Nematotoxic Activity Research Articles

Green fabrication of titanium dioxide nanoparticles via Syzygium cumini leaves extract: characterizations, photocatalytic activity and nematicidal evaluation

ABSTRACT The most significant plant parasitic nematode in terms of economic impact is root-knot nematode (Meloidogyne incognita), which severely harms essential crops used for agriculture. This study investigated green fabrication of titanium dioxide nanoparticles (TiO2 NPs) utilizing Syzygium cumini (SC) leaf extract and evaluated its nematotoxic activity against M. incognita in vitro and in pots. During experiments, second-stage juveniles (J2s) and egg masses of M. incognita were treated with different concentrations (125, 250, 375 and 500 ppm) of SC-TiO2 NPs. After 48 h and five days of exposure, respectively, lowest J2s mortality and per cent inhibition in egg hatching was discovered at 125 ppm, and highest J2s mortality and maximum per cent inhibition in egg hatching determined at 500 ppm. The treatment of SC TiO2 NPs considerably reduced M. incognita infection and enhanced carrot plants' growth and physiological characteristics. The characterization of green synthesized SC-TiO2 NPs made by X-ray diffraction, Photoluminescence, FTIR, SEM, UV-Visible Diffuse Reflectance Spectrophotometer, TEM, and photocatalytic activity also examined. In conclusion, it can be said that SC-TiO2 NPs possess nematode-toxic potential against M. incognita and are used as substitutes for high-risk chemical nematicides without causing any phytotoxicity.

Crude Aqueous Root Extracts of Solanum spp. Show Nematotoxic Activity Against Meloidogyne incognita

Brazilian crops are strongly affected by the root-knot nematode Meloidogyne incognita, which compromises food production and causes serious economic losses around the world. Synthetic nematicides are still some of the main control strategies for this pathogen, despite the high risk to human health and the environment. Therefore, sustainable biotechnology represents a viable alternative for nematode control. Plants of the Solanaceae family are present in several biomes and are a source of secondary metabolites of various chemical classes. In this context, the nematotoxic effect of crude aqueous root extracts (CAREs) of three Solanum species was investigated: Solanum americanum, S. subinerme and S. lycocarpum. CARE showed high nematicidal activity (94.6±9.2% mortality, p ≤ 0.001) against second-stage juveniles (J2) of M. incognita, even after heating to 50 °C. These CAREs also inhibited hatching of juveniles (98.4±0.7% of mortality, p ≤ 0.001) in addition to presenting a mean lethal dose of 336 μg ml-1. No toxic activity was observed against non-target organisms, such as fungi (Trichoderma harzianum and Trichoderma asperellum), yeast (Candida maltosa), nitrogen-fixing bacteria (Bradyrhizobium diazoefficiaens, B. japonicum), two strains of Azospirillum brasilense, AbV 5 and AbV 6 and Bacillus [2538 Ba]. Bioassay performed in a greenhouse demonstrated that CAREs of these species were effective in reducing the number of egg masses and galls (> 85%, p ≤ 0.0001), and did not show a phytotoxic effect on soybean seedlings. Our findings indicate that CAREs from plants of the Solanaceae family, especially S. lycocarpum, demonstrated nematotoxic activity on J2 and inhibited the hatching of Meloidogyne incognita eggs, representing an ecologically appropriate tool to control nematode infection in crops worldwide.

Structure-function relationship of a novel fucoside-binding fruiting body lectin from Coprinopsis cinerea exhibiting nematotoxic activity.

