Toxicity of plumbagin and juglone to the eggs of the cotton stainer,Dysdercus koenigii

Toxicity of two plumbaginoids viz., plumbagin and juglone to the eggs of the cotton stainer,Dysdercus koenigii was studied by a residual film technique. Of these two, plumbagin showed the higher toxicity against different-age eggs with LC₅₀ ranging from 0.0044 to 0.0066%. Eggs showed low susceptibility in the middle of embryogenesis. The toxicity of plumbaginoids, especially plumbagin, is discussed in relation to mode of action and prospects of its use as an ovicide in control of the insect.     

Autoradiographic localization of [22,23-(3)H(2)] dihydroazadirachtin binding sites in desert locust testes and effects of Azadirachtin on sperm motility

Localization of [22,23-(3)H(2)] dihydroazadirachtin binding sites in locust (Schistocerca gregaria) testes was investigated by in vitro autoradiography. Preferential binding of the ligand at concentrations of 10(-8) M was located in the testes follicles, localized on the tail portions of developing sperm. This binding was fully displaceable with an excess of unlabelled ligand. The results indicate that Azadirachtin binds preferentially to sites on the organelles associated with maturing locust sperm tails. Azadirachtin, at concentrations of 10(-4)M and above, caused a time-dependent reduction in the motility of eupyrene sperm bundles liberated from the accessory glands of mature male S. gregaria. The effect of azadirachtin on boar sperm motility was also time- and concentratiion-dependent. Forward motility was significantly reduced by concentrations above 10(-5)M at each time interval (P<0.05).     

Insect growth regulating effects of neem extract and azadirachtin on aphids

Neem,Azadirachta indica (A. Juss.), seed oil (NSO) applied to leaf discs at a concentration of 1.0% resulted in 94% to 100% mortality of second instar nymphs of currant-lettuce aphid,Nasonovia ribis-nigri (Mosley), and green peach aphid,Myzus persicae (Sulzer), after nine days. The equivalent amount of pure azadirachtin (AZA) (≈40 ppm), the principle active ingredient of neem, was as effective as NSO. The survival of adult aphids was unaffected by NSO or AZA, but the survival of offspring from treated adultM. persicae andN. ribis-nigri was reduced significantly. The lethal concentration of AZA resulting in 50% mortality of second instar nymphs of nine species of aphids ranged from 2.4 ppm forM. persicae on pepper to 635.0 ppm for the strawberry aphid,Chaetosiphon fragaefolii (Cockerell), on strawberry. ForM. persicae, the growth regulating effect of AZA was influenced by the host plant and the nymphal instar treated.     

Interaction of cotton tissue culture cells andVerticillium dahliae

Summary Elicitation of sesquiterpenoid aldehyde phytoalexins inGossypium arboreum cell suspension cultures was confirmed by thin layer chromatography, high performance reverse phase liquid chromatography, and an aniline-reaction assay after inoculation with heat-treated conidia ofVerticillium dahliae A 2.3X mean increase in total terpenoids was observed. Component phytoalexins varied, with either hemigossypol and gossypol being detected or the O-methylated terpenoids hemigossypol-6-methyl ether and related compounds. Long-termGossypium suspension cultures were mixoploid with an increase in chromosome number and mean DNA content. Addition ofV. dahliae elicitor(s) to the medium for embryo-proliferating callus ofG. hirsutum inhibited growth and embryo production with a linear correlation (r=−0.87;P<0.01) between the elicitor concentration and the number of embryos. Addition of¹⁴C-labeled NaOAc to suspension cells gave 30% incorporation, and from¹³C-NaOAc addition, labeled sesquiterpenoid aldehydes were recovered. The cotton-Verticillium system is another case of secondary metabolite elicitation in plant tissue culture and might be used for basic studies of hostpathogen interaction as well as for a selection tool to obtain resistance to an important disease.     

