the duration of activity of the natural C-11 and C-12 fatty acids. The optimum chain length for the esters has been determined and a highly active analog has been produced and shown to be more active than DEET in the Klun & Debboun (K&D) module bioassay system. Additional evaluations were performed in the Ali & Khan (A&K) bioassays this year. In-vivo evaluations of the most active compounds against Ae. aegypti were performed following the large scale synthesis of the analogs. In the A&K assay, undecanoic acid appears to be the most active compound in this group tested while butyl undecanoate was the most active ester analog synthesized. Both of these highly effective compounds are more effective than DEET and warrant further investigations as mosquito repellents. C2. Development and optimization of the chromene derivative 131-1 as a biopesticide. We have synthesized C2, C3, C4 and halogenated analogs of the patented chromene derivative 131-1 and identified the more effective repellent as a halogenated analog of 131-1. We were in the process of changing our strategy in developing non halogenated analogs so that these products will be acceptable and user friendly as public health insect repellent.
Impacts
(N/A)
Publications
- Semerdjieva, I.B., Zheljazkov, V.D., Cantrell, C.L., Koleva-Valkova, L., Maneva, V., Radoukova, T., Astatkie, T., Kacániová, M., Slavov, S.B., Atanasova, D., Borisova, D. 2024. Phytochemical composition and biopesticidal potential of Pinus mugo Turra essential oil. Industrial Crops and Products. 209:118019. https://doi.org/10.1016/j.indcrop.2023. 118019.
- Kurmanbayeva, A., Ospanov, M., Tamang, P., Shah, F., Ali, A., Cantrell, C. L., Dinara, S., Datkhayev, U., Khan, I.A., Ibrahim, M.A. 2023. Regioselective Claisen-Schmidt Adduct of 2-undecanone from Houttuynia cordata Thunb as insecticides against Solenopsis invicta and repositioning plant fungicides against Colletotrichum fragariae. Molecules. https://doi. org/10.3390/molecules28166100.
- Ali, A., Shah, F.M., Radwan, M.M., Elhendawy, M.A., Elsohly, M.A., Khan, I. A. 2023. Curcuma longa essential oils:toxicity and repellency against hybrid imported fire ants (Formicidae: Hymenoptera). Journal of Medical Entomology. 61:191-200. https://doi.org/10.1093/jme/tjad151.
- Temeyer, K.B., Schlechte, K.G., Coats, J.R., Cantrell, C.L., Rosario-Cruz, R., Lohmeyer, K.H., Perez De Leon, A.A., Li, A.Y. 2024. In vitro evaluation of essential oils and saturated fatty acids for repellency against the old-world sandfly, Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae). Insects. https://doi.org/10.3390/insects15030155.
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Progress 10/01/22 to 09/30/23
Outputs
PROGRESS REPORT Objectives (from AD-416): 1. Develop biopesticides to improve control of arthropods. 1.1. Discover novel bioinsecticide active ingredients from crude plant extract screening and bioassay-directed fractionation. 1.2. Discovery of fungal compounds with activity against permethrin- resistant and susceptible mosquitoes. 2. Develop biorepellents to improve control of arthropods. 2.1. Discover novel arthropod repelling biopesticides from mosquito repelling folk remedies and plant essential oils. 2.2. Development and optimization of the chromene derivative 131-1 as a biopesticide. 3. Develop natural product synthetic analogs for management and control of arthropods. 3.1. Use synthetic methods to develop and optimize fatty acids as natural topical and spatial arthropod repellents. 3.2. 131-1 Development into a commercial mosquito repelling product. Approach (from AD-416): A bioassay-directed investigative approach will be performed on bioactive extracts in efforts to discover bioinsecticides and repellents for use against arthropod pests. General procedures for isolation of active compounds from plants and microbes will be utilized. Solvent extractions, liquid-liquid partitioning, column chromatography and thin layer chromatography will be employed as needed. Extracts, fractions and pure compounds will be tested for insecticidal and insect repellent activity in assays being carried out routinely at collaborator's laboratories. Identification of active compounds will be done using spectroscopic methods including mass spectroscopy (EI, CI, and high resolution ESI), nuclear magnetic resonance (one- and two-dimensional). Chemical modification will be performed on compound(s) identified as "highly active" to improve activity or bioavailability. For Objective 1, develop biopesticides to improve control of arthropods, milestones have been met and significant progress has been made. Streptomyces is a type of bacteria commonly found in soil and known for its ability to produce various secondary metabolites, some of which have larvicidal properties. Metabolites serve as a defense mechanism against other microorganisms and predators in their natural habitat. Microbial larvicides derived from Streptomyces have been shown to be effective against many species of mosquitoes, including those that are major vectors of human diseases such as malaria, dengue fever, and Zika virus. They are also considered to have minimal impact on non-target organisms and the environment, making them a promising candidate for sustainable mosquito control methods. This accomplishment relates to the discovery of microbial bio-insecticide compositions for controlling Aedes mosquito larvae, which are formulated from microbial formulations and/or metabolic products of S. orinoci, including neoantimycin. The potent larvicidal activity of Streptomyces metabolites, combined with their low environmental impact and selectivity, makes them a promising source for the development of effective and sustainable tools for mosquito control. We discovered larvicidal compounds against Ae. aegypti from S. orinoci. The active compound was identified as neoantimycin, and LC50 values of the crude extract of S. orinoci and neoantimycin demonstrated 1.20 and 1. 