Evaluation of neem, Azadirachta indica, with and without water management, for the control of culicine mosquito larvae in rice-fields

Applications of neem, Azadirachta indica (Meliaceae), to rice-fields were evaluated with the dual objective of controlling the culicine mosquito vectors of Japanese encephalitis virus (JEV) and enhancing the grain yield. Since crude neem products deteriorate under improper storage conditions, a laboratory bioassay was developed to screen neem cake powder against mosquito larvae, Culex quinquefasciatus. Only samples of neem giving over 90% bioassay mortality were used in field trials. When good quality neem cake powder was applied at the dose of 500 kg/ha, either alone or coated over urea, there was a striking reduction in the abundance of late instar culicine larvae and pupae. Only fourteen pupae were obtained over a period of 13 weeks in neem cake powder treated plots, and four in those treated with neem coated urea, compared with 101 in control plots. Both treatments were significantly less than the control, but on par with one another. In another field trial, neem cake coated urea was tested at 500 and 250 kg neem/ha in combination with water management practices. No reduction in efficacy was noted at the lower dose. Larval abundance in plots under water management alone did not differ significantly from the controls, but was significantly reduced when water management was combined with neem products. Two stable formulations, 'Neemrich-I' (lipid rich) and 'Neemrich-II' (azadirachtin rich), also gave good suppression of immature culicines. All the treatments with neem also gave higher grain yield than the control.(ABSTRACT TRUNCATED AT 250 WORDS)     

SYSTEMIC INSECTICIDAL ACTION OF AZADIRACHTIN, NEEM SEED AND CHINABERRY SEED EXTRACTS APPLIED AS SOIL DRENCHES TO POTTED PLANTS

Abstract  The systemic insecticidal activities of: 1) azadirachtin, a potent insect antifeedant and growth regulator isolated from seeds of the neem tree, Azadirachta indica , 2) a neem seed extract containing 0. 30 quantitative fraction of azadirachtin, and 3) a methanolic extract of seeds from the chinaberry tree, Melia azedarach , a close relative of neem were determined. Soil drenches of azadirachtin or the neem seed extract at 1 and 2. 5 mg to potted potato ( Solanum tuberosum ) and tomato ( Lycopersicon esculentum ), remarkably retarded the growth of Manduca sexta larvae and strongly disrupted the pupation of Leptinotarsa decemlineata larvae, respectively. However, five to ten times as much azadirachtin or neem seed extract was needed to reduce the amount of tomato and potato foliage consumed. On sorghum ( Surghum biocolor ), much higher quantity (≥25 mg) of azadirachtin or the neem seed extract was necessary to slow the population growth of Schizaphis graminum , and prolong the survival of treated plants. But methanolic extracts of chinaberry seeds ( M. azedarach ) from two sources (Guangdong, PRC and Hurricane, Utah, USA) at 150 mg per pot were ineffective in reducing aphid population growth and preventing plant damage.     

南蛇藤假种皮形成过程中萜类合成基因的表达分析

为探究南蛇藤（Celastrus orbiculatus Thunb.）假种皮发育过程中萜类物质合成相关基因的表达差异,本文选取3个不同发育时期（盛花期、落花期和幼果期）的南蛇藤雌花为实验材料,构建3个cDNA文库,通过Illumina HiSeq^TM 2000平台进行转录组测序,共获得87600个unigene序列,平均长度为453bp,N50/N90比值为4.64。通过3个不同发育阶段基因差异表达的比较及功能富集分析,鉴定出16个萜类合成相关基因,其中8个基因的编码蛋白参与调控7个萜类合成代谢位点。还确定了与萜类合成相关的7个转录因子家族的成员数量以及相对表达量最高的基因。     

Identification and expression analysis of key enzymes of the terpenoids biosynthesis pathway of a liverwort Plagiochasma appendiculatum by EST analysis

