The ribosomal intergenic spacer (IGS) in the potato and tobacco cyst nematodes, Globodera pallida, G. rostochiensis and G. tabacum.

The potato cyst nematodes Globodera pallida and G. rostochiensis (PCN), and tobacco cyst nematode (TCN), G. tabacum, are the most important parasitic nematodes of potato and tobacco worldwide. Ribosomal DNA provides useful molecular data for diagnostics, the study of polymorphisms and for evolutionary research in eukaryotic organisms including nematodes. Here we present data on the structure and organization of a rarely studied part of the intergenic spacer (IGS) region of the PCN and TCN genome of cyst nematodes. This region has shown potential for diagnostic purposes and population studies in other organisms including nematodes. In nematodes, the ribosomal RNA gene cluster comprises three genes: 5.8S, 18S and 28S rRNA, which are separated by spacer regions: the intergenic spacer (IGS), non-transcribed spacer (NTS), externally transcribed spacer (EST) and the internally transcribed spacer (ITS). The intergenic spacer (IGS) region consists of an external transcribed spacer (ETS) and a non-transcribed spacer (NTS) which is located between the 28S of one repeat and the 18S gene of the next repeat within the rRNA genes cluster. In this study, the first flanking portion of the IGS was amplified, cloned and sequenced from PCN and TCN. Primers were then designed to amplify the whole IGS sequence. PCR amplification of IGS from G. tabacum, G. pallida, and G. rostochiensis yielded respectively: a single amplicon of 3 kb, three amplicons sized 2.5, 2.6 and 2.9 kb, and two amplicons sized 2.8 and 2.9 kb. Results showed that Globodera spp. has more than one variant copy of the IGS, with both long and short repetitive DNA elements. An approximately 400 bp long region without any internal repetitive elements, were identified in a position between the two repetitive regions suggesting that there is a 5S gene in the IGS of these species.

Effect of spray volume on the deposition, viability and infectivity of entomopathogenic nematodes in a foliar spray on vegetables.

BACKGROUND: Spray volume can influence the amount of free water on the leaf surface and subsequently the ability of entomopathogenic nematodes (EPNs) to move. In this study, an investigation was made of the effect of spray volume (548, 730 and 1095 L ha(-1) ) on the deposition, viability and infectivity of EPNs against Galleria mellonella on savoy cabbage, cauliflower and leek. RESULTS: Increasing spray volume decreased nematode deposition on 7.1 cm2 leek leaf discs at a 15° angle with the spray nozzle. Although the number of living nematodes observed on leek after 240 min of exposure was not significantly different between the low-volume application (548 L ha(-1) ) and the high-volume application (1095 L ha(-1) ), a greater infectivity was obtained in the latter application. The higher number of droplets deposited on the leek discs in the high-volume application may have stimulated nematode movement. No significant effect of spray volume was observed on the relative deposition of Steinernema carpocapsae on the bottom side of cauliflower and savoy cabbage leaf discs. In spite of the low S. carpocapsae deposition on the bottom side of the savoy cabbage discs, high infectivity was obtained against G. mellonella. Using the lowest spray volume on savoy cabbage, infectivity decreased with increasing exposure time, while infectivity was not affected by exposure time when a spray volume of 730 L ha(-1) or more was used. CONCLUSION: Spray volume is an important application parameter, as it affects nematode infectivity. Future research should investigate the effect of spray volume in the field and its influence on the effect of adjuvants.

Quantitative detection of the potato cyst nematode, Globodera pallida, and the beet cyst nematode, Heterodera schachtii, using Real-Time PCR with SYBR green I dye.

The potato cyst nematode Globodera pallida and the beet cyst nematode Heterodera schachtii are major nematode pests in world agriculture. Precise identification and knowledge about the number of nematodes in field soil are necessary to develop effective integrated pest control. Here we report the results of the Real-Time PCR assay for the rapid detection and quantification of G. pallida and H. schachtii. Using species specific primers and SYBR green I dye, we were able to detect a single second stage juvenile of cyst forming nematodes in samples. The specificity of the reaction was confirmed by the lack of amplification of DNAs from other Heterodera or Globodera species. Validation tests showed a rather high correlation between real numbers of second stage juveniles in a sample and expected numbers detected by Real-Time PCR. Reasons for observed differences in sensitivity and reliability of quantification detection for two species as well as other problems of Real-Time PCR are discussed. The Real-Time PCR assay with SYBR green I dye targeting fragments of the ITS-rDNA provided a sensitive means for the rapid and simultaneous detection and quantification of juveniles of these pests.