Bioactive compounds from neem tissue cultures and screening against insects

Hairy root cultures have been derived from neem (Azadirachta indica A Juss, Family Meliacceae) using Agrobacterium rhizogenes and have been studied for the production of compounds with antifeedant effects on insects. Six-week-old hairy root cultures were extracted, and HPLC yielded fractions ranging from polar to non-polar compounds. High antifeedancy levels against the desert locust were observed in fractions(F) 2, 3 and 4 whilst F1 and F5 were not significantly antifeedant. Interestingly F3 did not contain any of the well-known neem chemicals while F2 contained azadirachtin and 3-tigloylazadirachtol and F4 nimbin and salannin. © 1999 Society of Chemical Industry     

Comparison of anti-feedant and insecticidal activity of nimbin and salannin photo-oxidation products with neem (Azadirachta indica) limonoids

Photo-oxidation of the neem limonoids nimbin and salannin with UV light in the presence of oxygen gives two isomeric lactone products per limonoid, nimbinolide and isonimbinolide, and salanninolide and isosalanninolide, respectively. When compared in insect tests with the important limonoids of neem seeds, azadirachtin, nimbin and salannin, isonimbinolide and isosalanninolide show activity greater than that of nimbin or salannin and in some respects show activity approaching that of azadirachtin. The photo-oxidation products were tested for anti-feedant activity and toxicity against larvae of three species of Lepidoptera, Spodoptera littoralis (Boisd), Spodoptera frugiperda (FE Smith) and Helicoverpa armigera (Hübner) and nymphs of the locusts Schistocerca gregaria (Forsk?l) and Locusta migratoria (L).     

Microbiological and Chemical Analysis of Neem (Azadirachta indica) Extracts: New Data on Antimicrobial Activity

The antimicrobial effects of extracts of neem seed (Azadirachta indica A. Juss.) were investigated using microbial growth inhibition assays. A laboratory-prepared neem seed extract along with a commercially available formulated product, were characterized using HPLC, and shown to be effective against a range of bacteria in an agar diffusion assay. The active ingredient,i.e., the unformulated seed extract of the commercial product, also showed activity and this was further investigated in a biochromatogram, using the sensitive bacteriumBacillus mycoides. Results showed antibacterial activity as three discrete inhibition zones that did not correspond to the R_f of the major neem metabolites, azadirachtin, nimbin and salannin. This suggests that these compounds were not antibacterial. The colony radial growth rates of the fungal pathogens that cause ‘take-all’ and ‘snow mould’ disease were both significantly affected when the commercial, unformulated, neem seed extract was incorporated into the growth medium. Experiments in liquid culture suggested that the effect was fungistatic. Conidial germination of the commercially important obligate pathogenSphaerotheca fuliginea (powdery mildew) was reduced to 11%. The results show that neem seed extracts possess antimicrobial activity with notable effects on some fungal phytopathogens. This Work demonstrates that neem seed extracts have potential for controlling both microbial and insect pests. http://www.phytoparasitica.org posting Sept. 16,2001.     

Nimbolide is the Principal Cytotoxic Component of Neem-Seed Insecticide Preparations

Several neem-seed extracts, some used for preparing commercial azadirachtin-containing insecticides, are cytotoxic to N1E-115 murine neuroblastoma cells with IC₅₀ values of 20–200 μg extract ml⁻¹ culture medium. Bioassay-directed fractionation by reversed-phase HPLC shows that the toxicity to N1E-115 cells is associated primarily with a single minor component identified by isolation and NMR and MS as nimbolide with an IC₅₀ of 1·5 μg ml⁻¹ (3·2 μM ). The difference in quantity of nimbolide in seven neem extract sources generally correlates with their overall cytotoxicity. Three other limonoids (epoxyazadiradione, salannin and possibly deacetylsalannin) but not azadirachtin, nimbin and deacetylnimbin contribute in small part to the cytotoxicity. Reconstituted neem extract with only nimbolide removed is less cytotoxic than the original extract. It therefore appears that nimbolide is the principal cytotoxic component of the neem extracts examined and that such minor constituents may warrant consideration in safety evaluations.     

