a b s t r a c tThe purpose of this study was to develop and characterize biodegradable nanospheres containing essential oils from Zanthoxylum rhoifolium leaves and evaluate its insecticidal effect in Bemisia tabaci populations. The essential oil from Z. rhoifolium leaves exhibited an average yield of 0.03% and presented ␤-elemene (31.26%), D-germacrene (18.16%), ␤-caryophyllene (12.09%), ␦-elemene (7.63%), ␤-cedrene (6.69%), bicyclogermacrene (4.57%) and E-caryophyllene (3.63%) as main components. The PCL nanospheres containing this essential oil exhibited encapsulation efficiency higher than 96%, pH close to 5, particle diameter smaller than 500 nm and zeta potential values of approximately −20 mV. The in vitro release profile was characteristic of biphasic release, i.e., a fast initial release, followed by slow release for 12 h or more. The biological assay results were also significant and as such both the in natura and nanoencapsulated essential oils resulted in reductions as high as 95% in the number of eggs and nymphs compared with our control. Collectively these results suggest that both in natura and nanoencapsulated essential oils from Z. rhoifolium leaves can potentially be used in B. tabaci control in association with integrated pest management practices.

Short title: Seed-specific saponin biosynthesis in Medicago One-sentence summary: The discovery of a seed-specific regulator of hemolytic saponin biosynthesis in Medicago truncatula led to the identification of the missing P450 of the hemolytic saponin biosynthesis branch.

New control methods are necessary for stored grain pest management programs due to both the widespread problems of insecticide-resistance populations and the increasing concerns of consumers regarding pesticide residues in food products. Thus, this study evaluated the bioactivity of extracts and fractions obtained from different structures (leaves, branches, and seeds) of Annona mucosa (Annonaceae) against Sitophilus zeamais (Coleoptera: Curculionidae), which is a primary insect pest of stored cereals in tropical conditions. In the screening assay, the most promising treatments were extracts prepared from the seeds of Annona mucosa in hexane and dichloromethane (LC 90 values of 259.31 and 425.15 mg kg À1 , respectively) and, to a lesser extent, an extract prepared from the leaves in hexane (LC 90 of 1047.15 mg kg À1). Based on these results and the chromatographic profile of the bioactive crude extracts, the extract prepared from the seeds in hexane was fractionated by liquideliquid partitioning. The dichloromethane and hydroalcoholic fractions exhibited insecticidal activity against S. zeamais, and no significant difference was observed between these two fractions. The chemical analyses (1 H NMR, HPLC, and TLC) showed the presence of alkaloids and acetogenins in the bioactive fractions, which are likely related to the observed bioactivity. Thus, A. mucosa, particularly its seeds, is a promising source of compounds that can be used as a prototype model and/or a biorational insecticide for the control of S. zeamais in stored cereals.

The native Brazilian plant Maytenus ilicifolia accumulates a set of quinone methide triterpenoids with important pharmacological properties, of which maytenin, pristimerin and celastrol accumulate exclusively in the root bark of this medicinal plant. The first committed step in the quinone methide triterpenoid biosynthesis is the cyclization of 2,3-oxidosqualene to friedelin, catalyzed by the oxidosqualene cyclase friedelin synthase (FRS). In this study, we produced heterologous friedelin by the expression of M. ilicifolia FRS in Nicotiana benthamiana leaves and in a Saccharomyces cerevisiae strain engineered using CRISPR/Cas9. Furthermore, friedelin-producing N. benthamiana leaves and S. cerevisiae cells were used for the characterization of CYP712K4, a cytochrome P450 from M. ilicifolia that catalyzes the oxidation of friedelin at the C-29 position, leading to maytenoic acid, an intermediate of the quinone methide triterpenoid biosynthesis pathway. Maytenoic acid produced in N. benthamiana leaves was purified and its structure was confirmed using high-resolution mass spectrometry and nuclear magnetic resonance analysis. The three-step oxidation of friedelin to maytenoic acid by CYP712K4 can be considered as the second step of the quinone methide triterpenoid biosynthesis pathway, and may form the basis for further discovery of the pathway and heterologous production of friedelanes and ultimately quinone methide triterpenoids.