Liverworts are known to be a rich source of terpenoids and phenolic compounds whose biochemical and molecular biosynthesis properties are not well understood. To evaluate the biosynthetic processes of such compounds found in a liverwort Plagiochasma appendiculatum, a total of 5,024 clones were sequenced from a normalized cDNA library from leaves of P. appendiculatum. This produced 4,384 high-quality ESTs with a mean length of 550 bp. Cluster analysis indicated the presence of 704 contigs and 2,720 singletons, generating 3,424 unique sequences. A total of 1,180 sequences were functionally classified using gene ontologies (GO). Based on the homology to sequences present in GenBank, our EST collection was found to contain orthologs for known prenyl transferases and for genes involved in the 2-C-methyl-d-erythritol 4-phosphate (MEP) and the mevalonic acid (MVA) pathways, both of which are involved in terpenoid biosynthesis. Informatic analysis revealed that all of the genes involved in terpenoid biosynthesis showed extensive homology with tracheophyte genes. Phylogenetic analysis indicated that the topology of the phylogenetic trees constructed using genes MEP, DXR and HMGS was in good agreement with the traditional taxonomic classification, but the one constructed using gene FPS was not. Real-time PCR showed that the expression level of these genes was enhanced after the MeJA treatment, as in vascular plants.     

Effects of a neem extract on blood feeding, oviposition and oocyte ultrastructure in Anopheles stephensi Liston (Diptera: Culicidae)

Secondary metabolites of the neem tree (Azadirachta indica A. Juss., Meliaceae) exhibit a wide range of biological activities in insects. However, few studies have addressed the effects of neem extracts or compounds in arthropods of medical importance. In this study, a laboratory strain of Anopheles stephensi was used to assess the effects of a commercial formulation (Neem Azal) (NA)), containing azadirachtin A at 34%, on blood feeding, oviposition and oocyte ultrastructure. Oral administration of Neem Azal) to A. stephensi females through artificial blood meals did impair blood intake and oviposition in a concentration dependent manner. Similar results were obtained on females, which had consumed Neem Azal) in sucrose solution before taking a blood meal of plain blood. Neem treated females displayed a delay in oocyte development in both the phase of vitellogenesis and the phase of choriogenesis. The ultrastructural studies on ovaries from Neem Azal) treated females revealed distinct structural modifications indicative of: (i) a complete block of oogenesis, (ii) impairment of vitellogenesis and vitelline envelope formation, (iii) a severe degeneration of follicle cells. In agreement with results obtained in other insects, this study indicates that Neem Azal) impairs hormone control of oogenesis and exerts a cytotoxic effect on both follicular cells and oocytes of the Asian malaria vector A. stephensi.     

A COMPREHENSIVE STUDY ON APOPTOSIS INDUCTION BY AZADIRACHTIN IN Spodoptera frugiperda CULTURED CELL LINE Sf9

The induction of apoptosis by azadirachtin, a well‐known botanical tetranortriterpenoid isolated from the neem tree (Azadirachta indica A. Juss) and other members of the Meliaceae, was investigated in Spodoptera frugiperda cultured cell line (Sf9). Morphological changes in Sf9 cells treated by various concentrations of azadirachtin were observed at different times under light microscopy. Morphological and biochemical analysis indicated that Sf9 cells treated by 1.5 μg/mL azadirachtin showed typical morphological changes, which were indicative of apoptosis and a clear DNA ladder. The flow cytometry analysis showed the apoptosis rate reached a maximum value of 32.66% at 24 h with 1.5 μg/mL azadirachtin in Sf9 cells. The inhibition of Sf9 cell proliferation suggested that the effect of azadirachtin was dose dependent and the EC₅₀ at 48 and 72 h was 2.727 × 10⁻⁶ and 6.348 × 10⁻⁹ μg/mL, respectively. The treatment of azadirachtin in Sf9 cells could significantly increase the activity of Sf caspase‐1, but showed no effect on the activity of Topo I, suggesting that the apoptosis induced by azadirachtinin Sf9 cells is through caspase‐dependent pathway. These results provided not only a series of morphological, biochemical, and toxicological comprehensive evidences for induction of apoptosis by azadirachtin, but also a reference model for screening insect cell apoptosis inducers from natural compounds.     

