Nicole Viaene

Root-knot nematodes (Meloidogyne spp.) in Europe

In Europe, root-knot nematodes are increasingly important. Out of more than 90 Meloidogyne species currently described, 23 have been found on the continent. In the cooler climates, Meloidogyne hapla , M. naasi , M. chitwoodi and M. fallax are prevalent. Meloidogyne arenaria , M. javanica and M. incognita are the most common species in warmer conditions of southern Europe, but also in glasshouses in northern Europe. Morphological identification of root-knot nematodes is difficult and time consuming; therefore, many research groups have been developing molecular techniques for identification of Meloidogyne species. Meloidogyne chitwoodi and M. fallax are quarantine organisms and subject to regulations, and the highly aggressive M. enterolobii has been added to the EPPO alert list. Differences between temperate and tropical Meloidogyne species and their prevalence in Europe imply the need for different management strategies in south and north Europe. Possible crop rotations for the control of root-knot nematodes are limited due to the wide host range of several important species. The banning of methyl bromide and restrictions on other fumigant pesticides in the EU have increased the application of biofumigation significantly in south Europe. The egg-parasitising fungus Paecilomyces lilacinus is commercialised in Germany and applied as dispersible granules for application in water. Intensive research is conducted on the egg-parasitising fungus Pochonia chlamydosporia , and the obligate parasitic bacterium Pasteuria penetrans. European research has paid much attention to resistance breeding and selection. The Mi gene of tomato is widely used but resistance-breaking populations of M. incognita and M. javanica have been reported in different countries.

Management of Meloidogyne hapla on lettuce in organic soil with sudangrass as a cover crop

Host suitability for Meloidogyne hapla of six cover crops was tested in the greenhouse. Sudan-grass cv. Trudan 8 and rye (mixture of cultivars) were nonhosts; oat cv. Porter was a poor host; and phacelia cv. Angelia, oilseed radish cv. Renova, and yellow mustard cv. Martigena were maintenance hosts. When incorporated as a green manure before planting of lettuce cv. Mon-tello, sudangrass was the most effective of the cover crops in reducing egg production of M. hapla. Soil amendment with all parts of sudangrass resulted in lower reproduction of M. hapla on lettuce than soil amendment with only roots of sudangrass. Soil incorporation of 2-month-old (or younger) tissues of sudangrass was more effective in reducing nematode reproduction on subsequent lettuce plants than incorporation of 3-month-old tissues. Sudangrass was grown as a cover crop after lettuce for three growing seasons in field microplots and incorporated as a green manure before the first fall frost. Weight of lettuce heads was significantly higher and reproduction of M. hapla was significantly lower in sudangrass-amended microplots compared with those left fallow between lettuce crops, but results varied with year and nematode infestation level.

Long-term efficacy of Pochonia chlamydosporia for management of Meloidogyne javanica in glasshouse crops

Long-term efficacy of Pochonia chlamydosporia, a fungal parasite of root-knot nematodes, was tested in two cropping systems: one consisting of three consecutive lettuce crops and another consisting of one tomato crop followed by two lettuce crops. Plants were either grown in pots in soil inoculated with 5000 chlamydospores of P. chlamydosporia per cm3 soil or in soil without chlamydospores. Fifty or 25 second-stage juveniles (J2) of Meloidogyne javanica per 100 cm3 soil were applied in Tests 1 and 2, respectively. The high nematode inoculum density of Test 1 resulted in considerable plant death, showing that the fungus was unable to control high nematode levels. At the harvest of most crop cycles, fewer J2 were found in soil or roots or fewer egg masses per root system were counted in pots with P. chlamydosporia compared with pots without P. chlamydosporia. A one-time application of P. chlamydosporia was able to slow down the build-up of the M. javanica population for at least 5-7 months.