Activity of neem (Azadirachta indica A. Juss) seed kernel extracts against the mustard aphid,Lipaphis erysimi

An aqueous, an ethanolic and a hexane extract obtained from neem(Azadirachta indica A. Juss) seed kernels were tested for insecticidal activity against the mustard aphid,Lipaphis erysimi Kalt. The hexane extract, which exhibited a much higher activity than the two other extracts, had an LC50 of 0.674%. When the hexane extract was partitioned with ethanol, the ethanol-soluble fraction had an LC₅₀ of 0.328%, whereas the ethanol-insoluble part showed no activity even at 1%. Column chromatography of the ethanol-soluble fraction yielded eight compounds: nimbin, epinimbin, desacetylnimbin, salannin, desacetyl-salannin, azadirachtin and two unidentified compounds — a salannin derivative and a nonterpenoid. Of these, only five could be tested. Nimbin and epinimbin exhibited no toxicity at 0.3%, whereas salannin, a salannin derivative and the non-terpenoid gave LC₅₀ values of 0.055, 0.096 and 0.104%, respectively.     

Response ofSpodoptera littoralis (Boisd.) andEarias insulana (Boisd.) larvae to azadirachtin and salannin

The effect of azadirachtin and salannin, two triterpenoids isolated from seeds of neem (Azadirachta indica A. Juss), on the feeding response ofSpodoptera littoralis (Boisd.) andEarias insulana (Boisd.) larvae, was investigated. Styropor (foamed polystyrene) lamellae were painted on both sides with a mixture of 5% sucrose with different concentrations of either azadirachtin or salannin dissoled in methanol-water (3∶7). Azadirachtin strongly suppressed feeding inS. littoralis larvae even at 0.001%, whereas salannin showed some antifeedant activity at 0.005% and above. Larvae ofE. insulana were deterred from feeding on azadirachtin-treated lamellae even at 0.005%, whereas salannin was effective only at 0.01% and above. Azadirachtin applied on cotton leaves deterred larvae ofS. littoralis from feeding at all concentrations ranging between 0.001 and 0.02%.     

The toxicity and physiological effect of neem limonoids on Cnaphalocrocis medinalis (Guenée) the rice leaffolder

The effect of neem (Azadirachta indica) limonoids azadirachtin, salannin, deacetylgedunin, gedunin, 17-hydroxyazadiradione, and deaceytlnimbin on gut enzyme activity of the rice leaffolder larvae was investigated. When fed a diet of rice leaves treated with limonoids in bioassays, gut tissue enzymes—acid phosphatases (ACP), alkaline phosphatases (ALP), and adenosine triphosphatases (ATPase) activities of rice leaffolder (Cnaphalocrocis medinalis) larvae are affected. Azadirachtin was most potent in all experiments. Larvae that were chronically exposed to limonoids showed a reduction in weight (59-89%) and exhibited a significant reduction in ACP, ALP, and ATPase activities. These results indicate neem limonoids affects gut enzyme activities. These effects are most pronounced in early instars.     

Antifungal fractions and compounds from uncrushed Green Leaves ofAzadirachta indica

Using the groundnut rust disease (causal agentPuccinia arachidis Speg.) as the bioassay system, two limonoids from the neem tree (Azadirachta indica A. Juss.) which evinced antifungal activity, were isolated through extraction, solvent fractionation and HPLC. A polar extract derived through solvent partitioning reduced the disease intensity considerably. The polar extract and the impure HPLC fractions were more effective than the pure compounds in reducing the pustule numbers and, consequently, the disease severity.     