93 ppm in the lab-susceptible Orlando (ORL) 1952 strain of Ae. aegypti, respectively. Notably, neoantimycin also exhibited a LC50 value of 2.03 ppm in the pyrethroid-resistant Puerto Rican strain (PR) of Ae. aegypti. This accomplishment relates to the discovery of microbial bio-active compositions for controlling Aedes mosquito larvae and/or phytopathogenic fungi, which were previously unexplored Streptomyces species and their secondary metabolites. Our recent report indicates that Streptomyces distallicus and its extracts are a promising source of microbial biolarvicides. We evaluated six additional Streptomyces species, namely, S. ardus, S. kentuckensis, S. eurocidicus, S. salmonis, S. hachijoensis, and S. septatus, which are related to S. distallicus at the DNA level, for their possible application as microbial biopesticides, plant protectants, or biochemical biopesticide precursors with agricultural implications. We subjected the culture filtrates of these Streptomyces species to ethyl acetate extraction and conducted a comprehensive analysis using cutting-edge technology. We then subjected the resulting potent bioactive compounds to various in-house assays to evaluate their effectiveness against phytopathogenic fungi and larvae of Aedes aegypti. From the initial screening, LC50 values of the crude extract of S. ardus and S. kentuckensis demonstrated 1.64 and 7.27 ppm in the lab-susceptible Orlando (ORL) 1952 strain of Ae. aegypti, respectively. Notably, S. ardus also exhibited an LC50 value of 6.48 ppm in the pyrethroid-resistant Puerto Rican strain (PR) of Ae. aegypti. Moreover, S. ardus, S. eurocidicus and S. septatus showed portent phytopathogenic antifungal activity against Colletotrichum fragraiae. Apiaceae (formerly Umbelliferae), commonly known as parsley or carrot family is one of the largest plant families that consists of about 3780 species distributed throughout the globe. The Apiaceae family includes a wide variety of vegetables and herbs such as celery (Apium graveolens), parsley (Petroselinum crispum), dill (Anethum graveolens), fennel (Foeniculum vulgare) and cumin (Cuminum cyminum). Apiaceae plants have storage glands for essential oils giving rise to unique aroma and flavors. Apiaceae essential oils have activity against pests and microbes. Plants produce secondary metabolites as part of defense mechanisms against biotic or abiotic stress. These secondary metabolites protect plants from pests such as fungi, insects and other plants that compete for survival. Thus, these plant extracts and secondary metabolites are known to have various bioactivities, such as herbicidal, insecticidal, and antimicrobial activities. We have extracted seeds of Celery (Apium graveolens), parsley (Petroselinum crispum), dill (Anethum graveolens), fennel (Foeniculum vulgare), cumin (Cuminum cyminum) and shown to posses larvicide activity in our laboratory bioassay at the collaborator⿿s laboratory. We have isolated and identified sufficient quantities to carry out repellent studies. Cumin aldehyde, cumic alcohol, apiol, carvone, anethol, carvacrol, and cumic alcohol are some of these compounds. We have synthesized various analogs of the naturally occurring constituents and these are being evaluated for larvicide activity against permethrin susceptible and resistant Aedes aegypti mosquitoes and repellent activity against the same species of female mosquitoes. For Objective 2, develop biorepellents to improve control of arthropods, milestones have been met and significant progress has been made. Hypericum species and especially H. perforatum are well known for their therapeutic applications. The present study assessed the essential oils (EO) composition, antifungal and aphid suppression activity of seven Bulgarian Hypericum species. The EOs were analyzed by GC⿿MS-FID analyses to determine the chemical constituents present. H. perforatum EO had significant repellent and insecticidal activity against two aphid species, the bird cherry-oat aphid and the English grain aphid. The tested EOs did not show significant activity against selected economically important agricultural fungal pathogens Fusarium spp., Botrytis cinerea, Colletotrichum spp., Rhizoctonia solani and A. niger. The EO of the Hypericum species found in the Bulgarian flora could be utilized for the development of new biopesticides for aphid control. Phlebotomus papatasi, is a major vector for Leishmania major in the Middle East that significantly impacted U.S. Military operations and personnel, demonstrating the need for additional sand fly control and repellent options. We report measurement of spatial repellency and avoidance to essential oils of Lippia graveolens (Mexican oregano), Pimenta dioica (allspice), Amyris balsamifera (amyris), Nepeta cateria (catnip), Mentha piperita (peppermint), Melaleuca alternifolia (tea tree), the 9-12 carbon saturated fatty acids (nonanoic acid, decanoic acid, undecanoic acid, and dodecanoic acid), and the synthetic repellents DEET and IR3535 to adult female Phlebotomus papatasi sand flies in a static air olfactometer. Materials tested at 1% application exhibited varying levels of activity but were not significantly different compared to DEET and IR3535 in mean repellency and avoidance to female sand flies except for nonanoic acid. Some materials produced sand fly mortality, particularly nonanoic and undecanoic acids. Different trends were noted in mean repellency over increasing exposure time: 1) Pimenta dioica oil, tea