Lectins are non-immunoglobulin-type proteins that bind to specific carbohydrate epitopes and play important roles in intra- and inter-organismic interactions. Here, we describe a novel fucose-specific lectin, termed CML1, which we identified from fruiting body extracts of Coprinopsis cinerea. For further characterization, the coding sequence for CML1 was cloned and heterologously expressed in Escherichia coli. Feeding of CML1-producing bacteria inhibited larval development of the bacterivorous nematode Caenorhabditis tropicalis, but not of C. elegans. The crystal structure of the recombinant protein in its apo-form and in complex with H type I or Lewis A blood group antigens was determined by X-ray crystallography. The protein folds as a sandwich of 2 antiparallel β-sheets and forms hexamers resulting from a trimer of dimers. The hexameric arrangement was confirmed by small-angle X-ray scattering (SAXS). One carbohydrate-binding site per protomer was found at the dimer interface with both protomers contributing to ligand binding, resulting in a hexavalent lectin. In terms of lectin activity of recombinant CML1, substitution of the carbohydrate-interacting residues His54, Asn55, Trp94, and Arg114 by Ala abolished carbohydrate-binding and nematotoxicity. Although no similarities to any characterized lectin were found, sequence alignments identified many non-characterized agaricomycete proteins. These results suggest that CML1 is the founding member of a novel family of fucoside-binding lectins involved in the defense of agaricomycete fruiting bodies against predation by fungivorous nematodes.

Tomato Divinyl Ether-Biosynthesis Pathway Is Implicated in Modulating of Root-Knot Nematode Meloidogyne javanica's Parasitic Ability.

The role of the 9-lipoxygenase (9-LOX)-derived oxylipins in plant defense is mainly known in solanaceous plants. In this work, we identify the functional role of the tomato divinyl ether synthase (LeDES) branch, which exclusively converts 9-hydroperoxides to the 9-divinyl ethers (DVEs) colneleic acid (CA) and colnelenic acid (CnA), during infection by the root-knot nematode Meloidogyne javanica. Analysis of LeDES expression in roots indicated a concurrent response to nematode infection, demonstrating a sharp increase in expression during the molting of third/fourth-stage juveniles, 15 days after inoculation. Spatiotemporal expression analysis using an LeDES promoter:GUS tomato line showed high GUS activity associated with the developing gall; however the GUS signal became more constricted as infection progressed to the mature nematode feeding sites, and eventually disappeared. Wounding did not activate the LeDES promoter, but auxins and methyl salicylate triggered LeDES expression, indicating a hormone-mediated function of DVEs. Heterologous expression of LeDES in Arabidopsis thaliana rendered the plants more resistant to nematode infection and resulted in a significant reduction in third/fourth-stage juveniles and adult females as compared to a vector control and the wild type. To further evaluate the nematotoxic activity of the DVEs CA and CnA, recombinant yeast that catalyzes the formation of CA and CnA from 9-hydroperoxides was generated. Transgenic yeast accumulating CnA was tested for its impact on M. javanica juveniles, indicating a decrease in second-stage juvenile motility. Taken together, our results suggest an important role for LeDES as a determinant in the defense response during M. javanica parasitism, and indicate two functional modes: directly via DVE motility inhibition effect and through signal molecule-mediated defense reactions to nematodes that depend on methyl salicylate.

Identification, heterologous production and bioactivity of lentinulin A and dendrothelin A, two natural variants of backbone N-methylated peptide macrocycle omphalotin A

Backbone N-methylation and macrocyclization improve the pharmacological properties of peptides by enhancing their proteolytic stability, membrane permeability and target selectivity. Borosins are backbone N-methylated peptide macrocycles derived from a precursor protein which contains a peptide α-N-methyltransferase domain autocatalytically modifying the core peptide located at its C-terminus. Founding members of borosins are the omphalotins from the mushroom Omphalotus olearius (omphalotins A-I) with nine out of 12 L-amino acids being backbone N-methylated. The omphalotin biosynthetic gene cluster codes for the precursor protein OphMA, the protease prolyloligopeptidase OphP and other proteins that are likely to be involved in other post-translational modifications of the peptide. Mining of available fungal genome sequences revealed the existence of highly homologous gene clusters in the basidiomycetes Lentinula edodes and Dendrothele bispora. The respective borosins, referred to as lentinulins and dendrothelins are naturally produced by L. edodes and D. bispora as shown by analysis of respective mycelial extracts. We produced all three homologous peptide natural products by coexpression of OphMA hybrid proteins and OphP in the yeast Pichia pastoris. The recombinant peptides differ in their nematotoxic activity against the plant pathogen Meloidogyne incognita. Our findings pave the way for the production of borosin peptide natural products and their potential application as novel biopharmaceuticals and biopesticides.