樟树5种化学类型叶片转录组分析

樟树(Cinnamomum camphora )是樟科植物的一个代表种, 具有材用、药用、香料、油用和生态环境建设等多种用途。叶精油中富含利用价值极高的樟脑、芳樟醇、1,8-桉叶油素、异-橙花叔醇和右旋龙脑等萜类化合物。依据叶精油中主要成分的种类和含量, 可将樟树划分为脑樟、芳樟、油樟、异樟、龙脑樟5种化学类型。文章采用Illumina HiSeq™ 2000高通量测序技术, 对5种化学类型叶片转录组进行测序, 对测序得到的所有Unigene进行GO(Gene Ontology)、COG(Clusters of Orthologous Groups)和KEGG(Kyoto Encyclopedia of Genes and Genomes)分类, 给出功能注释和Pathway注释, 并预测Unigene蛋白编码区(Coding sequence, CDS)。De novo组装共获得156 278个Unigene, 序列平均长度584 bp, N50(覆盖50%所有核苷酸的最大Unigene长度)为1 023 bp。通过与其他核酸、蛋白数据库的Blast搜索比对, 共有55 955条Unigene获得了基因注释, 占所有Unigene的35.80%。其中, 有24 717条Unigene得到GO注释, 有21 806条Unigene得到COG注释。KEGG pathways分析结果表明, 共有3 350条基因(10.19%)注释到次生代谢生物合成途径, 其中参与单萜、二萜、倍半萜和萜类骨架合成的Unigene有424个。在单萜合成的代谢通路中, 有9条Unigene可能编码芳樟醇合成酶基因, 且表达分析结果显示, 芳樟醇合成酶基因在芳樟化学类型中优势表达, 在油樟化学类型中表达水平较低。这些注释信息的完成为樟树功能基因及相关候选基因的发掘提供了基础数据和重要依据。     

Identification of a putative azadirachtin-binding complex from Drosophila Kc167 cells

Cell-proliferation in Drosophila Kc167 cells was inhibited by 50% when cell cultures contained 1.7 x 10(-7) M azadirachtin for 48 h (a tertranortriterpenoid from the neem tree Azadirachta indica). Drosophila Kc167 cells exhibited direct nuclear damage within 6-h exposure to azadirachtin (5 x 10(-7) M and above) or within 24 h when lower concentrations were used (1 x 10(-9) M). Fractionation of an extract of Drosophila Kc167 cells combined with ligand overlay technique resulted in the identification of a putative azadirachtin binding complex. Identification of the members of this complex by Peptide Mass Fingerprinting (PMF) and N-terminal sequencing identified heat shock protein 60 (hsp60) as one of its components.     

中粒咖啡萜类合成酶基因家族的生物信息学分析

萜类化合物具有重要的生理、生态作用和药用价值,萜类合成酶(TPS)是合成萜类化合物的关键酶。通过整合中粒咖啡(Coffee canephora)的基因组和转录组数据,利用生物信息学方法,鉴定出43个萜类合成酶全长基因,并对这些基因的分子进化、结构、复制、表达及功能分化的机理进行了探究。结果表明,中粒咖啡萜类合成酶基因可以分为5个亚家族(a、b、c、e/f、g),不同亚家族的基因结构差异很大;串联复制是基因家族扩增的主要原因;表达分析结果表明,萜类合成酶基因在不同组织中的表达差异明显;中粒咖啡萜类合成酶基因启动子区的顺式调控元件可能与基因的功能分化相关;不同亚家族之间的功能差异主要由亚家族特异的氨基酸决定。     

Genomic analysis of the terpenoid synthase (<Emphasis Type="Italic">AtTPS</Emphasis>) gene family of<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

A family of 40 terpenoid synthase genes ( AtTPS) was discovered by genome sequence analysis in Arabidopsis thaliana. This is the largest and most diverse group of TPS genes currently known for any species. AtTPS genes cluster into five phylogenetic subfamilies of the plant TPS superfamily. Surprisingly, thirty AtTPS closely resemble, in all aspects of gene architecture, sequence relatedness and phylogenetic placement, the genes for plant monoterpene synthases, sesquiterpene synthases or diterpene synthases of secondary metabolism. Rapid evolution of these AtTPS resulted from repeated gene duplication and sequence divergence with minor changes in gene architecture. In contrast, only two AtTPS genes have known functions in basic (primary) metabolism, namely gibberellin biosynthesis. This striking difference in rates of gene diversification in primary and secondary metabolism is relevant for an understanding of the evolution of terpenoid natural product diversity. Eight AtTPS genes are interrupted and are likely to be inactive pseudogenes. The localization of AtTPS genes on all five chromosomes reflects the dynamics of the Arabidopsis genome; however, several AtTPS genes are clustered and organized in tandem repeats. Furthermore, some AtTPS genes are localized with prenyltransferase genes ( AtGGPPS, geranylgeranyl diphosphate synthase) in contiguous genomic clusters encoding consecutive steps in terpenoid biosynthesis. The clustered organization may have implications for TPS gene evolution and the evolution of pathway segments for the synthesis of terpenoid natural products. Phylogenetic analyses highlight events in the divergence of the TPS paralogs and suggest orthologous genes and a model for the evolution of the TPS gene family.