tree oil, decanoic acid, undecanoic acid, DEET and IR3535 exhibited increasing mean repellency over time; 2) catnip oil, amyris oil, peppermint oil and dodecanoic acid exhibited relatively constant mean repellency over time; and 3) Lippia graveolens oil and nonanoic acid exhibited a general decrease in mean repellent activity over time. Essential oils of catnip, amyris, and peppermint were effective spatial repellents at reduced concentrations compared to DEET. Additional research is needed to elucidate the modes of action and potential synergism repellents, and essential oils and their components responsible for observed activity of the oils, and chemical pesticides. C. For Objective 3, develop natural product synthetic analogs for management and control of arthropods, milestones have been met and significant progress has been made in the previous year to meet this objective's milestone. Plumbagin, a derivative of naphthoquinone, is widely recognized for its insecticidal properties and is commonly found in natural sources such as the Plumbago plant genus, as well as the carnivorous plant genera Drosera and Nepenthes. This accomplishment relates to synthesizing various naphthoquinone derivatives and assessing their effectiveness as larvicidal agents. Furthermore, molecular docking investigations were conducted using two Aedes aegypti proteins, namely an odor-binding protein and a saliva protein, revealing that the newly developed naphthoquinone derivatives exhibit favorable binding affinities. The most potent derivative, 6-methyl-5,8-dioxo-5,8-dihydronaphthalen-1-yl hexanoate, showed LC50 values of <6.95 ppm and <8.66 against the ORL 1952 strain and PR strain of Ae. aegypti, respectively. Artificial Intelligence (AI)/Machine Learning (ML) No artificial intelligence or machine learning methods were used. ACCOMPLISHMENTS 01 Streptomyces orinoci microbial formulations as a bioinsecticide. Streptomyces is a type of bacteria commonly found in soil and known for its ability to produce various secondary metabolites, some of which have larvicidal properties. Metabolites serve as a defense mechanism against other microorganisms and predators in their natural habitat. Microbial larvicides derived from Streptomyces have been shown to be effective against many species of mosquitoes, including those that are major vectors of human diseases such as malaria, dengue fever, and Zika virus. They are also considered to have minimal impact on non-target organisms and the environment, making them a promising candidate for sustainable mosquito control methods. This accomplishment relates to the discovery of microbial bio-insecticide compositions for controlling Aedes mosquito larvae, which are formulated from microbial formulations and/or metabolic products of S. orinoci, including neoantimycin. The active compound was identified as neoantimycin, and LC50 values of the crude extract of S. orinoci and neoantimycin demonstrated 1.20 and 1.93 ppm in the lab-susceptible Orlando (ORL) 1952 strain of Ae. aegypti, respectively. Notably, neoantimycin also exhibited a LC50 value of 2.03 ppm in the pyrethroid-resistant Puerto Rican strain (PR) of Ae. aegypti. ARS researchers in Oxford, Mississippi, have submitted the invention disclosure based on the efficacy of neoantimycin that is mentioned above. 02 Poncirus trifolia root extract against Aedes aegypti larvae. In the search for effective and environmentally friendly mosquito control agents, ARS researchers in Oxford, Mississippi, have examined natural sources such as microbes and plants and the synthetic analogs of natural products. These plants and microbes have evolved in their ecological niches to produce defensive compounds against other competing organisms in their surroundings such as microbes, plants, and insects to enhance their survival. Thus, some of these plants and microbes have bioactive compounds against insects. In our previous research, we have been very successful in isolation, synthetic modification, and total synthesis of bioactive constituents from natural sources. We have carried out synthetic modifications and total synthesis of marginally active isolated compounds to achieve significantly higher active compounds. We have focused on plants in the Rutaceae family as the members of this family are known to possess bioactive compounds with insecticidal activities. During last year we were successful in isolation of larvicidal coumarins from Poncirus trifoliata (Rutaceae) root extract. Among the five coumarins isolated, xanthoxyletin and demetylsuberosin have shown significantly high larvicide activity against ORL1952 strain of Aedes aegypti larvae.
Impacts
(N/A)
Publications
- Semerdjieva, I., Zheljazkov, V.D., Dincheva, I., Piperkova, N., Maneva, V., Cantrell, C.L., Astatkie, T., Stoyanova, A., Ivanova, T. 2023. Essential oil composition of seven Bulgarian Hypericum species and its potential as biopesticide. Plants. https://doi.org/10.3390/plants12040923.
- Kim, S., Cantrell, C.L., Avula, B., Chen, J., Schrader, K., Santo, S., Ali, A., Khan, I.A. 2022. Streptomyces distallicus, a potential microbial biolarvicide. Journal of Agricultural and Food Chemistry. https://pubs.acs. org/doi/10.1021/acs.jafc.2c03537.
- Zulfiqar, F., Ali, A., Ali, Z., Khan, I.A. 2022. Bioassay-guided isolation of iridoid glucosides from Stenaria nigricans, their biting deterrence against aedes aegypti (Diptera: Culicidae) and repellency potential against imported fire ants (Hymenoptera: Formicidae). Molecules. https:// doi.org/10.3390/molecules27207053.
- Meepagala, K.M., Estep III, A.S. 2023. Larvicidal constituents from Poncirus trifoliata root extracts. Journal of Medical Entomology. https:// doi.org/10.1093/jme/tjad086.