The effects of phytochemical pesticides on the growth of cultured invertebrate and vertebrate cells

A range of cultured cells of invertebrate and vertebrate origin was grown in the presence of a number of phytochemical pesticides to test the effect of the latter on cell proliferation. The main observation was that azadirachtin was a potent inhibitor of insect cell replication, with an EC50 of 1.5 x 10(10) M against Spodoptera cells and of 6.3 x 10(9) M against Aedes albopictus cells, whilst affecting mammalian cells only at high concentrations (> 10(-4) M). As expected, the other phytochemical pesticides, except for rotenone, had little effect on the growth of the cultured cells. Rotenone was highly effective in inhibiting the growth of insect cells (EC50:10(-8) M) but slightly less toxic towards mammalian cells (EC50:2 x 10(-7) M). Neem terpenoids other than azadirachtin and those very similar in structure significantly inhibited growth of the cell cultures, but to a lesser degree. The major neem seed terpenoids, nimbin and salannin, for example, inhibited insect cell growth by 23% and 15%, respectively.     

Effects of neem (Azadirachta indica) extracts onLeptocorisa chinensis under choice and no-choice conditions

Two extracts from neem (Azadirachta indica A. Juss. (Meliaceae)) seeds, azadirachtin and oil, and a mixture of neem oil and abamectin, were tested on second-instar nymphs of the rice bugLeptocorisa chinensis (Dallas) (Hemiptera: Alydidae). To clarify the effect of spraying coverage on bioefficacy of test materials, experiments were conducted under choice and no-choice conditions in field cages. In a choice test, treatment with the mixture of neem oil and abamectin was most effective in reducing the survival ofL. chinensis, followed by azadirachtin at 60 ppm, 30 ppm and 3% neem oil, whereas all treatments except neem oil caused 100% mortality within 3 weeks in a no-choice test. When second-instar nymphs had choices of treated and untreated plants within a treatment, no differences in yield and sum of dead and stained grains were found between those two choices, indicating that nymphs neither caused significant reduction in yield nor reduced the quality of untreated plants. Regardless of treatment, the difference in overall yield between treated and untreated plants under choice conditions was not statistically significant (P>0.05). Our results indicate that neem-based formulations, used alone or in combination with abamectin, have the potential to be integrated into the existing programs to control the rice bug. http://www.phytoparasitica.org posting Aug. 28, 2005.     

Wirkung von Neem-Produkten auf höhere Pflanzen und ihre mögliche Nutzung zur Reduzierung der Seitentriebe von Kulturpflanzen sowie zur Unkrautbekämpfung – eine Bestandsaufnahme

The neem tree (Azadirachta indica A. Juss) offers many possibilities of usage. In plant protection mostly the insecticidal activity of neem products is important. In different parts of the plant several biologically active substances occur. One is the active ingredient azadirachtin, which already is commercially used as an insecticide against insect pests. Some active ingredients of neem may be systemically taken up by the treated plants. Often a rapid decomposition occurred which was enhanced by UV radiation. Therefore formulated neem products often need a UV blocker to elongate their activity. After the application of neem products within plant protection sometimes phytotoxic effects could be observed on different plant species. This phytotoxicity can be used to reduce suckers on tobacco plants and recently also on eucalyptus plants. The existing trials to use neem products for weed control mostly concentrated on the direct treatment of some parasitic weeds like Cuscuta, Orobanche and Striga as well as on a few other weeds (e.g. Echinochloa crus-galli). Mostly the growth of the weeds which were often repeatedly treated with high dosages was reduced, but many plants remained still alive. At present the potential of neem products for an effective weed control is not sufficiently investigated. As a consequence of some already known insecticidal and microbiocidal activities of many neem products also side effects on parts of the epigeic and soil fauna and on the nitrification should be considered after usage for weed control.     