Effect of some crude and azadirachtin-enriched neem (Azadirachta indica) seed kernel extracts on larvae of Aedes aegypti

Bioassays against larvae of Aedes aegypti were conducted with neem seed kernel extracts obtained by extraction with water and organic solvents. Permanent exposure of fourth instar larvae to treated water resulted in a conspicuous growth-disrupting effect, mainly during imaginal development. The effectiveness of the extracts increased with decreasing polarity of the solvents used for extraction. Three neem seed kernel extracts caused an extreme prolongation of the larval period when first instar larvae were continuously exposed to treated water until adult emergence. The time necessary for lethal action of neem seed kernel extracts to set in was similar to that reported for some synthetic IGR's.

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Zusammenfassung Zehn mit Wasser und verschiedenen organischen Lösungsmitteln hergestellte Niem-Samen-Extrakte wurden auf ihre Wirkung auf Larven der Gelbfiebermücke Aedes aegypti untersucht. Dauerhaltung der Viertlarven in behandeltem Wasser führte zu einer beträchtlichen wachstumshemmenden Wirkung, die hauptsächlich während der Imaginalentwicklung in Erscheinung trat. Der Wirkungsgrad der Extrakte steigerte sich im biotest mit abnehmender Polarität der bei der Extraktion verwendeten Lösungsmittel. Drei Extrakte (ANSKE, AZT-VR-K-E und MTB/H₂O-K-NR-E) deren Wirkung auch auf Erstlarven untersucht wurde, verursachten erhebliche Entwicklungsverzögerungen wenn die Larven bis zum Erscheinen der Imagines behandeltem Wasser ausgesetzt waren. Die von den Extrakten verursachte Mortalität trat zu einem ähnlichen Zeitpunkt ein, wie er für einige synthetische Insektenwachstumshemmer berichtet wird.

     

Natural enemies of the pumpkin caterpillarDiaphania indica [Lepidoptera: Pyralidae] in Tamil Nadu

The survey for the natural enemies associated with the pumpkin caterpillar,Diaphania indica revealed the presence of 20 species of parasitoids, predators and pathogens. Of these, 16 were parasitoids belonging to the familiesBraconidae, Ichneumonidae, Bethylidae, Elasmidae andChalcididae. Except for 3 species the remaining parasitoids were new records forD. indica. The predators recorded were ants and spiders. A microsporidia also was recorded for the first time onD. indica.      

Efficacy of spider and ant predators on the cotton fleahopper [Hemiptera: Miridae]

The efficacy of predators of immature cotton fleahoppers,Pseudatomoscelis seriatus (Reuter), was calculated using field and laboratory cage confinement tests for consumption rate. The predators tested were the striped lynx spider,Oxyopes salticus Hentz; the black and white jumping spider,Phidippus audax (Hentz); the celer crab spider,Misumenops celer Hentz; and the red imported fire ant,Solenopsis invicta Buren. The spider predators were evaluated in a cotton field using predator-prey confinement cages on cotton plants. Average percent control (sensuAbbott 1925) of fleahoppers byO. salticus, P. audax, andM. celer were 42%, 66% and 32% respectively. The rate of fleahopper consumption by red imported fire ants was measured in the laboratory using various numbers of ants and fleahoppers. Daily percent control by ants ranged from 0.5% (single ant and fleahopper) to 100% (colony linked). The functional response of the 4 arthropod species to different prey numbers is illustrated and discussed as is the relative potential usefulness of natural enemies to suppress fleahoppers on cotton.      

Insect growth regulating and antifeedant effects of neem extracts and azadirachtin on two aphid species of ornamental plants

Leaf disc choice test bioassay demonstrated that formulated neem seed extracts were highly deterrent and growth regulatory to rose aphid,Microsiphum rosae (L.) and Chrysanthemum aphid,Macrosiphoniella sanbornii (Gillete). Effective concentrations to produce 50% feeding deterrence was 0.80 and 0.84% respectively for 2nd instar nymphs irrespective of bioassay duration. The disruption of aphid feeding was related to the presence of azadirachtin concentration in the extract. The toxicity on contact from the leaf surface or via topical application due to azadirachtin was significantly different and topical treatment was at least 7 times more effective for both species. Thus growth regulatory effects of azadirachtin were influenced by the host plant and the stage of treatment. Field evaluation with formulated neem extracts revealed the effect to be more of growth regulatory nature thereby showing that azadirachtin is a physiological toxin for aphid species. Neem seed extracts reduced the population of aphid on respective host plants significantly, EC₅₀ values being 0.88 and 0.96% forM. rosae andM. sanbornii respectively.     