- Mccomic, S.E., Duke, S.O., Burgess Iv, E.R., Swale, D.R. 2023. Defining the toxicological profile of 4-hydroxyphenylpyruvate dioxygenase-directed herbicides to Aedes aegypti and Amblyomma americanum. Pesticide Biochemistry and Physiology. https://doi.org/10.1016/j.pestbp.2023.105532.
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Progress 10/01/21 to 09/30/22
Outputs
PROGRESS REPORT Objectives (from AD-416): 1. Develop biopesticides to improve control of arthropods. 1.1. Discover novel bioinsecticide active ingredients from crude plant extract screening and bioassay-directed fractionation. 1.2. Discovery of fungal compounds with activity against permethrin- resistant and susceptible mosquitoes. 2. Develop biorepellents to improve control of arthropods. 2.1. Discover novel arthropod repelling biopesticides from mosquito repelling folk remedies and plant essential oils. 2.2. Development and optimization of the chromene derivative 131-1 as a biopesticide. 3. Develop natural product synthetic analogs for management and control of arthropods. 3.1. Use synthetic methods to develop and optimize fatty acids as natural topical and spatial arthropod repellents. 3.2. 131-1 Development into a commercial mosquito repelling product. Approach (from AD-416): A bioassay-directed investigative approach will be performed on bioactive extracts in efforts to discover bioinsecticides and repellents for use against arthropod pests. General procedures for isolation of active compounds from plants and microbes will be utilized. Solvent extractions, liquid-liquid partitioning, column chromatography and thin layer chromatography will be employed as needed. Extracts, fractions and pure compounds will be tested for insecticidal and insect repellent activity in assays being carried out routinely at collaborator's laboratories. Identification of active compounds will be done using spectroscopic methods including mass spectroscopy (EI, CI, and high resolution ESI), nuclear magnetic resonance (one- and two-dimensional). Chemical modification will be performed on compound(s) identified as "highly active" to improve activity or bioavailability. For Objective 1, develop biopesticides to improve control of arthropods, milestones have been met and significant progress has been made. An infected mosquito from the genus Aedes, such as Aedes aegypti or Aedes albopictus has become the world's most influential factor for morbidity and death each year. This study describes the isolation and identification of six secondary metabolites from a soil-derived isolate of Streptomyces distallicus using the GNPS (Global Natural Product Social Molecular Networking) platform, ToF-DART HRMS exact mass determinations, and spectroscopic characterization via bioassay-guided isolation approaches. In addition, these metabolites were evaluated for Ae. aegypti mosquito larvicidal activity and antifungal activity against Colletotricum sp. Two isomeric compounds, aureothin and allo-aureothin, showed larvicidal activity against Ae. aegypti with LC50 values of 1.5 and 3.1 ppm for 24 h post-treatment, respectively, and 3.8 and 7.4 ppm for 48 h post-treatment, respectively. The crude extract of S. distallicus demonstrated potent larvicidal activity with LC50 values of 1. 46 and 1.2 ppm for 24 and 48 h post-treatment, respectively. Deoxyaureothin, a furan ring reduced form of aureothin, showed no activity against Ae. aegypti. Thus, the study discovered that S. distallicus and crude extracts thereof are promising sources of potential microbial larvicides. A review article was published as a result of the congressional funds that pass through NPURU to the University of Mississippi. Briefly, natural products (NPs) have a long history as sources of compounds for crop protection. Perhaps a more important role for NPs has been as models and inspiration for the discovery and development of synthetic crop protection compounds. NPs and their synthetic mimics account for 18% of all crop protection compounds, whereas another 38% of all crop protection compounds have a NP that could have served as a model. Because NPs are often complex molecules, have limited availability, or possess structural features that constrain their suitability for use in agricultural settings, a key element in NP-inspired compounds is the simplification of the NP structure to provide a synthetically accessible molecule that possesses the physicochemical properties needed for use in crop protection. We reviewed a series of examples of NP mimics that demonstrate the structural or synthetic simplification of NPs as a guide for the discovery of future NP-inspired agrochemicals focused on fungicides, herbicides, and insecticides. The genus Salvia includes around 900 species distributed throughout the world and is the largest genus in the economically and medicinally important plants of the Lamiaceae family. The secondary compounds of Salvia species have been extensively studied for numerous biological activities. The genus Salvia contains several allelopathic species and extracts from three of these species, Salvia miltiorrhiza, S. hispanica and S. columbariae, that were tested in this study for phytotoxicity. Although there are several reports of phytotoxins from Salvia, there are no reports of phytotoxins against higher plants from Salvia miltiorrhiza. The abietane diterpenes tanshinone I and tanshinone IIA were identified as the compounds responsible for phytotoxicity. Neither compound was toxic to mosquito (Aedes aegypti) at 125 ppm (ca. 0.435 mM). For Objective 2, develop biorepellents to improve control of arthropods., milestones have been met and significant progress has been made. Curcuma longa L. belongs to family Zingiberaceae produced in warm wet regions of the world. Commonly known as turmeric, C. longa is commonly used in Indian folk medicines. Turmeric is especially popular because of its attractive culinary, cosmetic, and medicinal uses. This is an essential spice used in many dishes for good color and aroma. Turmeric contains odoriferous oils and the rhizomes have been reported to possess biological actives including the toxicity and repellency against Ae. aegypti. Essential oils of Curcuma longa rhizome and leaf were evaluated for their repellency and toxicity against imported fire ants. The samples of essential oils used in this study were reported to have ar-turmerone constituents of 36.9%, and 24.9 % respectively. Ar-turmerone was the major compound of the rhizome oil (36.9%) and leaf