Location: Natural Products Utilization Research2021 Annual Report
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.
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.
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.