Transcriptomic analysis of Bursaphelenchus xylophilus treated by a potential phytonematicide, punicalagin.

Punicalagin showed significant nematotoxic activity against pine wood nematode (PWN), Bursaphelenchus xylophilus, in the authors’ previous research. The authors performed high-throughput transcriptomic sequencing of punicalagin-treated nematodes to generate clues for its nematotoxic mechanism of action. The authors identified 2,575 differentially expressed genes, 1,428 of which were up-regulated and 1,147 down-regulated. Based on a comprehensive functional in silico analysis, the authors speculate that PWN may respond to the stimulus of punicalagin through phagosome, endocytosis, peroxisome and MAPK signaling pathways. In addition, punicalagin could greatly affect PWN energy metabolism including oxidative phosphorylation. The genes encoding twitchin and a nematode cuticular collagen could be crucial regulation targets of punicalagin, which might contribute to its nematotoxic activity against PWN.

Nematotoxic activity of essential oils from Monarda species

Bioactivity of essential oils (EOs) from Monarda species has never been investigated on phytoparasitic nematodes. In this study, the EOs from two Italian ecotypes of Monarda didyma and M. fistulosa and their main compounds, carvacrol, γ-terpinene, o-cymene, and thymol, were evaluated for their in vitro activity on the infective stages of phytoparasitic nematodes Meloidogyne incognita and Pratylenchus vulnus, as well as on M. incognita egg hatch. Soil treatments with the two EOs were also investigated for their suppressiveness on M. incognita on tomato. Both EOs were strongly active on M. incognita juveniles, as a only 1.0 μL mL−1 LC50 value was evaluated after a 24-h exposure to both EOs, whereas a lower activity was recorded on P. vulnus (15.7 and 12.5 μL mL−1 LC50 values for M. didyma and M. fistulosa EOs, respectively). Among the EOs’ main compounds, carvacrol was highly active also at a short exposure in low concentrations, whereas γ-terpinene and thymol were much less active on both nematode species and o-cymene showed a discrete activity on P. vulnus only at the highest concentration. Hatch percent of M. incognita eggs treated with M. didyma and M. fistulosa EOs was always significantly lower than in water or in Tween 20 and Oxamyl solutions. In the experiment in soil, the multiplication of M. incognita and gall formation on tomato roots was significantly reduced by soil treatments with both EOs. The strong nematicidal activity of both Monarda EOs may suggest them as potential sources of new sustainable nematicidal products.

Toxicidad in vitro de Micromicetos del Trópico Mexicano en Juveniles Infectivos de Meloidogyne incognita

Filtrados de cultivos y extractos de micelio (acetato de etilo y metanol) de nueve cepas fúngicas Mexicanas tropicales se evaluaron en condiciones <em>in vitro</em> contra juveniles de segundo estadio (J<sub>2</sub>)de <em>Meloidogyne incognita</em>. Los micromicetos <em>Acremonium kiliense</em> TA31, <em>Aspergillus</em> sp. 2XA5, <em>Gliocladium</em> sp. MR41, <em>Selenosporella</em> sp. MR26, <em>Stagonospora</em> sp. TA34 y cuatro cepas no identificadas (TA13, 2TA6, 2TA7 y 2XA7) se cultivaron en medio Czapeck-Dox durante 14 días y la masa micelial se separó por filtración para la extracción de metabolitos. <em>Aspergillus</em> sp. 2XA5 y <em>Selenosporella</em> sp. MR26 mostraron la mayor actividad nematóxica, tanto en filtrados de cultivo (100% de mortalidad) como en extractos metanólicos (> 90% de mortalidad). La CE<sub>50</sub> más baja (0.08 mg mL<sup>-1</sup>) se obtuvo con el extracto de metanol de la cepa 2XA7 no identificada. Los resultados obtenidos indican que la micobiota tropical tiene potencial como agentes de control biológico de nematodos fitopatógenos.

Systemic acquired resistance activation in solanaceous crops as a management strategy against root-knot nematodes.