An evaluation of the implications of virulence in non-European populations of Globodera pallida and G. rostochiensis for potato cultivation in Europe

The potato cyst nematodes Globodera pallida and G. rostochiensis are listed in the EU Plant Health Directive 2000/29/EC and are also subject to the new EU Council Directive 2007/33/EC on the control of potato cyst nematodes, requiring unilateral suppression of these pests in Europe. At the same time there is also pressure to increase world trade in potatoes. Such pressure has to be balanced by the risks involved in the associated spread of these pests and subsequent problems in management. Populations of the potato cyst nematodes from outside Europe, in particular South America, which is considered the origin of G. pallida and G. rostochiensis, pose a risk to those European countries where limited genetic variability of these nematode species has been recorded. The development and usage of resistant cultivars under such conditions has formed a pivotal role in integrated management programmes in Europe. Molecular studies have shown that populations of G. pallida and G. rostochiensis from South America have a different genetic composition from those in Europe. The introduction of such populations would pose a threat to the use of resistant cultivars as a major tool in reducing the potential spread and damage caused by these species. At present, an inability to link precisely genetic variability to the virulence characteristics of a specific nematode population, and quickly identify the virulence status of intercepted populations for inspection purposes, strengthens the case for using plant health legislation to prevent their introduction.

Responses of banana and plantain cultivars, lines and hybrids to the burrowing nematode Radopholus similis

Twenty-eight genotypes (bred diploids, seed-fertile triploids and tetraploid hybrids) of Musa spp. were evaluated in pot tests in Honduras for resistance and tolerance to Radopholus similis through comparison with reference genotypes (Grand Nain as susceptible and Pisang Jari Buaya and Yangambi Km5 as resistant). Eleven tests were carried out, each with seven to 12 Musa genotypes. Tissueculture plants (TC) or plants grown from a corm (Co) were inoculated with 1000 or 3500 nematodes per plant, respectively. Data on nematode population densities, fresh root weight, percentages dead roots, root necrosis and root bases on the corm with lesions were taken 13-23 weeks after inoculation. In most tests, Gros Michel and Highgate were as susceptible to R. similis as Grand Nain. Resistance was shown by both TC and Co plants of SH-3142, SH-3362, SH-3648 and SH-3723, and by Co plants of SH-2095, SH-3624, the female parents Calcutta 4 and Prata Enana, and the hybrid FHIA-01. Moderate resistance was shown by TC plants of SH-3624 and Co plants of SH-3437, the female parent Pelipita and the hybrids FHIA-18 and FHIA-23. The other genotypes were susceptible to R. similis although the male parents SH-3386 and SH-3640, and the hybrid FHIA-21 had some degree of resistance. FHIA-03 was susceptible but showed tolerance to R. similis.

Development of two species-specific primer sets to detect the cereal cyst nematodes Heterodera avenae and Heterodera filipjevi

Twelve Heterodera species are of major economic significance in wheat and barley. Of these, H. avenae, H. filipjevi and H. latipons are among the most important ones, and sometimes coexist. The identification of Heterodera species using morphological characteristics is time consuming, requires specialized skill and can be imprecise, especially when they occur mixed in field populations. Molecular techniques can provide a more accurate way for nematode identification. This study reports the results of experiments targeting the mitochondrial cytochrome oxidase subunit 1 (COI) gene to develop species-specific primers that could be used for the identification of H. avenae and H. filipjevi. The COI gene of 9 Heterodera spp. and Punctodera punctata was partially sequenced and the resultant sequences were aligned to find unique sites suitable for the design of primers. The alignment showed variability between H. avenae, H. filipjevi and other Heterodera species. Two sets of species-specific primers were identified for the identification of both species and the conditions for their use in PCR were optimised. The specificity of the designed primers was checked by comparison with one population of P. punctata and populations of 14 other Heterodera species, nine populations of H. avenae and 10 populations of H. filipjevi originating from different countries. To test the sensitivity, the PCR was run with DNA extracted from five second-stage juveniles (J2) of H. avenae or five J2 of H. filipjevi mixed with DNA extracted from varying numbers of J2 of H. latipons. It was possible to detect as few as five J2 of H. avenae or H. filipjevi among 100 J2 of H. latipons. The two primers sets allow the detection of H. avenae and H. filipjevi where they occur in mixed populations with other Heterodera spp.

