Isolation Of Systemic Antifeedant Ingredient From Brucea Javanica(L.) Merr.and Its Mechanism Of Action On Plutella Xylostella L.

Abstrast: The discovery of the good anticancer activity of Brucea javanica(L.)Mett and the successful development of its oil emulsion promoted the large-scale cultivation of B.javanica,and there was a serious oversupply situation,which caused a great impact on the local Chinese herbal medicine planting industry.Developing agricultural uses of B.javanica can balance the supply and restore planters’ confidence.At the same time,a large number of raw materials of B.javanica also provide guarantee for the development of pesticides derived from B.javanica.We intended to make full use of the Chinese medicinal herbal residues,and found that the compost of B.javanica fruit residues can not only promote the growth of flowering Chinese cabbage(Brassica campestris L.ssp.chinensis var.utilis Tsen et Lee),but also affected Plutella xylostella,suggesting that some active ingredients were translocated to leaves.Systemic insecticides can be absorbed by crop leaves or roots and transported to other parts of the crop to achieve a systematic distribution,giving the crop full protection.Systemic property improve insecticidal efficiencies and field duration.Systemic insecticides were suitable for granule,seed treatment,soil watering,and application drip irrigation.It is widely welcomed by planters.Nowadays,systemic insecticides presently used in the field are primarily synthetic,which pose an unacceptable risk to non-target organisms.As an ideal substitute for synthetic pesticides,the development of botanical systemic pesticides is particularly urgent.As a plant-derived used in China,the pesticide activity spectrum of B.javanica has not been systematically investigated.The aims of the present study were to systematically determine the bioactivities of B.javanica oil(BJO)and ethanol extracts of B.javanica residue(BJEA)against the pests and diseases that are harmful to South China crops(especially for vegetables),and designed a bioassay of plant extracts via root application against diamondback moth(DBM,Plutella xylostella L.),to isolate and identify the systemic active ingredients.The systemic and insecticidal activity of the ingredients were evaluated with multi-dimensional,and mechanism of action were then investigated.The main results of this study were as follows:1.The BJO and BJEA showed no inhibitory activity against Fusarium oxysporum f.sp.cubense,Magnaporthe oryzae,Thanatephorus cucumeris,Colletotrichum gloeosporioides,Ustilagrnoidea virens(Cke.)Tak.,Sclerotinia sclerotiorum(Lib.)de Bary,Ralstonia solanacearum.The insecticidal activity of BJO and BJEA against DBM,Spodoptera exigua Hübner,Tetranychus urticae,Diaphorina citri Kuwayama,Bemisia tabaci Gennadius,Solenopsis invicta Buren,Phyllotreta striolata Fabricius,Ditylenchus angustus(Bütler)Filipjev,and Bursaphelenchus xylophilus.The results showed that BJO had no biological activity on the tested pests.The BJEA exhibit satisfying insecticidal activity at a concentration of 5000 μg/m L against DBM,beet armyworm(Spodoptera exigua)and Ditylenchus angustus(Bütler)Filipjevrice,the corrected mortality rate was over 79% after 24 h treatment.The effect on Phyllotreta striolata Fabricius,Solenopsis invicta Buren and Bemisia tabaci Gennadius were relatively slow,and the mortality rate reached more than 74% with 48 h treatment.Other pests did not show significant activity.After 48 h coincubated with BJEA,the antifeedant activity index of the leaves of flowering Chinses cabbage against DBM was 81.42%.2.Bruceine D,a quassinoid,was identified in Brucea javanica(L.)Merr.through systemic activity tracking and displayed outstanding systemic properties and excellent antifeedant activity against DBM,beet armyworm and cotton leafworm(Spodoptera litura Fabricius),the 50 % antifeedant action were 0.11,0.091,0.13 μg/cm2 respectively.Its antifeedant effect on third instar larvae of of DBM was approximately 6.2-fold stronger than that of azadirachtin.3.When bruceine D was applied to roots at a concentration of 100 μg/m L for 24 h and 48 h,its concentration in flowering Chinese cabbage leaves were 38.69 μg/g(fresh weight,FW)and 108.45 μg/g(FW),respectively,the content of bruceine D in leaves was 1.8 and 2.7 fold than that for azadirachtin treatment.These concentrations could achieve 93.80 % and 96.83 % antifeedant effects,which were significantly greater than those of azadirachtin.The systemic antifeedant activity againt beet armyworm and cotton leafworm in cowpea [Vigna