Activators of systemic acquired resistance (SAR), such as salicylic acid (SA) and its synthetic functional analogues benzo(1,2,3)thiadiazole-7-carbothionic acid-S-methyl ester (BTH) and 2,6-dichloroisonicotinic acid (INA), were tested on tomato, eggplant and pepper for the control of the root-knot nematode Meloidogyne incognita. Effects on plant fitness, nematode reproduction and root galling were screened in relation to different methods of application, to different applied dosages of chemicals and to different plant growth stages. Dosages applied to plants were in relation to plant weights. These chemicals were also tested for their possible nematotoxic activity in vitro. Soil drenches of SA and INA and root dip application of SA and BTH inhibited nematode reproduction, at specific dosage ranges, without affecting plant growth. SA and INA were able to reduce root galling as well. Foliar sprays of both SA and BTH were ineffective against nematode attacks. Plants tolerated SA more than the other chemicals tested. BTH at elevated concentrations increased the mortality of nematode juveniles and reduced egg hatching in vitro. SAR activators at concentrations suitable for different plant growth stages and applied by the proper method can possibly be included in IPM programmes for nematode management.

Nematotoxic activity from essential oils and their hydrocarbons and oxygen-containing molecules fractions against the pinewood nematode Bursaphelenchus xylophilus

Nematotoxic activity from essential oils and their hydrocarbons and oxygen-containing molecules fractions against the pinewood nematode Bursaphelenchus xylophilus

Bioactivity against Bursaphelenchus xylophilus: Nematotoxics from essential oils, essential oils fractions and decoction waters

The Portuguese pine forest has become dangerously threatened by pine wilt disease (PWD), caused by the pinewood nematode (PWN), Bursaphelenchus xylophilus. Synthetic chemicals are the most common pesticides used against phytoparasitic nematodes but its use has negative ecological impacts. Phytochemicals may prove to be environmentally friendly alternatives. Essential oils (EOs) and decoction waters, isolated from 84 plant samples, were tested against B. xylophilus, in direct contact assays. Some successful EOs were fractionated and the fractions containing hydrocarbons or oxygen-containing molecules tested separately. Twenty EOs showed corrected mortalities ⩾96% at 2 μL/mL. These were further tested at lower concentrations. Ruta graveolens, Satureja montana and Thymbra capitata EOs showed lethal concentrations (LC100)<0.4μL/mL. Oxygen-containing molecules fractions showing corrected mortality ⩾96% did not always show LC100 values similar to the corresponding EOs, suggesting additive and/or synergistic relationships among fractions. Nine decoction waters (remaining hydrodistillation waters) revealed 100% mortality at a minimum concentration of 12.5μL/mL. R. graveolens, S. montana and T. capitata EOs are potential environmentally friendly alternatives for B. xylophilus control given their high nematotoxic properties. Nematotoxic activity of an EO should be taken in its entirety, as its different components may contribute, in distinct ways, to the overall EO activity.

Antimicrobial and nematicidal screening of anamorphic fungi isolated from plant debris of tropical areas in Mexico

Forty seven fungal strains were isolated from plant debris in the tropical regions of Mexico, where fifteen of them were identified to species and twenty two to genus level. All isolates were grown in fermented rice and their EtOAc extracts screened against ten targets, four bacteria (Bacillus subtilis,Erwinia carotovora, Staphylococcus aureus and Xanthomonas campestris), the yeast Candida albicans, three phytopathogenic fungi (Alternaria tagetica, Colletotrichum gloeosporioides andFusarium oxysporum), the Oomycete Pythium aphanidermatum and the nematode Meloidogyne incognita. Antimicrobial activity was detected in 18 isolates against at least one of the target strains tested. Seven of these isolates with broad spectrum activity, which were defatted and their minimum inhibitory concentrations (MICs) were determined by microdilution assay. The greatest antagonistic action was produced by Cylindrium elongatum with broad spectrum activity while Corynespora cassiicola and Memnoniella sp. MR33 showed moderate antimicrobial properties. On other hand, in vitro nematotoxic activity was clearly detected only in Selenosporella sp. GH26 with 91 (LD50) and 147 µg/ml (LD90). This is the first report on the isolation and biological evaluation of anamorphic fungi from some Mexican tropical regions, demonstrating their potential as a source of biologically active natural metabolites for use in future applications in agriculture or pharmacy. Key words: Anamorphic fungi, antimicrobial, Corynespora, Cylindrium, fungal extracts, nematicide,Selenosporella, Yucatan peninsula.