oil (24.9%) essential oils. Essential oils of C. longa rhizome and leaf showed significantly higher repellency than DEET. Both the essential oils tested in this study showed very high repellency and appears to have a potential for field use. Whiteflies are a common arthropod pest in vegetable crops, and they attack horticultural plants in both fields and the environment. This research focused on a wild watermelon species, Citrullus ecirrhosus, that were offered resistance against the Bemisia tabaci MEAM1. The isolation was performed by liquid extraction, hydrodistillation and/or steam distillation from frozen Citrullus ecirrhosus, fresh Citrullus ecirrhosus plant material, and/or fruit. The biological evaluation was delayed multiple times as samples have been provided to an ARS researcher in Charleston, South Carolina. For Objective 3, develop natural product synthetic analogs for management and control of arthropods, milestones have been met and significant progress has been made in the previous year to meet this objectives milestone. There is a need for the development of natural product-based insect repellents and biting deterrents to manage important disease vectors such as Aedes aegypti, which transmits viral pathogens to humans, including yellow fever and dengue, both of which can cause severe human morbidity and mortality. Prior work by our group has focused on investigations of traditional insect repelling remedies used by indigenous groups as a source of natural product-based chemical solutions for managing disease vectors. These investigations lead to successful research on the Indian folk remedy plant, Jatropha curcas, as well as an investigation of breadfruit, Artocarpus altilis, which is used throughout Oceania. The usage of these plants in controlling biting mosquitoes was attributed to the presence of fatty acids and to a smaller extent to the presence of triglycerides. We report here an extension of our research and understanding of the role triglycerides may play in biting deterrency. Analogs of triglycerides are being investigated to understand their role in biting deterrency as well as repellency of both mosquitoes and fire ants. In search of biopesticides we have explored plants belonging to Rutaceae family. We have been very successful in finding highly active mosquitocidal compounds and their synthetic analogs. During this year we have been working on various plant parts (roots, leaves and fruits) of Poncirus trifoliata a member in the Rutaceae family also known as hardy orange or trifoliate orange. We found that the ethyl acetate and hexane extracts showed larvicidal and topical mosquitocidal activities but the extracts were not discriminant between insecticide resistant and susceptible mosquitoes on adulticide activity. When tested for larvicide activity, the ethyl acetate extract and the hexane extracts showed higher activities towards the insecticide resistant Orlado strain Ae. Aegypti larvae. Bioassay guided fractionation led to the isolation and identification of four pyanocoumarins Xanthoxyleti, dentatin and nordentatin and ponfolin, We have also carried out isovalaryl, acetate and benzoate analogs of nordentatin. Several of these analogs have been reported to possess anticancer activity in the literature. At present these isolated and synthesized derivatives are being tested on dose dependent larvicide assay at USDA, ARS Gainesville laboratory. We have been investigating phytopathogens as sources of biopesticides. We were able to isolate, identify and evaluate phytopathogens belonging to various genera such as Phoma, Diaporthe,Curvularia, Didymella, Pantoea, and Ascochyta. We successfully isolated and synthesized analogs of active principles from some of these pathogens belonging to isochromenes, macrolides and lactones. We isolated a fungus growing in Ipomoea aquatica plants in Oxford, Mississippi. The ethyl acetate extract of PDB (Potato dextrose broth) culture broth has showed larvicidal activity. We are carrying out large scale growth of this fungus and bioassay guided isolation of active metabolites. In our previous work on plants belonging to the Rutaceae family, we were able to isolate and identify a chromene amide. Based on this compound we were able to synthesize potent chromene derivatives that have higher activity and three times longer duration of protection than DEET against mosquito bites. These analogs have now shown activity against Fall army worms, and we are synthesizing various analogs to be tested against fire ants and house flies. Plumbagin is a naphthoquinone derivative that is well known for its biological activity as an insecticide. It is also commonly found in natural sources, including the plant genus Plumbago, the carnivorous plant genera Drosera, and Nepenthes. This study is a synthesis of naphthoquinone derivatives and evaluates their larvicidal activity. In addition, molecular docking studies performed with two Ae. aegypti proteins, an odor-binding protein, and a saliva protein, show that the novel naphthoquinone derivatives possess a reliable binding affinity. Twenty different naphthoquinone derivatives have been synthesized, and the potential biological effects of these compounds are being evaluated. ACCOMPLISHMENTS 01 Streptomyces distallicus as a potent microbial biolarvicide. Mosquitoes are widely prevalent species of vectors capable of spreading diseases such as malaria, dengue fever, filariasis, yellow fever, etc. Mosquitos also affect humans by causing localized angioedema as well as skin and systemic allergic reactions. A relatively new threat, the Zika virus, is transmitted to humans via an infected mosquito from the genus Aedes, such as Ae. aegypti or Ae. albopictus. Consequently, infected mosquitos from the genus Aedes have become one of the world's most influential contributors to human morbidity and death. Microbial insecticides are currently a principal portion of the biopesticide industry. To explore new biopesticides with activity against Ae. aegypti, Streptomyces distallicus was investigated by ARS researchers in Oxford, Mississippi. The present study evaluated S. distallicus for the discovery of larvicidal compounds against Ae. aegypti for the first time. We reported the isolation and characterization of six compounds from S. distallicus, as well as their larvicidal activity against Ae. aegypti. Thus, the study discovered that S. distallicus and crude extracts thereof are promising sources of potential microbial larvicides.