Fungitoxicity of 5-aminoisoxazole-4-thiocyanate derivatives

3-Methyl-5-(acyl/alkyl)aminoisoxazole-4-thiocyanates were prepared by rhodanation of N-acyl/alkyl-3-methyl-5-isoxazolamines. These products were tested for antifungal activity against a series of phytopathogenic fungi of different taxonomic classes. Some of the compounds showed interesting in-vitro activity. The more active compound in the in-vitro test displayed a moderate preventive activity against Plasmopara viticola, Alternaria solani and Pyricularia recondita.     

枯草芽胞杆菌NCD-2菌株抑菌蛋白的分离及鉴定

枯草芽胞杆菌NCD-2菌株对灰葡萄孢菌具有较强的抑菌活性,抑菌蛋白是其产生的抑菌物质之一。本研究为明确NCD-2菌株所产抑菌蛋白的作用方式和特性,采用牛津杯法测定抑菌蛋白的抑菌活性及其对病原菌菌丝生长的影响,采用混合培养法测定其对病原菌分生孢子萌发的影响,同时采用阴离子交换层析和非变性聚丙烯酰胺凝胶电泳技术对其进行分离纯化,并利用MALDI-TOF-MS进行鉴定。结果表明,NCD-2菌株产生的粗蛋白能够显著抑制灰葡萄孢菌分生孢子的萌发和菌丝生长,并导致病原菌菌丝分枝增多、局部膨大肿胀。通过分离纯化获得具抑菌活性的蛋白组分D1-3,经鉴定该蛋白的肽指纹图谱与leucyl aminopeptidase[Bacillus subtilis](WP_041057629.1)的氨基酸序列匹配率最高,达到65%,相对分子质量为53.936 k Da,功能分析表明组分D1-3可能是一种新的抑菌蛋白。     

Fungicidal activity of N-(2-cyano-2-methoximinoacetyl)amino acids and their derivatives

More than 20 N-(2-cyano-2-methoximinoacetyl)amino acids and derivatives were synthesised and tested for antifungal activity against grape downy mildew, caused by Plasmopara viticola (de Bary) Berl. & de Toni, and rape downy mildew, Peronospora parasitica Fr. Two of the compounds containing a free carboxylic acid group, a moiety which has been shown to confer phloem mobility on compounds, showed high activity, especially against P. parasitica, in protectant tests. These results indicate that there is no incompatibility between the acid function and fungicidal activity. A number of the esters showed activity comparable with that of cymoxanil in the protectant tests, and the tert-butyl esters of the methionine derivative and its sulfone were more than ten times as active as the commercial compound.     

The antifeedant effect of neem seed kernel extract and azadirachtin on nymphs ofEyprepocnemis plorans

Acridids belonging to different species and families exhibit large differences in their response to neem components. In this context the antifeedant effect of a methanolic neem seed kernel extract (NSKE) and of azadirachtin (AZA) on fourth-instar nymphs of the acrididEyprepocnemis plorans Charpentier (Saltatoria:Acrididae) was investigated. Nymphs were offered either saccharose-impregnated filter paper disks or leaves of broad beans, treated with neem components. The amount of substrate consumed was determined by weighing the filter paper or by measuring the leaf area. On filter paper both NSKE and AZA were highly active down to the 10⁻⁴% treatment. In the leaf treatment, however, AZA was definitely more active than NSKE, with 100% deterrence at 10⁻⁴% and 10⁻²%, respectively. The methanolic NSKE was somewhat more active than the commercial preparation ‘Neemark’.     