Xyloglucan breakdown during cotton fiber development

Cotton (Gossypium herbaceum L.) fibers elongated almost linearly up to about 20 days post anthesis. The molecular mass of xyloglucans in fiber cell walls decreased gradually during the elongation stage. When enzymatically active (native) cell wall preparations of fibers were autolyzed, the molecular mass of xyloglucans decreased. The decrease was most prominent in wall preparations obtained from the rapidly elongating fibers. The xyloglucan-degrading activity was recovered from the fiber cell walls with 3 mol/L NaCl, and the activity was high at the stages in which fibers elongated vigorously. These results suggest the possible involvement of xyloglucan metabolism in the regulation of cotton fiber elongation.     

Analysis of cell-wall polymers during cotton fiber development

Although the fibers of cotton (Gossypium hirsutum L.) are single cells with a secondary wall composed primarily of cellulose, the cell-wall polymers of the fibers are technically difficult to characterize with respect to molecular weights. This limitation hinders understanding how the fiber wall composition changes during development, particularly with respect to genotypic variations, and how the molecular composition is related to physical properties. We analyzed cell-wall polymers from cotton fibers (cultivar, Texas Marker-1) at several developmental stages (8–60 days post-anthesis; DPA) by gel-permeation chromatography of components soluble in dimethyl acetamide and lithium chloride. This procedure solubilizes fiber cell-wall components directly without prior extraction or derivatization, processes that could lead to degradation of high-molecular-weight components. Cellwall polymers from fibers at primary cell-wall stages had lower molecular weights than the cellulose from fibers at the secondary wall stages; however, the high-molecularweight cellulose characteristic of mature cotton was detected as early as 8 DPA. High-molecular-weight material decreased during the period of 10–18 DPA with concomitant increase in lower-molecular-weight wall components, possibly indicating hydrolysis during the later stages of elongation.Abbreviations DMAC dimethyl acetamide - DP degree of polymerization - DPA days post anthesis - GPC gel-permeation chromatography - MW molecular weight - MWD molecular-weight distribution - TM-1 Texas Marker 1     

Membranes and calcium sequestration during spermiogenesis in the cotton seed bug (Dysdercus intermedius: Heteroptera)

Summary By use of osmium ferricyanide (OsFeCN) staining the fate of cytoplasmic membranes was followed during spermiogenesis in the cotton seed bug (Dysdercus intermedius). During early spermiogenesis interzonal lamellae of endoplasmic reticulum become aggregated as a stack of membranes traversing the entire cell body from the nucleus to the cytoplasmic bridge connecting neighbouring spermatids. Cisternae of endoplasmic reticulum ensheath the acroblast from which vesicles of different sizes are pinched off into the cytoplasm. The oxalate method was used to show that acroblast and associated vesicles are calcium-sequestering sites in spermatids. Membrane profiles with dense calcium oxalate precipitate derived from the acroblast form an uninterrupted membranous sheath at the apical side of the nucleus where the proacrosome will be attached. With further development of the spermatids, the vesicles derived from the acroblast also participate in forming a calciumsequestering sheath enveloping the axoneme and the mitochondrial nebenkern derivatives.     

Heterologous gap junctions between oocytes and follicle cells of an insect,Dysdercus intermedius,and their potential role as ion current pathways

