Purification, characterization and molecular cloning of a monocot mannose-binding lectin from Remusatia vivipara with nematicidal activity

A mannose-binding lectin (RVL) was purified from the tubers of Remusatia vivipara, a monocot plant by single-step affinity chromatography on asialofetuin-Sepharose 4B. RVL agglutinated only rabbit erythrocytes and was inhibited by mucin, asialomucin, asialofetuin and thyroglobulin. Lectin activity was stable up to 80A degrees C and under wide range of pH (2.0-9.3). SDS-PAGE and gel filtration results showed the lectin is a homotetramer of Mr 49.5 kDa, but MALDI analysis showed two distinct peaks corresponding to subunit mass of 12 kDa and 12.7 kDa. Also the N-terminal sequencing gave two different sequences indicating presence of two polypeptide chains. Cloning of RVL gene indicated posttranslational cleavage of RVL precursor into two mature polypeptides of 116 and 117 amino-acid residues. Dynamic light scattering (DLS) and gel filtration studies together confirmed the homogeneity of the purified lectin and supported RVL as a dimer with Mr 49.5 kDa derived from single polypeptide precursor of 233 amino acids. Purified RVL exerts potent nematicidal activity on Meloidogyne incognita, a root knot nematode. Fluorescent confocal microscopic studies demonstrated the binding of RVL to specific regions of the alimentary-tract and exhibited a potent toxic effect on M. incognita. RVL-mucin complex failed to interact with the gut confirming the receptor mediated lectin interaction. Very high mortality (88%) rate was observed at lectin concentration as low as 30 A mu g/ml, suggesting its potential application in the development of nematode resistant transgenic-crops.

Nematicidal activity of Solanum nigrum and S. sisymbriifolium extracts against the root-lesion nematode Pratylenchus goodeyi and its effects on infection and gene expression

The control of Pratylenchus goodeyi a common nematode parasite of banana crop in Madeira Island can benefit from searching for natural nematicides through plants extracts. With this aim we submitted Solanum nigrum and S. sisymbriifolium dried plants to a sequential extraction in the solvent sequence of dichloromethane, acetone, ethanol and water, and to na aqueous extraction of the fresh and dried plants. Analyses with the extracts at several concentrations were used to assess mobility and mortality on P. goodeyi. Results showed that the water extract and aqueous extracts from both plants at a concentration of 10 mg/mL affected nematode mobility and caused mortality but the acetone extract from S. nigrum was the most efficient, causing 100% mortality whereas dichloromethane had no effect on P. goodeyi. Determination of the lipophilic and phenolic compounds present in the two most effective Solanum extracts (acetone and water) and in dichloromethane extract revealed that some of these compounds had nematicidal activity. S. nigrum acetone extract (10 mg/mL) was used to find out the nematicidal potential following the effect at gene expression level and nematode behaviour. Genes coding for calreticulin and beta-1,4- endoglucanase related to parasitism and translocon-associated protein putatively connected to stress were obtained and its relative expression assessed in nematodes exposed to the extract. Results revealed that expression of Pg-CRT decreased showing to influence the infection, Pg-ENG remained steady and Pg-TRAPδ was induced over time exposure. Biological assays showed that P. goodeyi mobility and ability to infect the banana roots were affected as a decrease in the number of nematodes that reached the roots was obtained with the increased exposure time to the extract being implicated in the infection success. The information obtained from this thesis showed that S. nigrum has potential to be used for the development of a new control strategy against plant-parasitic nematodes.

Molecular and biotechnological approaches to essential oils production in thymus caespititius

Tese de doutoramento, Biologia (Biotecnologia), Universidade de Lisboa, Faculdade de Ciências, 2014

Suppression of root-knot nematode through innovative mustard biofumigation

The nematicidal activity of mustard plant against hatching, migration and mortality of the root-knot nematode Meloidogyne javanica was investigated. In vitro test confirmed that mixing the sandy clay soil mixture with mustard as 4% as a biofumigant significantly reduce the percentage of egg hatching at all different incubation periods 24, 48, 72, 96 and 168 h, compared to control treatment (un-amended mixture soil and eggs in free water). Results indicate that the percentage of egg hatching reduction was 88.5, 90, 81.4, 74 and 69.4%, respectively. Mustard mixed with soil as a biofumigant led to high percentage of larval mortality at the different intervals periods in vitro. The percentage of larval mortality was 94, 100, 90.5, 90.5, and 79.4%, respectively compared to control. Laboratory results confirmed that the highest reduction in egg hatching and larval mortality was obtained after incubation period for 48 h. In vivo experiment reveals that the incorporation of the soil pots with mustard at all different doses used 3, 5% (48 h before nematode inoculation, or soil infestation with nematode), and 5% (one week before nematode inoculation or 7% of soil weight) significantly reduces all the nematode parameters compared to plant treated nematode alone. All nematode parameters i.e. the number of galls per root system, gall index, number of egg masses per root system, as well as number of juveniles per 250g soil showed high reduction with mixing the soil pots with mustard at 5% (one week before nematode inoculation), followed by the same treatment for 48h before nematode inoculation. Mustard application, one week before nematode inoculation, reduced the nematode parameters by 97, 64, 97, and 93%, respectively, compared to control. The percent of chemical components i.e. total sugars, total amino acids and total phenols were markedly enhanced compared to positive and negative control. The highest percentage was obtained with mustard at 5% one week before nematode inoculation by 68.7, 57.3 and 45%, respectively. Finally, we have to conclude that this modified technology is an innovative and can be used efficiently to control Root-knot nematode under organic agriculture and Global GAP agricultural systems instead of these carcinogenic nematicides.

First report on Meloidogyne chitwoodi hatching inhibition activity of essential oils and essential oils fractions

The Columbia root-knot nematode (CRKN), Meloidogyne chitwoodi, is an EPPO A2 type quarantine pest since 1998. This nematode causes severe damage in economically important crops such as potato and tomato, making agricultural products unacceptable for the fresh market and food processing. Commonly used nematicidal synthetic chemicals are often environmentally unsafe. Essential oils (EOs) may constitute safer alternatives against RKN. EOs, isolated from 56 plant samples, were tested against CRKN hatching, in direct contact bioassays. Some of the most successful EOs were fractionated and the hydrocarbon molecules (HM) and oxygen-containing molecules (OCM) fractions tested separately. 24 EOs displayed very strong hatching inhibitions (≥90 %) at 2 µL mL−1 and were further tested at lower concentrations. Dysphaniaambrosioides, Filipendula ulmaria, Ruta graveolens, Satureja montana and Thymbra capitata EOs revealed the lowest EC50 values (<0.15 µL mL−1). The main compounds of these EOs, namely 2-undecanone, ascaridol, carvacrol, isoascaridol, methyl salicylate, p-cymene and/or γ-terpinene, were putatively considered responsible for CRKN hatching inhibition. S. montana and T. capitata OCM fractions showed hatching inhibitions higher than HM fractions. The comparison of EO and corresponding fractions EC50 values suggests interactions between OCM and HM fractions against CRKN hatching. These species EOs showed to be potential environmentally friendly CRKN hatching inhibitors; nonetheless, bioactivity should be considered globally, since its HM and OCM fractions may contribute, diversely, to the full anti-hatching activity.