Impacts
(N/A)
Publications
- Mccomic, S.E., Meepagala, K.M., Swale, D. 2021. Characterization of toxicological and neurophysiological effects of natural product based chromenes to Fall Armyworm, Spodoptera frugiperda. Journal of Economic Entomology. https://doi.org/10.1093/jee/toab172.
- Da Cruz-Silva, C.T., Cantrell, C.L., Nobrega, L.P., Ali, A., Duke, S. 2021. Bioassay-guided isolation of phytotoxins from three salvia species. Allelopathy Journal. 54(1):13-24. https://doi.org/10.26651/allelo.j/2021- 54-1-1344.
- Sparks, T.C., Duke, S.O. 2021. Structure simplification of natural products as a lead generation approach in crop protection compound discovery. Journal of Agricultural and Food Chemistry. 69(30):8324-8346. https://doi.org/10.1021/acs.jafc.1c02616.
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Progress 10/01/20 to 09/30/21
Outputs
PROGRESS REPORT Objectives (from AD-416): 1. Develop biopesticides to improve control of arthropods. 1.1. Discover novel bioinsecticide active ingredients from crude plant extract screening and bioassay-directed fractionation. 1.2. Discovery of fungal compounds with activity against permethrin- resistant and susceptible mosquitoes. 2. Develop biorepellents to improve control of arthropods. 2.1. Discover novel arthropod repelling biopesticides from mosquito repelling folk remedies and plant essential oils. 2.2. Development and optimization of the chromene derivative 131-1 as a biopesticide. 3. Develop natural product synthetic analogs for management and control of arthropods. 3.1. Use synthetic methods to develop and optimize fatty acids as natural topical and spatial arthropod repellents. 3.2. 131-1 Development into a commercial mosquito repelling product. Approach (from AD-416): A bioassay-directed investigative approach will be performed on bioactive extracts in efforts to discover bioinsecticides and repellents for use against arthropod pests. General procedures for isolation of active compounds from plants and microbes will be utilized. Solvent extractions, liquid-liquid partitioning, column chromatography and thin layer chromatography will be employed as needed. Extracts, fractions and pure compounds will be tested for insecticidal and insect repellent activity in assays being carried out routinely at collaborator's laboratories. Identification of active compounds will be done using spectroscopic methods including mass spectroscopy (EI, CI, and high resolution ESI), nuclear magnetic resonance (one- and two-dimensional). Chemical modification will be performed on compound(s) identified as "highly active" to improve activity or bioavailability. Actinomycetes, especially Streptomyces species, are widely recognized as industrially important microorganisms studied as a source of new biochemical biopesticides and new microbial pesticides. One focus of this project is the discovery of new metabolic products from Streptomyces distallicus for controlling insect pests while avoiding negative impacts on the environment and public safety hazards in agriculture. After developing and optimizing the fermentation methodology through HPLC analysis and in-vitro assay, we isolated five compounds. The Aedes aegypti larvicidal activity of these compounds was evaluated, and only two showed larvicidal activities against A. aegypti with potent LC50 values of 1.5 ppm and 3.1 ppm for 24 h post-treatment, respectively, and 3.8 ppm and 7.4 ppm for 48 h post-treatment, respectively. An invention disclosure pertaining to the above results was filed but a patent will not be pursued at the present time. Extensions to the above study of Streptomyces distallicus: GNPS (global natural products social molecular networking) platform showed the initial fermentation method did not produce many metabolic products from Streptomyces distallicus at detectable concentrations. To solve this problem, we applied adsorption techniques to the fermentation of Streptomyces distallicus. As a result, we were able to enhance secondary metabolic production. Isolation and identification are in progress for the new products. We conducted a study to assess the potential of another Strepromyces species, S. orinoci, as a source of new microbial bioinsecticides. For developing the fermentation methodology, we tested and optimized several different fermentation conditions for S. orinoci through HPLC analysis and in-vitro assays. Following the successful development of the fermentation methodology, three crude extractions showed potent larvicidal activities. Five compounds were isolated, and one of them was identified which showed potent larvicidal activity against Aedes aegypti. Structure elucidation of the remaining compounds are in progress together with biological evaluations. This is the first study to explore novel products from Streptomyces orinoci with the aim of developing microbial bioinsecticides to be used in the management of Aedes mosquito larvae. Juniper (Juniperus L.) species are some of the most widely distributed plants on the planet. Most junipers contain essential oil (EO) with specific aroma, while some also contain podophyllotoxin. The EO of some juniper species extracted from leaves, wood, or berries (galbuli) is used as a major aromatic agent in numerous consumer products. The EO profile and podophyllotoxin concentration depend on juniper species, sex (most junipers are dioecious), but may also depend on