Summary Plant roots are specialized belowground organs that spatiotemporally shape their development in function of varying soil conditions. This root plasticity relies on intricate molecular networks driven by phytohormones, such as auxin and jasmonate (JA). Loss‐of‐function of the NOVEL INTERACTOR OF JAZ (NINJA), a core component of the JA signaling pathway, leads to enhanced triterpene biosynthesis, in particular of the thalianol gene cluster, in Arabidopsis thaliana roots. We have investigated the biological role of thalianol and its derivatives by focusing on Thalianol Synthase (THAS) and Thalianol Acyltransferase 2 (THAA2), two thalianol cluster genes that are upregulated in the roots of ninja mutant plants. THAS and THAA2 activity was investigated in yeast, and metabolite and phenotype profiling of thas and thaa2 loss‐of‐function plants was carried out. THAA2 was shown to be responsible for the acetylation of thalianol and its derivatives, both in yeast and in planta. In addition, THAS and THAA2 activity was shown to modulate root development. Our results indicate that the thalianol pathway is not only controlled by phytohormonal cues, but also may modulate phytohormonal action itself, thereby affecting root development and interaction with the environment.

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The toxicity of organic plant extracts to Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) was assessed for three tropical plant species: branches, leaves, and seeds of Annona montana (Annonaceae), branches of Aristolochia paulistana (Aristolochiaceae), and leaves and branches of Casearia sylvestris (Salicaceae). The screening assay resulted that the extracts of A. montana seeds obtained with hexane (LC50 = 534.75 mg kg(-1) and LT50 = 6.10 days) and with dichloromethane (LC50 = 424.67 mg kg(-1) and LT50 = 5.03 days) were the most promising treatments, followed by the extract prepared from A. montana leaves with hexane (LC50 = 837.70 mg kg(-1) and LT50 = 4.90 days). Moreover, extracts (at 1,500 mg kg(-1)) prepared from C. sylvestris branches with dichloromethane and A. paulistana with hexane caused significant mortality (37% and 41.5%, respectively) beyond sublethal effects on S. zeamais. Therefore, based on the biological assays, extraction yield, and evaluation of the chromatographic profile of the crude extracts by TLC, the hexane extract of A. montana seeds was selected and fractioned using liquid-liquid partitioning. The hydroalcoholic fraction caused mortality of 55.5%, significantly superior to dichloromethane fraction, which caused 35.5% of mortality. Chemical analyses ((1)H NMR, HPLC, and TLC) were performed, and the results showed the presence of alkaloids and acetogenins in both active fractions, which have been associated with bioactivity. Therefore, extracts prepared from A. montana seeds (especially) is an interesting source of new compounds with promising grain-protectant properties.

Among tropical plant families, members of Annonaceae have great potential as a source of biopesticides. To develop an alternative tool for control of Helicoverpa armigera, efficacy of ethanolic extracts from seeds of five species belonging to the genus Annona (A. montana Macfad., A. mucosa Jacq., A. muricata L., A. reticulata L. and A. sylvatica A. St.-Hil.) and an acetogenin-based commercial bioinsecticide (Anosom Ò 1 EC, 10,000 ppm of annonin as the main active ingredient) were evaluated in a dietary exposure bioassays. In an initial screening, an ethanolic extract from A. mucosa seeds (LC 50 = 1479 ppm) and Anosom Ò 1 EC (LC 50 = 1151 ppm) were the most promising treatments. In addition to acute toxicity, pronounced inhibition of H. armigera larval growth was observed in both treatments. Using chromatographic techniques, bioguided fractionations were conducted and the acetogenin bis-tetrahydrofuran rolliniastatin-1 was isolated as the primary compound from the most active fractions of A. mucosa. At a concentration of 41.55 ppm, rolliniastatin-1 caused total mortality of H. armigera larvae after the fourth day of exposure. In greenhouse trials, extract of A. mucosa (as an emulsifiable concentrate formulation) and the botanical insecticide based on extract of Annona squamosa L. (Anosom Ò 1 EC), both at LC 90 values previously estimated, were compared with a diamide-based commercial insecticide (flubendiamide 480 SC) for mortality after 168 h of exposure to larvae on tomato plants; all treatments caused high larval mortality ([90%). Thus, the results of this study indicate that the derivatives of Annonaceae are a useful alternative for the integrated management of H. armigera. Keywords Annonaceae Á Acetogenin Á Botanical insecticides Á Old world bollworm Á Rolliniastatin-1 Á Chromatographic techniques Key message • Annonaceae species were screened to detect sources of insecticidal compounds. • An ethanolic extract from A. mucosa seeds caused strong mortality to Helicoverpa armigera. • The major compound present is the acetogenin bistetrahydrofuran rolliniastatin-1. • Ethanolic extract from A. mucosa seeds also caused high larval mortality in greenhouse trial. Communicated by M.B. Isman.