unguiculata(L.)Walp.] and Joseph’s coat(Amaranthus tricolor L.)were investigated.When incubated with 25 μg/m L bruceine D 48 h,the antifeedant index against beet armyworm and cotton leafworm in cowpea were 79.43 % and 66.11 %,and that for Joseph’s coat were 60.60 % and 55.47 %.4.The Ricinus communis seedlings were employed to evaluate the xylem and phloem mobility of burceine D and azadirachtin.The result showed that xylem mobility of bruceine D was better that azadirachtin.After 2 h incubation,the concentration of bruceine D and azadirachtin in xylem were 26.45,5.16 μg/m L,the concentration factor(CF)value of bruceine D was 0.25 which was 5-fold for azadirachtin.For phloem mobility test,the azadirachtin process a better property than bruceine D.the amount of bruceine D and azadirachtin were 29.13 and 14.70 μg/m L after 2 h incubation.The CF value for azadirachtin was 0.29,which was about 2.5 fold than bruceine D.5.Bruceine D exhibited significant inhibitory effect on the growth and development of DBM larvae.Feeding with 10 μg/g bruceine D for 12 h,the inhibition rate was 42.55 %;with 24 h,body weight did not increase,and the growth inhibition rate reached 253.39 %;20 μg/g treatment group the growth inhibition rates were 117.45 %,72.78 %,311.42 % and 83 %,at 6,12,24,48 h.Long-term feeding of bruceine D at lower concentrations showed that the pupation rates of the 2.5,5,and 10 μg/g treatment groups were 100 %,73.67 %,and 57.67 %,respectively.The collected pupas were observed continuously and record the eclosion rate.Results showed that no adults were observed for 10 μg/g treatment,and the eclosion rates of treatment group of 2.5,5 μg/g were 87 % and 42.97 %,respectively.Bruceine D significantly inhibited the pupation and eclosion of DBM larvae.Bruceine D management DBM effectively.When treated at a lower concentration of 5 μg/g it can still reduce 31.67 % of adults.6.Bruceine D has certain neurotoxicity against DBM.It was found by voltage clamp that Bruceine D has a significant inhibitory effect on the current induced by γ-aminobutyric acid(GABA)in Px RDL1,and inhibitory concentration(IC50)was measured by 111.30 μM.7.The RNA-Seq was used to determine the effect of bruceine D against DBM larvae.It was found that the genes related to the midgut membrane structure were significantly induced after bruceine D treatment.Seven peritrophic membrane factor-related genes were significantly down-regulated,and chitinase 8 abundantly expressed in the midgut,was significantly up-regulated.Two types of proteins bound to chitin: 4 CBP and 9 Mucin genes were significantly down-regulated;the genes involved in collagen and laminin were also significantly down-regulated,the results suggested that bruceine D affect the extracellular matrix,causing damage to the membrane structure in midgut.Pathological section of midgut of DBM larvae was observed after feeding with bruceine D for 48 h.The result showed that the peritrophic membrane of the midgut disappeared,the arrangement of brush border was sparse,the cells on the intestinal wall were detached from the muscle layer and squeezed into the lumen,the morphology of the columnar cells expanded and only a few food was found in the intestinal lumen.In summary,after 10 μg/g of bruceine D was fed to DBM larvae for 48 h,the membrane structure of larval midgut showed obvious lesions,which was consistent with the results predicted by the transcriptome.Bruceine D destroys the structure of the peritrophic membrane hindering the absorption and metabolism of nutrients which inhibits the growth and development of DBM larvae.The midgut may be the target organs of bruceine D.In this study,the systemic insecticide activity was used as an indicator,and bruceine D was isolated from the B.javanica fruit residues with excellent antifeedant activity and systemic property.The novel activity tracking-separation method inspired a novel method for new systemic pesticides discovering.The discovering of bruceine D offered support for the development and utilization of B.javanica fruit residues.Bruceine D poses excellent systemic property and muti-bioactivities,such as stomach poison,antifeedant activity and growth inhibition.The outstanding biological activities and abundant of plant raw materials provide guarantee for the commercial development of the bruceine D.The transcriptome analysis and hematoxylin-eosin staining were employed to reveal the mode of action of bruceine D against DBM,the result suggested that midgut was the action target.These results could promote to uncover the mechanism of action of bruceine D.