/pdf/migration-of-plant-parasitic-nematodes-towards-plant-roots-20ilu3fcer.pdf

Migration of plant-parasitic nematodes towards plant roots

The golden potato cyst nematode, Globodera rostochiensis (Woll) is responsible for large yield losses in potato crops in northern Portugal. Fumigation with synthetic chemicals controls this soil pest with possible negative impact on the environment. Thus, it is desirable to find environmentally friendly alternative measures to control this nematode species. Amending plant parts of Brassicaceae have been recognized as potential biofumigation practices. Active volatiles compounds released after enzymatic hydrolysis are responsible for the efficacy of biofumigants. The glucosinolate-myrosinase-isothiocyanates system appears to be controlling several soilborne pests and pathogens. The biological role of Brassicacea green manure amendments on potato cyst nematodes populations has not been reported in Portugal. In this study we evaluated the effects of two concentrations (0.2 and 0.05 µmoles 100 g−1 dry weight) of total glucosinolates of six plants extracts (broccoli, cauliflower, collards of Brassica rapa, collards of kale, Portuguese cabbage and watercress) on potato cyst nematodes population. The results were expressed as number of new cysts formed on bioassay Desiree potato roots. ANOVA analysis showed a highly significant effect (P < 0.001) of the plant extracts and concentrations tested on the suppression of potato cysts nematodes. The LSD test showed that the number of newly formed cyst on root system of bioassay plant was also related (P<0.05) to type and concentrations of glucosinolates extracts. The lowest numbers of cysts were recovered from potato roots that received extracts of watercress, cauliflower and B. Rapa, respectively. The cyst reduction appeared to be associated with high levels of 2-Phenthyl and 2-propenyl glucosinolates in these plants.

Suppressing Potato Cyst Nematode, Globodera rostochiensis, with Extracts of Brassicacea Plants

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.

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

Plant-parasitic nematodes are serious pests causing important crop losses worldwide. After extensive screening of non-tuber-bearing Solanaceae, a resistant trap crop, Solanum sisymbriifolium, with a high production level of hatching agents, seemed an ideal control method for potato cyst nematodes (PCN), Globodera spp. Recently, root-knot nematodes (RKN), Meloidogyne spp., were found coexisting with PCN. Therefore, it is important to find alternative methods to control both nematode genera. The chemical properties of S. sisymbriifolium turns this plant into an excellent candidate for further nematicidal studies and to develop new crop production models. Studies concerning the effects of this plant on plant-parasitic nematodes are presented. Pathogenicity studies with four S. sisymbriifolium cvs (Domino, Pion, Sis 4004 and Sharp) and five Meloidogyne species showed that all cultivars of S. sisymbriifolium studied were resistant to M. chitwoodi and hypersusceptible to M. arenaria and M. hapla. For M. hispanica only cv Pion was susceptible. M. javanica induced different responses: cvs Pion and Sharp were susceptible; cv Domino resistant and Sis 4004 hypersusceptible. The studies of the hatching effects of root exudates from these cvs showed that they had an influence on the hatching inhibition of second stage juveniles of the five Meloidogyne species tested.

Solanum sisymbriifolium - a new approach for the management of plant-parasitic nematodes

The reproduction of a Meloidogyne hispanica isolate from Portugal was evaluated in 63 plant species/cultivars, in pot assays at 25 ± 2.0°C, on the basis of root gall index (GI) and reproduction factor (Rf = final/initial egg density) at 60 days after inoculation. Cultivars of aubergine, bean, beetroot, broccoli, carnation, corn, cucumber, French garlic, lettuce, melon, onion, parsley, pea, potato, spinach, and tobacco and two of cabbage were susceptible (3 ≤ GI ≤ 5; 1.15 ≤ Rf ≤ 262.86). Cabbage cv. Bacalan, cauliflower cv. Temporao and pepper cv. Zafiro R2 were hypersusceptible or poor hosts (Rf   2) and pepper cvs. Aurelio and Solero were resistant (0.0 ≤ GI ≤ 0.4; 0.00 ≤ Rf ≤ 0.03). The response of the pepper cultivars and the Mi-1 resistant tomato cv. Rossol was also conducted in pots using two inoculum levels and four temperatures, three growth chamber (25 ± 2.7°C, 29.3 ± 1.8°C and 33.6 ± 1.2°C) and one outdoors (24.4 ± 8.2°C). At 24.4 ± 8.2°C and 25 ± 2.7°C, the reproduction on the resistant tomato was significantly lower compared to the susceptible cv. Easypeel. At all temperatures, resistance was evident for the pepper cultivars, despite the fact they were not found to contain any of the Me1, Me3, Me7 and N genes. The eggs obtained on cv. Aurelio at 33.6 ± 1.2°C were used to get a selected resistance breaking isolate of M. hispanica that was able to reproduce on the three pepper cultivars. Our results suggest that the initial M. hispanica isolate is a mixture of virulent and avirulent individuals. The pepper cultivars tested, have potential to reduce M. hispanica populations in agro-ecosystems under certain conditions, but they should be used as a part of an integrated management strategy in order to prevent the development of virulent populations.

