Temperature Effects on Expression Levels of hsp Genes in Eggs and Second-Stage Juveniles of Meloidogyne hapla Chitwood, 1949.

Meloidogyne hapla is one of the most important nematode pathogens. It is a sedentary, biotrophic parasite of plants that overwinters in the soil or in diseased roots. The development of M. hapla is temperature dependent. Numerous studies have been performed on the effect of temperature on the development of M. hapla, but only a few of them analyzed the heat shock protein (hsp) genes. The aim of the study was to perform expression profiling of eight hsp genes (Mh-hsp90, Mh-hsp1, Mh-hsp4, Mh-hsp6, Mh-hsp60, Mh-dnj19, Mh-hsp43, and Mh-hsp12.2) at two development stages of M. hapla, i.e., in eggs and second-stage juveniles (J2). The eggs and J2 were incubated under cold stress (5 °C), heat stress (35 °C, 40 °C), and non-stress (10 °C, 20 °C, and 30 °C) conditions. Expression profiling was performed by qPCR. It was demonstrated that only two genes, Mh-hsp60 and Mh-dnj19, have been upregulated by heat and cold stress at both development stages. Heat stress upregulated the expression of more hsp genes than cold stress did. The level of upregulation of most hsp genes was more marked in J2 than in eggs. The obtained results suggest that the Mh-hsp90 and Mh-hsp1 genes can be used as bioindicators of environmental impacts on nematodes of the Meloidogyne genus.

Effect of Vicia sativa L. on Motility, Mortality and Expression Levels of hsp Genes in J2 Stage of Meloidogyne hapla.

Assuming that the seeds of Vicia sativa L. have a stressful effect on J2 stage Meloidogyne hapla, we undertook research on the effect of these seeds on the motility and mortality of J2 and determined the expression levels of selected hsp genes in J2. The assessment of the effect of V. sativa seeds on the motility of M. hapla specimens consisted of observing the movement of J2 immersed in a seed diffusate or in a tomato root filtrate at temperatures of 10, 17, and 21°C. In J2 treated with V. sativa (cv. Ina) seed diffusates, the expression level of hsp genes was determined by qPCR. J2 exposed to V. sativa diffusates were found to lose their motility, while their mortality did not exceed 30%. J2 in the seed diffusate were characterized by an increase in the expression levels of the Mh-hsp90, Mh-hsp1, and Mh-hsp43 genes. It is suggested that the hsp90 gene may be a potential bioindicator of the environmental impact on Meloidogyne nematodes. The impaired ability to move in J2 of M. hapla is attributable to the occurrence of V. sativa seeds in their habitat. These studies may contribute to developing methods of reducing crop damage caused by M. hapla.

Description of Pratylenchoides ojcowensis sp. nov. (Nematoda: Merlinidae) from Polish Jurassic Highland.

A new species of the genus Pratylenchoides has been described. It was found in Polish Jurassic Highland, in Ojców National Park. Pratylenchoides ojcowensis sp. nov. was isolated from the soil located around tangled roots of Elymus sp. and Trifolium sp. This species is marked by a conical head in both females and males which is not separated from the body contour and has with 4-5 annuli; a relatively short stylet (20.3-21.3 µm females, 17.7-20.9 µm males) with oval knobs directed posteriorly; the dorsal pharyngeal nucleus located anterior to the cardia (the subventral pharyngeal nuclei located posterior; a pharyngeal lobe of length about two body widths (1.8-2.6); a lateral field with 6 lines in the middle part of body and sometimes with partially areolated outer bands; intestinal fasciculi present; round sperm in the spermatheca in females; a female tail with a maximum of 29 annuli, and an annulated tail terminus. The status of the new species has been verifiied by DNA sequencing and phylogenetic analysis of the 28S rDNA region. The results obtained in the study indicated that P. ojcowensis sp. nov. is most related to P. alkani, P. ritteri and P. nevadensis from which is distinguished by the shape of the female head (conoid vs rounded), shorter stylet in females (20.3-21.3 µm vs 22.0-25.0 µm, 21.0-25.0 µm, 22.0-26.0 µm) and differences in 28S rDNA sequences. In addition (as per the original descriptions Yüksel 1977, Sher 1970, Talavera Tobar 1996) it is distinguished from P. alkani by smaller number of male's head annuli (4-5 vs 7-9), from P. ritteri it is distinguished by posteriorly directed stylet knobs (vs directed laterally), from P. nevadensis it is distinguished by oval and posteriorly directed stylet knobs (vs rounded and directed laterally).

Suppression of NGB and NAB/ERabp1 in tomato modifies root responses to potato cyst nematode infestation.