Soil application of azadirachtin and 3-tigloyl-azadirachtol to control western flower thrips, Frankliniella occidentalis (Thysanoptera: Thripidae): translocation and persistence in bean plants

To study the systemic effects of active neem ingredients, the substrate of bean plants was treated with a 170 g kg(-1) azadirachtin (NeemAzal-U; Trifolio-M GmbH, Lahnau, Germany, registration pending). This product was used at a dose rate of 10 mg AZA (azadirachtin a) and 1.2 mg 3-tigloyl-azadirachtol (azadirachtin b) per treated bean plant. Afterwards, the translocation and persistence of AZA and 3-tigloyl-azadirachtol and the effects on western flower thrips, Frankliniella occidentalis (Pergande), were studied. Residues of AZA and 3-tigloyl-azadirachtol from substrates with different contents of organic matter [pure culture substrate (CS), CS-sand mixture] and from various plant parts were quantified by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The dissipation trends of AZA and 3-tigloyl-azadirachtol were similar within the same substrates. A slower decline of both active ingredients was measured with CS than with CS-sand mixture. Residue analysis of the bean plants showed that only small proportions of the initial amounts of AZA and 3-tigloyl-azadirachtol applied to the substrate were present in the plant (0.3-8.1%). Variable amounts of residues of the active components in relation to plant parts and time of analysis indicated a different translocation pattern for the two active ingredients. Higher residues of the active ingredients were found in roots and stems after neem application using CS, whereas higher residues were found in leaves after CS-sand mixture treatments. Mortality of F. occidentalis after NeemAzal-U soil applications reached up to 95% on CS-sand mixture, compared with 86% in CS.     

Structure–biological activity relationships in triterpenic saponins: the relative activity of protobassic acid and its derivatives against plant pathogenic fungi

BACKGROUND: Triterpenic saponins from Sapindus mukorossi Gaertn. and Diploknema butyracea JF Gmelin were evaluated for in vitro antifungal activity against four phytopathogenic fungi. The study of the structure–antifungal activity relationships of protobassic acid saponins was widened by including semi‐synthetic derivatives. RESULTS: Diploknema butyracea saponins exhibited significant antifungal activity against three fungi (ED₅₀ 230–455 µg mL^?1), whereas S. mukorossi saponin was effective against two fungi (ED₅₀ 181–407 µg mL^?1). The n‐butanol extract after preparative HPLC separation provided two saponins from D. butyracea saponin mixture: 3‐O‐[β‐D ‐glucopyarnosyl‐β‐D ‐glucopyranosyl]‐16‐α‐hydroxyprotobassic acid‐28‐O‐[arabinopyranosyl‐glucopyranosyl‐xylopyranosyl]‐arabinopyranoside (MI‐I), and 3‐O‐β‐D ‐glucopyranosyl‐glucopyranosyl‐glucopyranosyl‐16‐α‐hydroxyprotobassic acid‐28‐O‐[arabinopyranosyl‐xylopyranosyl‐arabinopyranosyl]‐apiofuranoside (MI‐III). The single saponin extracted from S. mukorossi saponin mixture was identified as 3‐O‐[O‐acetyl‐β‐D ‐xylopyranosyl‐β‐D ‐arabinopyranosyl‐β‐D ‐rhamnopyranosyl] hederagenin‐28‐O[β‐D ‐glucopyranosyl‐β‐D ‐glucopyranosyl‐β‐D ‐rhamnopyranosyl] ester (SM‐I). Monodesmosides resulting from the partial degradation of hederagenin and hydroxyprotobassic acid bisdesmosides exhibited significant reduction in antifungal effect. Further removal of sugar moiety yielded complete loss in activity. The antifungal activity of the triterpenic saponins was associated with their aglycone moieties, and esterification of the hydroxyl group led to change in antifungal activity. CONCLUSION: Sapindus mukorossi saponin, which is effective against Rhizoctonia bataticola (Taub.) Briton Jones and Sclerotium rolfsii Sacc., can be exploited for the development of a natural fungicide. A sugar moiety is a prerequisite for the antifungal activity of triterpenic saponin. Copyright © 2010 Society of Chemical Industry     