Summary In telotrophic insect ovaries, the oocytes develop in association with two kinds of supporting cells. Each ovary contains five to seven ovarioles. An ovariole consists of a single strand of several oocytes. At the apex of each ovariole is a syncytium of nurse cells (the tropharium), which connects by strands of cytoplasm (the trophic cords) to four or more previtellogenic oocytes. In addition, each oocyte is surrounded by an epithelium of follicle cells, with which it may form gap junctions. To study the temporal and spatial patterns of these associations, Lucifer yellow was microinjected into ovaries of the red cotton bug, Dysdercus intermedius. Freeze-fracture replicas were examined to analyze the distribution of gap junctions between the oocyte and the follicle cells. Dye-coupling between oocytes and follicle cells was detectable early in previtellogenesis and was maintained through late vitellogenesis. It was restricted to the lateral follicle cells. The anterior and posterior follicle cells were not dye-coupled. Freeze-fracture analysis showed microvilli formed by the oocyte during mid-previtellogenesis, and the gap junctions became located at the tips of these. As the microvilli continued to elongate until late vitellogenesis, gap junction particles between them and follicle cell membranes became arranged in long arrays. The morphological findings raise questions about pathways for the intrafollicular phase of the ion currents known to surround the previtellogenic and vitellogenic growth zones of the ovariole.Supported by the Deutsche Forschungsgemeinschaft (Schwerpunkt Differenzierung)     

Plant regeneration from different explants of neem

When different seedling explants, i.e. hypocotyl, epicotyl, cotyledonary node, root-shoot zone, cotyledon, leaves and roots from 7-day-old seedlings of neem were cultured on Murashige and Skoog's medium supplemented with 2 mg l⁻¹ benzyladenine and 0.1 mg l⁻¹indole-3-acetic acid, shoot buds were initiated from all the explants tested, with leaf explants producing the highest average number of shoots/explant. The regenerated shoots were further subcultured and later could be rooted on a medium supplemented with indole butyric acid (1 mg l⁻¹) and complete plants could be obtained. This revised version was published online in June 2006 with corrections to the Cover Date.     

Molecular analysis of micropropagated neem plants using aflp markers for ascertaining clonal fidelity

Summary The importance of neem (Azadirachta indica A. Juss.) as a medicinal tree species has been acknowledged worldwide. Superior trees with desired traits such as high azadirachtin content have been identified and micropropagated. Somaclonal variants that may arise in vitro, however, pose limitations to large-scale micropropagation. It is, therefore, imperative to establish genetic uniformity of such plantlets by ensuring strict quality checks at various stages of in vitro culture. This is the first study that evaluates the applicability of amplified fragment length polymorphism (AFLP) markers in establishing clonal fidelity of tissue culture(TC)-raised neem plants. Seven AFLP primer combinations generated a total of 334 amplified fragments across the mother plant, TC progenies, and other neem accessions that were included as controls. Two hundred and thirty-nine amplified fragments were monomorphic across the mother tree and its TC progenies. No extra band was detected in the TC plantlets that was absent in the mother tree, indicating that the TC plantlets regenerated through nodal explants are indeed true-to-type. Ninety-five AFLP fragments were detected in the controls, which allowed their discrimination from the elite mother tree and its TC progenies. Similarity matrix based on Jaccard's coefficient revealed that the pair-wise value between the mother tree and its TC plantlets was ‘1’, indicating perfect similarity. Phenetic dendrogram based on UPGMA (unweighted pair group method of arithmetic averages) analysis further confirmed the true-to-type nature of TC progenies, since a tie was observed between the mother tree and its TC plantlets. On the contrary, the control neem accessions were distinct from the mother and its TC progenies. AFLP markers proved to be an ideal tool for routine analysis and certification of genetic fidelity of micropropagated plants prior to commercialization, especially in tree species because of their long generation time.     

Studies on plant gums. Role of calcium in polysaccharide-protein interaction in the neem (Azadirachta indica) gum

The partial removal of tightly bound Ca²⁺ from dialysed neem (Azadirachta indica) gum, resulted in the release of a basic protein from a highly anionic polysaccharide-protein complex as evidenced by chromatographic studies on TEAE-cellulose. Complete removal of Ca²⁺ caused, in addition, the release of a minor heteropolysaccharide which was found in association with the basic protein. These processes were reversed on the addition of Ca²⁺. The gum, in addition, contained a protein-rich component accounting for 35% protein and 7.5% total carbohydrate. This component behaved as a distinct entity during ion-exchange chromatography of the native gum solutions, or which were either partially or completely depleted of bound Ca²⁺.     