subspecies/chemotype, the environment, the plant part from which it is extracted (leaves, galbuli, or wood), and the extraction procedure. As part of a larger study on Juniper essential oils, repellent and insecticidal action of the semi- commercial extraction EO of J. sabina and J. excelsa on aphids (Sitobion avenae, Rhopalosiphum padi) was determined. The aphids Rh. padi and S. avenae are economically important pests on cereals. The tested EOs demonstrated significant insecticidal effect 24 h after application on aphids. We investigated two plants Thamnomsa texana and Helietta parvifoli for active constituents against pests. Hopeyhopin, and some analogs of hopeyhopin, chalepin and analogs were isolated and identified by spectroscopic techniques. These are being evaluated for mosquito and Fall Army worms at USDA-ARS in Gainesville, Florida, and at Dept of Entomology at Louisiana State University (LSU). The fall armyworm (FAW), Spodoptera frugiperda (Lepidoptera: Noctuidae), is a polyphagous lepidopteran herbivore that has expanded their range from the Americas to nearly 100 countries and continues to threaten the global food supply. Thus, FAW is recognized as one of the most damaging agricultural pests of row crops with economic losses estimated to be at $6 billion dollars per year. We investigated three fungal culture broths for insecticidal constituents. The plant pathogenic fungi Diaporthe ceratozamiae, Chaetomium globosum, and Curvularia geniculate were cultured in PDB in the presence of Diaion HP-20. We isolated four azaphilones including a new azaphilone and a phenolic compound. The structures were elucidated by spectroscopic methods. These compounds are being evaluated against Fall Army worms and mosquito species in Gainsville, Florida, and at LSU, Department of Entomology. A7. We have also isolated an entomopathogenic fungus coexisting with a plant pathogen. This fungus was identified as a Beauveria species. This fungus is being tested on mosquitoes at the USDA in Gainesville to see the effectiveness. We are also trying to identify the metabolites, or the enzymes produced by this fungus when the insects are infected. For Objective 2, develop biorepellents to improve control of arthropods, milestones have been met and significant progress has been made. Whiteflies are a common arthropod pest in vegetable crops, and they attack horticultural plants in both fields and the environment. This research focused on a wild watermelon species, Citrullus ecirrhosus, that were offered resistance against the Bemisia tabaci MEAM1. The isolation was performed by liquid extraction, hydrodistillation and/or steam distillation from frozen Citrullus ecirrhosus, fresh Citrullus ecirrhosus plant material, and/or fruit. The biological evaluation is in progress for isolated products. In an effort to develop the fatty acid repellent technology, we worked directly with Thermacell from 2016 to 2020. Discussions led to numerous experiments directly utilizing fatty acids provided to them. Evaluations focused on determination of the efficacy of using the recommended fatty acids in spatial repellent delivery devices such as the MR300 Repeller. For Objective 3, develop natural product synthetic analogs for management and control of arthropods, milestones have been met and significant progress has been made. Plumbagin, a naphthoquinone derivative, is well known for its biological activities as an insecticide. It is also commonly found in natural sources, including the plant genus Plumbago, and carnivorous plant genera Drosera and Nepenthes. This study focused on the synthesis of naphthoquinone derivatives and evaluation of their mosquito larvicidal activities. Thirteen naphthoquinone derivatives were synthesized, and the biological evaluation is in progress. C2. Some chromene analogs including a novel CF3 moiety containing chromenes (13 compounds) were synthesized and tested for electro- physiological activities against Fall Army worms at LSU, Department of Entomology. We have shown that some of these chromene analogs are effective against these pests and the active chromene moiety can be used to develop more effective compounds. Record of Any Impact of Maximized Teleworking Requirement: The maximized telework situation has directly impacted the productivity of this project but fallen short of preventing us from meeting milestones. Publications are certainly reduced from prior years, meeting attendance has been reduced, international collaborations are reduced, and new MTA⿿s and other agreements are reduced.
Impacts
(N/A)
Publications
- Barreto, D.L., Nogueira De Azevedo, R., Carvalho, C., Ferreira, M.C., Cantrell, C.L., Duke, S.O., Rosa, L.H. 2021. Bioactive compounds produced by Neotropical endophytic fungi applied to agriculture. In: Rosa L.H. editors. Neotropical Endophytic Fungi. Springer, Cham: Springer International Publishing. p. 257-295.
- Perera, W.H., Meepagala, K.M., Duke, S.O. 2020. Sesquiterpene-a-amino acid quaternary ammonium hybrids from Stereum complicatum (Steraceae). Biochemical Systematics and Ecology. 93,104176. https://doi.org/10.1016/j. bse.2020.104176.
- Zheljazkov, V.D., Cantrell, C.L., Semerdjieva, I., Radoukova, T., Stoyanova, A., Maneva, V., Kacaniova, M., Astatkie, T., Borisova, D., Dincheva, I., Salamon, I. 2021. Essential oil and bioactivity of two juniper species from Bulgaria and Slovakia. Molecules. 2021, 26, 3659.