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Host status of cultivated plants to Meloidogyne hispanica

Root-knot nematodes (RKN), Meloidogyne spp., are among the most economically important nematodes in agriculture and can cause significant losses in potato (Solanum tuberosum L.), in both warm and cool climates, depending upon the nematode species (Brodie et al., 1993; Greco, 1993). RKN have not yet been recognised as serious nematode pests in the potato-growing areas of Portugal. However, during the past 10 years, nematode damage has been found in some areas and it is clear that infestation of potato fields by RKN is becoming more widespread. Some of the RKN species attacking potato, e.g., M. chitwoodi, also have quarantine status and one isolate originating from Portugal has already been included in a molecular study (Powers et al., 2005). The aim of this work was to provide data on the identification of Meloidogyne spp. on potato in Portugal. During a survey for potato cyst nematodes (PCN), Globodera spp., RKN isolates were detected in tubers and in soil collected in 210 potato fields in various districts of Portugal (Table 1). When tubers infected with RKN were detected, egg masses were collected and susceptible tomato plants (Solanum lycopersicum L.) cv. Easypeel were inoculated with ten egg masses/pot. When only soil samples were taken, tomato plants with four true leaves were transplanted into the original soil. All plants were maintained in a glasshouse at 25-30◦C. After 45 days, plants were uprooted, their roots gently washed free of soil and females, when present, collected with forceps under a stereomicroscope. Morphological characterisation was based on a minimum of 20 perineal patterns (Taylor & Netscher, 1974; Hartmann & Sasser, 1985). Their char-

Root-knot nematodes, Meloidogyne spp., on potato in Portugal

Populations of potato cyst nematode (PCN) were assembled from all of the potato growing regions of Portugal, either by direct sampling of fields or from cyst samples sent to the University of Coimbra for identification. When 30 or more cysts were available, isoelectric focusing of species-specific protein bands was used to identify which PCN species were present. PCN was present in 35% of a set of 226 soil samples. PCN was also identified in a larger set of 423 populations. Of the 423 populations, 83% were pure Globodera rostochiensis, 8% were pure G. pallida and 9% were mixtures of the two. PCN was found in all of the main potato growing areas but G. pallida was not detected in Coimbra, Setubal or Viana do Castelo districts.

Characterisation of potato cyst nematode populations from Portugal

The morphology of the perineal pattern in the posterior body region of female rootknot nematodes is one of the most reliable characters for differentiating Meloidogyne species. Light microscopy has been used extensively to observe this character. Several recent descriptions of Meloidogyne species have shown that the cuticular surface features of patterns are emphasized and better resolved by a scanning electron microscope (SEM). Few techniques have been described for preparing perineal patterns for SEM observation. Mulvey et al. (4) and Khan and Thames (3) used the whole female to study the perineal pattern, whereas Yik and Birchfield (6) and Eisenback et al. (2) used the excised posterior end. Our objective is to provide a rapid technique for preparing perineal patterns for SEM. Two Portuguese populations (M. hispanica Hirschmann and an undescribed Meloidogyne sp.) were used in this study. Perineal patterns were prepared from

/pdf/a-technique-for-preparing-perineal-patterns-of-root-knot-3k75jr7vfg.pdf

A Technique for Preparing Perineal Patterns of Root-knot Nematodes for Scanning Electron Microscopy.

Identification of species and virulence groups of potato cyst nematodes (PCN), Globodera pallida and G. rostochiensis , present in field populations is important in the control of these nematodes by means of resistant cultivars. In order to characterize the virulence of Globodera spp. isolates from Portugal, 43 G. rostochiensis and three G. pallida isolates were evaluated by measuring their multiplication rates on a susceptible potato cultivar and five differential potato genotypes in a growth chamber pot experiment. Principal Component Analysis and Hierarchical Cluster Analysis showed that the reproduction rates were different in terms of both the numbers of eggs and the numbers of cysts produced. Portuguese isolates of PCN were more virulent on genotypes derived from Solanum vernei than on genotypes derived from other Solanum resistance sources, and there was a significant nematode isolate × host genotype interaction. The virulence bioassay clearly distinguished the two PCN species but failed to differentiate isolates into pathotypes. There was a wide and continuous range of virulence to the resistant genotypes, especially in G. rostochiensis isolates.

Virulence assessment of Portuguese isolates of potato cyst nematodes (Globodera spp.)

Nematologists need correct species identification to carry out research, teaching, extension and other
activities. Therefore, nematode taxonomy must be pursued diligently at all levels. The identification of plant-parasitic
nematodes is not always easy and that of some species is especially difficult. Most of the information that nematologists
use when characterizing and identifying specimens is based on morphological and morphometrical characters. Although
these characters are of primary importance, in the last three decades they have been supplemented by biochemical/
molecular characters. Biochemical approaches include the separation of proteins (general proteins and
isozymes) by one-dimensional gel electrophoresis, isoelectric focusing, two-dimensional gel electrophoresis, and sodium
dodecyl sulphate-capillary gel electrophoresis. Serology has also been found effective in the identification and
quantification of nematodes, monoclonal antibodies being a more useful immunological tool than polyclonal antibodies.
Identification based on the direct examination of DNA is potentially a more powerful method to characterize
inter- and intra-specific variability. The development of techniques such as the polymerase chain reaction, restriction
fragment length polymorphism, randomly amplified polymorphic DNA, and amplified fragment length polymorphism
has increased the accuracy and speed of nematode characterization/identification. Progress continues to be
made and more and more nematologists are using molecular techniques for diagnostic purposes and to assess genetic
variation.

M. Susana N. de A. Santos

Papers

Root-knot nematodes, Meloidogyne spp., on potato in Portugal

Characterisation of potato cyst nematode populations from Portugal

A Technique for Preparing Perineal Patterns of Root-knot Nematodes for Scanning Electron Microscopy.

Virulence assessment of Portuguese isolates of potato cyst nematodes (Globodera spp.)

Biochemical and molecular characterization of plant-parasitic nematodes