Fungi associated with the Egyptian cotton leafwormSpodoptera littoralis Boisdoval

A total of 28 species representing 15 fungal genera were found to be associated with the all different stages of the Egyptian cotton leafworm,Spodoptera littoralis Boisd., its faeces and the air of the containers where it was reared on Czapek Dox agar medium at 28°C.Aspergillus was the most common genus and was frequently isolated from all substrates.A. flavus was the only fungus associated with all insect stages. At least two well known facultative pathogens were obtained:Beauveria alba andCephaliophora tropica. Nearly all fungi recovered from insect faeces or its environment were also encountered from different insect stages. Forty-two isolates were tested for their abilities for growth on a synthetic medium (nitrogen- and carbon-free) containing partially purified chitin. Most (76.2%) had moderate growth rates.     

Genetic transformation of green-colored cotton

Summary This study reports an Agrobacterium-mediated transformation of green-colored cotton (Gossypium hirsutum L.). A tissue culture procedure was optimized to induce callus formation from hypocotyl explants and subsequent differentiation into the embryogenic type. Callus formation could be induced by growing explants on Murashige and Skoog medium containing 2,4-dichlorophenoxyacetic acid and kinetin. Among the four genotypes studied, embryogenic calli and plant regeneration were observed only in var. G9803. Agrobacterium-mediated transformation of G9803 with the fiber-specific expansin gene GhExpl was achieved based on the establishment of these tissue culture methods. A total of 32 individual regenerants resistant to kanamycin were generated within 7 mo., with a transformation frequency of 17.8%. Transformation was confirmed by Southern blot analysis and RT-PCR. These results represent the first step towards genetic manipulation of the colors and fiber quality of green-colored cottons by biotechnology. These authors contributed equally to this work     

Defined conditions for the initiation on growth of cotton callus in vitro I.Gossypium arboreum

Summary Defined in vitro conditions for callus initiation byGossypium arboreum L. were determined, and different tissues were evaluated as explant sources. Environmental conditions tested included light versus dark, and low light versus high light. Different nutrient media as well as carbohydrate sources were examined. Our data show that hypocotyl tissue was superior to cotyledon or leaf tissue as the explant source for callus proliferation; the Murashige-Skoog inorganic formulation with (in mg per 1) 100 myo-inositol, 0.4 thiamine·HCl, 2 indoleacetic acid (IAA), 1 kinetin, and 3% glucose solidified by agar was the best medium to initiate callus. Cultures with sucrose as a carbohydrate source browned rapidly. Callus proliferation was superior under high light (8000 to 9000 lux) conditions at 29±1°C. Various combinations of auxins and cytokinins were tested for their ability to improve callus proliferation and subsequent growth of subcultures. Although the MS medium containing IAA and kinetin was found superior for obtaining rapid proliferation of callus from hypocotyl explants, a second medium containing 2 mg per 1 naphthalenacetic acid (NAA) and 0.5 to 1 mg per 1 benzyladenine (BA) was found necessary for vigorous growth of subcultured callus. A MS medium with 5 to 10 mg per 1 {ie329-1} (2iP) and 1 mg per 1 NAA was also favorable for continued subculturing. Technical Article 12485 from the Texas Agricultural Experiment Station.     

Induced resistance against spider mites in cotton: Field verification

Cotton plants that had been damaged by spider mites (Tetranychus spp.) or by mechanical abrasion at the cotyledon stage were less likely to develop infestations of spider mites compared to controls early in the season. These are the first field results to demonstrate that induced resistance can reduce pest populations in an agricultural system. Differences in mite populations early in the season caused by induced resistance did not translate into differences in plant growth or cotton yield at harvest.

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Vérification au champ de la résistance du coton induite par les acariens

Résumé Les plants de coton qui ont été endommagées, au stade cotylédons, par des acariens ou par une abrasion mécanique, ont moins de chance que les témoins, de présenter ultérieurement des dégats dus aux acariens. C'est la première fois que des résultats montrent qu'une résistance induite peut réduire les populations dans un agrosystème. Les différences ultérieures dans les populations d'acariens, ne transparaissent pas au niveau de la croissance du coton ou de la production au moment de la récolte.