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Progress 10/01/19 to 09/30/20
Outputs
Progress Report Objectives (from AD-416): 1. Develop biopesticides to improve control of arthropods. 1.1. Discover novel bioinsecticide active ingredients from crude plant extract screening and bioassay-directed fractionation. 1.2. Discovery of fungal compounds with activity against permethrin- resistant and susceptible mosquitoes. 2. Develop biorepellents to improve control of arthropods. 2.1. Discover novel arthropod repelling biopesticides from mosquito repelling folk remedies and plant essential oils. 2.2. Development and optimization of the chromene derivative 131-1 as a biopesticide. 3. Develop natural product synthetic analogs for management and control of arthropods. 3.1. Use synthetic methods to develop and optimize fatty acids as natural topical and spatial arthropod repellents. 3.2. 131-1 Development into a commercial mosquito repelling product. Approach (from AD-416): A bioassay-directed investigative approach will be performed on bioactive extracts in efforts to discover bioinsecticides and repellents for use against arthropod pests. General procedures for isolation of active compounds from plants and microbes will be utilized. Solvent extractions, liquid-liquid partitioning, column chromatography and thin layer chromatography will be employed as needed. Extracts, fractions and pure compounds will be tested for insecticidal and insect repellent activity in assays being carried out routinely at collaborator's laboratories. Identification of active compounds will be done using spectroscopic methods including mass spectroscopy (EI, CI, and high resolution ESI), nuclear magnetic resonance (one- and two-dimensional). Chemical modification will be performed on compound(s) identified as "highly active" to improve activity or bioavailability. Actinomycetes, especially Streptomyces species are widely recognized as industrially important microorganisms that are studied of bio-pesticides, including fungi, actinomycetes, bacteria, viruses, and nematodes, have been reported as potent materials for biological control. The present study has been undertaken to isolate, characterize, and evaluate the larvicidal (Aedes aegypti) compounds produced by Streptomyces distallicus. For developing the fermentation methodology, we tested and optimized several different fermentation conditions for S. distallicus through High Performance Liquid Chromatography (HPLC) analysis and in-vitro assay. While successfully developing the fermentation methodology, novel bioactive products were discovered and isolated. The most active compound was determined to be an aureothin analog. This is the first report on exploring novel products from Streptomyces distallicus for developing microbial bioinsecticides to be used in the management of Aedes mosquito larvae. Building from the discovery above, we conducted a study to assess the potential of Streptomyces orinoci as a source of new microbial bioinsecticides. For developing the fermentation methodology, we tested and optimized several different fermentation conditions for S. orinoci through HPLC analysis and in-vitro assays. While successfully developing the fermentation methodology, three crude extractions showed potent larvicidal activities. Further studies toward the identification of specific bioactive compounds and their detailed biochemical pathways and physiological studies are currently underway. This is the first study to explore novel products from Streptomyces orinoci for developing microbial bioinsecticides to be used in the management of Aedes mosquito larvae. Stureja pilosa Velen. is a Balkan endemic plant that is not well characterized and is found on rocky outcrops of limestone base in Stara Planina (the Balkan Mountains) and the Rhodope Mountains. In collaboration with colleagues at the University of Mississippi, this study was initially implemented to assess the variability of essential oil (EO) content and composition of S. pilosa collected at 33 locations across the Balkan and Rhodope Mountains in Bulgaria. Biological evaluation of the essential oil revealed five different chemotypes which exhibited larvicidal and mosquito repellent activity against Ae. aegypti. Additional plant extracts have been evaluated for repellent activity against Ae. aegypti and investigations are underway to isolate the bioactive constituents. Esters of C-11:0 and C-12:0 fatty acids were synthesized last fiscal year in an effort to both improve the mosquito repellency and improve the duration of activity of the natural C-11 and C-12 fatty acids. The optimum chain length for the esters has been determined and a highly active analog has been produced and shown to be more active than DEET in the Klun & Debboun (K&D) module bioassay system. Additional evaluations were performed in the Ali & Khan (A&K) bioassays this year. In-vivo evaluations of the most active compounds against Ae. aegypti were performed following the large scale synthesis of the analogs. In the A&K assay, undecanoic acid appears to be the most active compound in this group tested while butyl undecanoate was the most active ester analog synthesized. Both of these highly effective compounds are more effective than DEET and warrant further investigations as mosquito repellents. In collaboration with a CRADA partner, the mosquito repellent 131-1 has been produced in large quantities. This is the first synthetic batch produced outside of the unit in an attempt to transfer the technology to the private sector. The product produced is being evaluated for Ae. aegypti repellency by collaborators at the Center for Medical, Agricultural, and Veterinary Entomology (CMAVE) in Gainesville, Florida.
Impacts
(N/A)
Publications
- Semerdjieva, I.B., Zheljazkov, V.D., Cantrell, C.L., Astatkie, T., Ali, A. 2020. Essential oil yield and composition of the Balkan endemic Satureja pilosa Velen. (Lamiaceae). Molecules. 25(4):827.
- Liu, X., Yu, W., Min, L., Wedge, D., Tan, C., Weng, J., Wu, H., Cantrell, C.L., Bajsa Hirschel, J.N., Hua, X., Duke, S.O. 2020. Synthesis and pesticidal activities of new Quinoxalines. Journal of Agricultural and Food Chemistry. 68:7324-7332.
- Cantrell, C.L., Zaki, M., Reichley, A.C., Kim, S.J. Sink, M.C., Ali, A. 2020. Biting deterrency of Undecanoic acid and Dodecanoic acid ester analogs against Aedes aegypti. Pest Management Science.
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