The investigation of nematocidal activity inStenotrophomonas maltophiliaG2 and characterization of a novel virulence serine protease

The Gram-negative bacterium Stenotrophomonas maltophilia G2 was isolated from a soil sample and was found to have high nematotoxic activity against a free-living nematode, Panagrellus redivivus, and a plant-parasitic nematode, Bursaphelenchus xylophilus. The analysis of virulence factors revealed that although the small molecular metabolites participated in nematode killing, the crude extracellular protein extract from the bacterial culture supernatant contributed significantly to its nematocidal activity. An extracellular protease was purified by chromatography, and its effects on degrading purified nematode cuticle and killing living nematodes were confirmed experimentally. Characterization of this purified protease revealed that the application of phenylmethylsulphonyl fluoride, an inhibitor of serine proteases, could completely abolish its proteolytic activity. The results from N-terminal amino acid sequencing showed no similarity with any known serine protease in S. maltophilia, suggesting a novel virulence serine protease was obtained. Our study is the first to show the nematocidal activity of S. maltophilia, and we identified a novel serine protease as an important pathogenicity factor.

Bacillus sp. B16 kills nematodes with a serine protease identified as a pathogenic factor

An endospore-forming bacterium, strain B16, was isolated from a soil sample and identified as a Bacillus sp. The strain presented remarkable nematotoxic activity against nematode Panagrellus redivivus. The crude extracellular protein extract from culture supernatant of the bacteria killed about 80% of the tested nematodes within 24 h, suggesting the involvement of extracellular proteases. A homogeneous extracellular protease was purified by chromatography, and the hypothesis of proteinaceous pathogeny in the infection of B16 strain was confirmed by the experiments of killing living nematodes and by the degradation of purified nematode cuticle when treated with the homogenous protease. The gene for the virulence protease was cloned, and the nucleotide sequence was determined. The deduced amino acid sequence showed significant similarity with subtilisin BPN' but low homology with the other cuticle-degrading proteases previously reported in fungi. Characterization of the purified protease revealed the molecular mass of 28 kDa and the optimum activity at pH 10, 50 degrees C. The purified protease can hydrolyze several native proteinaceous substrates, including collagen and nematode cuticle. To our knowledge, this is the first report of a serine protease from a Bacillus genus of bacteria that serves as a pathogenic factor against nematodes, an important step in understanding the relationship between bacterial pathogen and host and in improving the nematocidal activity in biological control.

Bacillus nematocida sp. nov., a novel bacterial strain with nematotoxic activity isolated from soil in Yunnan, China

An endospore-forming bacterium, designated strain B-16 T, was isolated from a forest soil sample in Yunnan, China. The isolate presented remarkable nematotoxic activity against nematode Panagrellus redivivus. The organism was strictly aerobic, motile, spore forming and rod shaped, catalase- and oxidase-positive. The predominant isoprenoid quinone was menaquinone 7 (MK-7). The major cellular fatty acid profiles were anteiso-C 15:0 (48.67%), iso-C 15:0 (13.45%), C 16:0 (9.06%) and anteiso-C 17:0 (8.29%). The DNA G+C content was 46%. Phylogenetic analyses based on 16S rDNA sequence revealed that isolate belongs to the genus Bacillus. Strain B-16 T exhibited high 16S rDNA similarity with its closest neighbors Bacillus vallismortis (99.79%), B. subtilis (99.43%), B. atrophaeus (99.43%), B. amyloliquefaciens (99.36%), B. licheniformis (98.0%) and less than 97.0% with all the other relative type strains in the genus Bacillus. The phenotypic and genotypic characteristics and DNA–DNA relatedness data indicate that strain B-16 T should be distinguished from all the relative species of genus Bacillus. Therefore, on the basis of the polyphasic taxonomic data presented, a new species of the genus Bacillus, B. nematocida, with the type strain B-16 T (=CGMCC 1128 T) is proposed. The GenBank accession number for the sequence reported in this paper is AY820954.