Plant-parasitic nematodes cause significant damage to major crops throughout the world. The small number of genes conferring natural plant resistance and the limitations of chemical control require the development of new protective strategies. RNA interference or the inducible over-expression of nematicidal genes provides an environment-friendly approach to this problem. Candidate genes include NGB, which encodes a small GTP-binding protein, and NAB/ERabp1, which encodes an auxin-binding protein, which were identified as being up-regulated in tomato roots in a transcriptome screen of potato cyst nematode (Globodera rostochiensis) feeding sites. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization confirmed the localized up-regulation of these genes in syncytia and surrounding cells following nematode infection. Gene-silencing constructs were introduced into tomato, resulting in a 20%-98% decrease in transcription levels. Nematode infection tests conducted on transgenic plants showed 57%-82% reduction in the number of G. rostochiensis females in vitro and 30%-46% reduction in pot trials. Transmission electron microscopy revealed a deterioration of cytoplasm, and degraded mitochondria and plastids, in syncytia induced in plants with reduced NAB/ERabp1 expression. Cytoplasm in syncytia induced in plants with low NGB expression was strongly electron translucent and contained very few ribosomes; however, mitochondria and plastids remained intact. Functional impairments in syncytial cytoplasm of silenced plants may result from NGB's role in ribosome biogenesis; this was confirmed by localization of yellow fluorescent protein (YFP)-labelled NGB protein in nucleoli and co-repression of NGB in plants with reduced NAB/ERabp1 expression. These results demonstrate that NGB and NAB/ERabp1 play important roles in the development of nematode-induced syncytia.

A fast and sensitive method for the simultaneous identification of three important nematode species of the genus Ditylenchus.

BACKGROUND: Nematodes of the genus Ditylenchus are parasites of a wide range of hosts, including higher plants. The most destructive of these species are D. dipsaci and D. destructor, two frequently quarantined pests. No rapid molecular method is available for unambiguous detection and distinguishing of these species from each other or from D. gigas, a pest of Vicia faba, either by multiplex PCR or real-time PCR. RESULTS: By aligning all D. dipsaci, D. destructor and D. gigas rDNA sequences, the authors found a constant-sequence region that could be used as a universal 5' primer and constant regions in the ITS1 regions of the rDNAs that could be used as species-specific 3' primers for PCR detection of these nematodes. A standardised protocol was developed for both singleplex- and triplex-mode PCR that yields a single product of distinct length for each of the species. The PCR protocol has also been adapted for real-time PCR. CONCLUSION: The present diagnostic PCR protocol is the only method that can identify all three species with the use of a triplex and/or a singleplex PCR assay. Importantly, the 3' primer for D. destructor ITS1 rDNA was designed so that it would hybridise all haplotypes.

[Legionella contamination of hospital hot water supply systems in the light of research conducted in 2008-2010 as part of supervision by the Country Sanitary Inspector in Bydgoszcz]. / Kolonizacja szpitalnych sieci wody cieplej Legionella sp. w swietle badan prowadzonych w latach 2008 - 2010 w ramach nadzoru przez Panstwowego Powiatowego Inspektora Sanitarnego w Bydgoszczy.

The aim of the study was to assess the level of Legionella sp. contamination in the hot water supply systems at the premises of inpatient healthcare facilities. In the years 2008-2010 the State Poviat Sanitary Inspector in Bydgoszcz tested the hot water supply systems in 8 hospitals. A total of 88 samples of hot water were collected in the years 2008-2010. The analysis involved temperature measurements and microbiological testing of the hot water. Contamination levels exceeding the applicable standards were discovered in 6 hospitals. The corrective measures introduced allowed for a significant improvement in each case. The hospital hot water systems revealed Legionella sp. contamination levels considerably exceeding the approved standards. Of the 88 water samples tested, 37 contained excessive numbers of Legionella sp. bacteria (i.e. above 100 CFU in 100 ml of water), which constituted 63.6% of the samples tested. In 6 of the 8 investigated hospitals the Legionella sp. contamination of the hot water supply system was found to be on the medium or high level. The analysis of temperature measurements revealed that the lowest temperature readings were associated with high bacterial colonization of the plumbing system. After the implementation of corrective measures, 50 control samples were collected, and in 37 of them the bacterial levels were below 100 CFU per 100 ml of water. The Legionella sp. contamination was found to be associated with low temperature of the hot water.

TaqMan REAL-Time PCR-based approach for differentiation between Globodera rostochiensis (golden nematode) and Globodera artemisiae species.

Cyst nematodes from the genus Globodera are common, and widely distributed parasites of Solaraceae. Intact cysts persist in soil even up to 10 years without detriment. Out of two Globodera species occurring in Poland, Globodera rostochiensis is considered by European and Mediterranean Plant Protection Organization (EPPO) as a quarantine pest, while Globodera artemisiae is not. Therefore, the distinction between these two species is crucial. Classic methods of detection and differentiation are laborious and time-consuming. Instead, application of molecular biology techniques allows obtaining of rapid and reliable results. The aim of this study was to establish detection and differentiation method of two species, G. rostochiensis and G. artemisiae, based upon real-time polymerase chain reaction with the use of TaqMan probes. In reaction with primers and probes specific for the nematodes' ribosomal DNA (rDNA), the samples used were DNAs isolated from the two species, alone or in mixture, as well as crushed single cysts. Applied probes enable not only to identify the species in DNA mixtures but also in a single cyst. The use of a crushed cyst eliminates long-lasting procedure of DNA isolation and reduces costs of analysis.