Nematoxic effect of essential oils and their fractions against the pinewood nematode, Bursaphelenchus xylophilus

The pinewood nematode (PWN) Bursaphelenchus xylophilus is a highly pathogenic plant parasite that greatly affects pine forests. In Portugal, the most affected species is Pinus pinaster Aiton. Despite great efforts, since its fist detection in 1999, the PWN has spread through the country, including Madeira Island, having been recently detected in Spain [1,2]. Containing this pest is of the utmost importance for European pine forest safeguard. Since most synthetic chemicals used to control phytoparasites are toxic to humans and animals, and can accumulate in the soil and in food plants [3], in the present work, the nematoxic potential of over 80 essential oils (EOs), isolated from the Portuguese flora, were assessed against the PWN. EOs were isolated by hydrodistillation and analysed by GC and GC-MS [3]. EOs hydrocarbon and oxygen-containing fractions were obtained as in [4]. Direct-contact assays, adapted from [3], were performed by adding EOs/methanol stock-solutions to 50-100 mixed-stage PWN suspensions. After 24h in darkness, dead and live nematodes were counted under an inverted microscope. Assays were repeated at least 10 times in two series. Mortalities ≥96% were obtained with 2μL/mL of the EOs isolated from Cymbopogon citratus, Eucalyptus citriodora, Mentha arvensis, Origanum virens, Origanum vulgare, Ruta graveolens, Satureja montana, Syzygium aromaticum, Thymbra capitata, Thymus caespititius (carvacrol and/or thymol-rich), Thymus vulgaris and Thymus zygis. These EOs were further tested at 1, 0.5 and 0.25μL/mL. Minimum lethal concentrations (LC100) <0.4μL/mL, were obtained for the 2-undecanone-rich R. graveolens EO and the carvacrol and γ-terpinene-rich S. montana and T. capitata EOs. Assays with EO fractions revealed that the monoterpene-rich nematoxic EOs control PWN through their combined hydrocarbon and oxygen-containing fractions through additive and/or synergic relations. As complex mixtures of active components, EOs may prove to be effective nematoxic age nts

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

In vitro co-culture of Solanum tuberosum hairy roots with Meloidogyne chitwoodi: structure, growth and production of volatiles

Meloidogyne spp., commonly known as root- knot nematodes (RKNs), are economically important plant sedentary endoparasites that cause galls on susceptible hosts. The Columbia root-knot nematode (CRKN), M. chitwoodi, is a quarantine A2 type pest by the European and Mediterranean Plant Protection Organization since 1998. This nematode has been found associated with economi- cally important crops such as potato and tomato, causing severe damage and making the agricultural products unac- ceptable for the fresh market and food processing. In vitro co-culture of host and parasite offers an advantageous experimental system for studying plant-RKN interactions. The structure, growth and production of volatiles of Sola- num tuberosum hairy roots (HR) and of S. tuberosum HR/ CRKN co-cultures were compared. HR were induced by inoculation of aseptic potato tuber segments with Rhizo- bium rhizogenes. Co-cultures were initiated by inoculating HR with sterilized CRKN eggs. Infection with CRKN induced the RKN symptomatology in the HR and several nematode life stages were observed by light and scanning electron microscopy. Potato HR and HR/CRKN co-culturesexhibited similar growth patterns, evaluated by measuring fresh and dry weight and by the dissimilation method. Volatiles, isolated by distillation–extraction and analyzed by gas chromatography (GC) and gas chromatography coupled to mass spectrometry, revealed that palmitic acid (37–52 %), n–pentadecanal (10–16 %) and linoleic acid (2–16 %) were the main constitutive components of S. tu- berosum HR, and of the HR/CRKN co-cultures (24–44, 8–22 and 4–18 %, respectively). S. tuberosum HR/CRKN co-cultures can be considered a suitable biotechnological tool to study RKN infection mechanism by mimicking what occurs under field conditions

Bias in the prediction of genetic gain due to mass and half-sib selection in random mating populations

The prediction of gains from selection allows the comparison of breeding methods and selection strategies, although these estimates may be biased. The objective of this study was to investigate the extent of such bias in predicting genetic gain. For this, we simulated 10 cycles of a hypothetical breeding program that involved seven traits, three population classes, three experimental conditions and two breeding methods (mass and half-sib selection). Each combination of trait, population, heritability, method and cycle was repeated 10 times. The predicted gains were biased, even when the genetic parameters were estimated without error. Gain from selection in both genders is twice the gain from selection in a single gender only in the absence of dominance. The use of genotypic variance or broad sense heritability in the predictions represented an additional source of bias. Predictions based on additive variance and narrow sense heritability were equivalent, as were predictions based on genotypic variance and broad sense heritability. The predictions based on mass and family selection were suitable for comparing selection strategies, whereas those based on selection within progenies showed the largest bias and lower association with the realized gain

In vitro co-cultures of Pinus pinaster with Bursaphelenchus xylophilus: a biotechnological approach to study pine wilt disease

Abstract Main conclusion Co-cultures of Pinus pinaster with Bursaphelenchus xylophilus were established as a biotechnological tool to evaluate the effect of nematotoxics addition in a host/parasite culture system. The pinewood nematode (PWN), Bursaphelenchus xylophilus, the causal agent of pine wilt disease (PWD), was detected for the first time in Europe in 1999 spreading throughout the pine forests in Portugal and recently in Spain. Plant in vitro cultures may be a useful experimental system to investigate the plant/nematode relationships in loco, thus avoiding the difficulties of field assays. In this study, Pinus pinaster in vitro cultures were established and compared to in vivo 1 year-old plantlets by analyzing shoot structure and volatiles production. In vitro co-cultures were established with the PWN and the effect of the phytoparasite on in vitro shoot structure, water content and volatiles production was evaluated. In vitro shoots showed similar structure and volatiles production to in vivo maritime pine plantlets. The first macroscopic symptoms of PWD were observed about 4 weeks after in vitro co-culture establishment. Nematode population in the culture medium increased and PWNs were detected in gaps of the callus tissue and in cavities developed from the degradation of cambial cells. In terms of volatiles main components, plantlets, P. pinaster cultures, and P. pinaster with B. xylophilus co-cultures were all b- and a-pinene rich. Cocultures may be an easy-to-handle biotechnological approach to study this pathology, envisioning the understanding of and finding ways to restrain this highly devastating nematode. Keywords Maritime pine ! Monoxenic culture ! Pinewood nematode ! Relative water content ! Shoots structure ! Volatiles Abbreviations BAP 6-Benzylaminopurine DAI Days after inoculation EPPO European and Mediterranean Plant Protectio