Effect of marigold (Tagetes patula) on population dynamics of Pratylenchus penetrans in a field

The effect of a sequence of host and non-host crops on the population dynamics of Pratylenchus penetrans was monitored for 2 years in a field. The sequence was pea (Pisum sativum, 25% cv. Colombia and 75% cv. Coca), French marigolds (Tagetes patula nana cv. Sparky); fallow; carrot (Daucus carota, cv. Amsterdamse bak); bean (Phaseolus vulgaris, cv. Avignon) and again fallow. Samples were taken every 21 days during the marigold crop and following fallow, and at planting and harvest for the other crops. From five 2 × 5 m2 plots, 15 cores were taken per plot to a depth of 70 cm and split into seven segments of 10 cm each. Within the plots, segments from corresponding depths were pooled and nematodes were estimated from a 200 g subsample. Densities of P. penetrans increased 2.2-fold under pea, but decreased by 90% after marigolds had been grown for 105 days. The P. penetrans population was reduced by about 90% in the soil layer above 40 cm while the reduction was about 80% below 50 cm after 105 days of marigold culture. Densities of P. penetrans declined exponentially in time under marigold and fallow. The effect of marigold was persistent as no increase in P. penetrans densities over the whole soil profile was noticed after two crop cycles of host plants.

Hatching of the root-lesion nematode, Pratylenchus penetrans, under the influence of temperature and host

The influence of temperature and root diffusates from different plants on the hatching behaviour of Pratylenchus penetrans was studied. Diffusates were obtained from maize (cv. Husar), carrot (cv. Masei), black salsify (cv. Omega), pea (cv. Alouette), bean (cv. Polder) and marigold (cv. Single Gold) at different plant ages. Soil leachate was used as a control treatment. Hatching was also examined in 10% dilutions of the root diffusates and the soil leachate. Hatching was monitored at 5-day intervals until 60 days at most. Test solutions were refreshed at the same intervals. Pratylenchus penetrans hatched more at 20°C than at 10, 15 or 25°C. Hatching was stimulated by all host diffusates, as well as marigold; it was highest in maize and bean diffusates (ca 50%). Except for carrot diffusate and soil leachate, hatching activity decreased with increasing plant age. It also decreased in 10% diluted root diffusates, except for pea, black salsify and soil leachate. There was no evidence for the presence of hatching inhibitors in the diffusate of any host.

Distribution of Meloidogyne chitwoodi in potato tubers and comparison of extraction methods

Meloidogyne chitwoodi and M. fallax are quarantine organisms in Europe. One measure to restrict the spread of these nematodes is careful inspection of potato tubers. The distribution of M. chitwoodi in heavily infected tubers was studied and several methods for extraction of these nematodes from tubers were compared. The majority of the nematodes (96%) were found in the first 5.25 mm of the tuber, corresponding to the depth of the vascular ring. About half of them were found between 1.75 and 3.50 mm deep. Incubation of small pieces of tuber on Baermann funnels in the misting chamber during 36 days yielded about 12 times fewer juveniles than mixing potato tissues and extracting them using zonal centrifugation, a process that took about 1 h. Enzymatic maceration of potato tissues for 24 or 48 h did not liberate more nematodes than 2 min of blending the tissues at high speed. More nematodes, with 95% consisting of eggs, were extracted by zonal centrifuging than by pouring the macerated suspension over a set of 500 μm, 250 μm and 20 μm sieves.

Development of a species-specific PCR to detect the cereal cyst nematode, Heterodera latipons

Summary ‐ Several Heterodera species can reduce the yield of wheat and barley, among which H. avenae, H. filipjevi and H. latipons are economically the most important. Their identification, based on morphological characteristics, is not straightforward but can be made easier using molecular techniques. In this study, we developed species-specific primers for the detection of H. latipons. The actin gene of eight Heterodera species was partially sequenced and, after purifying and sequencing the PCR products, all sequences were aligned to find unique sites. The alignment showed moderate to very high similarities between the species. However, a small fragment of the actin gene was suitable for the construction of a potentially useful species-specific primer for H. latipons. The optimised PCR was subsequently tested with several populations of 14 Heterodera species and a single population of Punctodera punctata. Heterodera latipons was represented by 16 populations originating from six different countries. The primer set (Hlat-act), designed using AlleleID 7.73, was shown to be very specific. To test its sensitivity further, the PCR was conducted on DNA extracted from five second-stage juveniles (J2) of H. latipons mixed with five or 100 J2 belonging to H. avenae. The PCR was able to detect up to 1:10 dilution of the DNA obtained from five J2. The results showed that a specific and sensitive H. latipons species-specific PCR was constructed.