Improving the pathogenicity of a nematode-trapping fungus by genetic engineering of a subtilisin with nematotoxic activity.

Nematophagous fungi are soil-living fungi that are used as biological control agents of plant and animal parasitic nematodes. Their potential could be improved by genetic engineering, but the lack of information about the molecular background of the infection has precluded this development. In this paper we report that a subtilisin-like extracellular serine protease designated PII is an important pathogenicity factor in the common nematode-trapping fungus Arthrobotrys oligospora. The transcript of PII was not detected during the early stages of infection (adhesion and penetration), but high levels were expressed concurrent with the killing and colonization of the nematode. Disruption of the PII gene by homologous recombination had a limited effect on the pathogenicity of the fungus. However, mutants containing additional copies of the PII gene developed a higher number of infection structures and had an increased speed of capturing and killing nematodes compared to the wild type. The paralyzing activity of PII was verified by demonstrating that a heterologous-produced PII (in Aspergillus niger) had a nematotoxic activity when added to free-living nematodes. The toxic activity of PII was significantly higher than that of other commercially available serine proteases. This is the first report showing that genetic engineering can be used to improve the pathogenicity of a nematode-trapping fungus. In the future it should be possible to express recombinant subtilisins with nematicidal activity in other organisms that are present in the habitat of parasitic nematodes (e.g., host plant).

Nematocidal Properties of Carboxylic-Acids and Derivatives

During research on nematocidal toxins produced by microorganisms, a metabolite has been found active and identified as a volatile fatty acid (monocarboxylic), namely acetic acid. That volatile fatty acid showed selective nematotoxic activity against some plant-parasitic nematodes, but linked to pH and with recovery effect. To improve its efficacy, a structure/activity study on some derivatives has been performed with biological tests on different nematode species. Adding a second carboxylic function and a double bond in the volatile fatty acid hydrocarbonic chain provided unsaturated dicarboxylic acids; these compounds exhibited a significant selective nematocidal activity in considerably shorter periods of time than the monocarboxylic acids, at lower concentrations, in a broad range of pH and without reversible effect of juvenile paralysis. The methyl esters of those dicarboxylic acids and cyclic derivatives exhibited toxicological activity without pH dependency. The importance of the spatial configuration of those molecules which triggered the appearance of significant nematocidal properties is discussed.

Acetic Acid: a Selective Nematicidal Metabolite From Culture Filtrates of Paecilomyces Lilacinus (Thom) Samson and Trichoderma Longibrachiatum Rifai

The nematotoxic activity, efficient against plant-parasitic species, found in the culture filtrates of two Hyphomycetes: Paecilomyces lilacinus (nematophagous fungus) and Trichoderma longibrachiatum was characterized. Purification was performed by distillation and liquid chromatography and identification by gas-chromatography coupled with a DCI system (dynamic headspace). The active substance has been identified as acetic acid which, produced abundantly during fungal growth in liquid medium (up to 0.044 mols of CH 3 COOH per culture filtrate litre), inhibited movement of infesting juvenile nematodes. These results were confirmed by comparative studies using the commercial molecule as for the 1 H NMR as the biological tests. The toxic activity appeared only when the carboxylic function was in its undissociated form, hence the real interest of pH when studying this nematicidal activity. A study of the literature confirmed the selective permeability of different cuticle zones of nematodes for non-charged substances (non-polar).

Preparation of [22,23‐3H2]‐dihydroavermectin B1a of high specific activity

Abstract[22,23‐3H2]‐Dihydroavermectin B1a of 2.13 TBq/mmo1 (57.7 Ci/mmol) specific activity was derived from avermectin B1a by selective tritiation using Wilkinson's homogeneous catalyst. Data on binding affinity and nematotoxic activity of the title compound are reported.