Antifungal Activity Toward Fusarium culmorum in Soluble Wheat Extracts

ABSTRACT This study investigated antifungal activity in soluble extracts from seed of a range of wheat cultivars differing in susceptibility to Fusarium head blight. Antifungal activity was assessed in terms of beta-D-glucuronidase (GUS) activity of a Fusarium culmorum GUS transformant using a sensitive laboratory assay. Significant antifungal activity was detected in seed extracts from WEK0609, CM 820036, and Arina. Initial characterization of the Arina seed extract indicated that it contained antifungal proteinaceous compounds. The Arina extract yielded two (60 and 80%) ammonium sulfate fractions containing inhibitory compounds. Gel filtration chromatography and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis of antifungal fractions showed that the antifungal activities detected in the Arina 60 and 80% ammonium sulfate fractions were associated with putative proteinaceous compounds with apparent molecular masses of approximately 60 and 28 kDa, respectively.     

Detection of viridiofungin A and other antifungal metabolites excreted by <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> active against different plant pathogens

Antibiosis is assumed to be an essential mechanism exerted by potential biocontrol agents (BCAs) of Trichoderma spp. Therefore, in the present study, we report for the first time on the elucidation and production of viridiofungin A (VFA) from T. harzianum isolate T23 cultures and investigate the antifungal potential of VFA and some other secondary metabolites purified from T. harzianum cultures against Fusarium moniliforme. The bioautography assay revealed that T. harzianum isolates T16 and T23 excreted several secondary metabolites with antifungal activity. Following isolation and purification of the antifungal zones, three fractions (F223, F323 and F423) from extracts of isolate T23 and two fractions (F416 and F516) from extracts of isolate T16 exhibited pronounced fungitoxic activity in the bioautography and antibiotic disk assays against Cladosporium spp. and F. moniliforme, respectively. The structure of the antifungal metabolite in fraction F323 was identified as viridiofungin A (VFA), the first report of production of VFA by isolate T23 of T. harzianum. Following cultivation of isolate T23 in PDB medium for 9 days, 94.6 mg l⁻¹ of VFA were determined. VFA and fraction F516 retarded the mycelial growth of F. moniliforme in the non-volatile phase assay by >90% for each 250 μg ml⁻¹ 7 days post-inoculation (dpi). While VFA and fraction F416 showed both volatile and non-volatile effects, fraction F516 seemed to exhibit mainly non-volatile activity. Microscopic examination revealed that hyphae of F. moniliforme grown on VFA-amended medium were less branched and appeared thicker than untreated hyphae. Furthermore, in the presence of VFA, formation of chlamydospores by F. moniliforme was increased. Finally, the antifungal spectrum of VFA towards various important plant pathogens was evaluated. Germination of propagules of a variety of fungal pathogens in vitro was differentially inhibited by VFA. While in the presence of 100 μg ml⁻¹ VFA conidial germination of V. dahliae was completely inhibited, a slightly higher concentration (150 μg ml⁻¹) of the inhibitor was required to suppress germination of Phytophthora infestans sporangia or sclerotia of Sclerotinia sclerotiorum. Contrary to several reports in the literature, VFA proved to be fungistatic rather than fungicidal. However, neither VFA nor the other Trichoderma metabolites, such as 6PAP, F416 and F516, exhibited any antibacterial activity against Gram-positive and Gram-negative bacteria.     

The antifungal activity of alkyl benzimidazol-2-ylcarbamates and related compounds

The fungistatic activity against Penicillium digitatum and Diplodia natalensis decreased slightly in ascending a homologous series of alkyl esters of benzimidazol-2-ylcarbamic acid from the methyl ester (carbendazim) to the pentyl ester; the hexyl and octyl esters were inactive. 2-(Acylamino)benzimidazoles were slightly less active than the analogous alkyl benzimidazol-2-ylcarbamates. Introduction of a methylene bridge between the benzimidazole ring and the 2-methoxycarbonylamino group abolished antifungal activity. Methylation of either the carbamate nitrogen or an imidazole nitrogen of carbendazim produced inactive compounds. Replacement of the benzimidazole ring of carbendazim with various other ring systems was accompanied by marked reduction in antifungal activity.