Interactions between the oomycete Pythium arrhenomanes and the rice root-knot nematode Meloidogyne graminicola in aerobic Asian rice varieties.

BACKGROUND: Aerobic rice fields are frequently infested by pathogenic oomycetes (Pythium spp.) and the rice root-knot nematode Meloidogyne graminicola. Here, the interaction between Pythium arrhenomanes and Meloidogyne graminicola was studied in rice roots of two aerobic rice varieties. In different experimental set-ups and infection regimes, plant growth, rice yield, Pythium colonization, as well as establishment, development and reproduction of M. graminicola were studied. RESULTS: In this study, it is shown that the presence of P. arrhenomanes delays the establishment, development and reproduction of M. graminicola compared to single nematode infected plants. The delay in establishment and development of M. graminicola becomes stronger with higher P. arrhenomanes infection pressure. CONCLUSIONS: Our data indicate that P. arrhenomanes antagonizes M. graminicola in the rice root and that the plant benefits from this antagonism as shown by the yield data, especially when either of the pathogens is present in high levels.

Comparing systemic defence-related gene expression changes upon migratory and sedentary nematode attack in rice.

Complex defence signalling pathways, controlled by different hormones, are known to be involved in the reaction of plants to a wide range of biotic and abiotic stress factors. Here, we studied the differential expression of genes involved in stress and defence responses in systemic tissue of rice infected with the root knot nematode (RKN) Meloidogyne graminicola and the migratory root rot nematode Hirschmanniella oryzae, two agronomically important rice pathogens with very different lifestyles. qRT-PCR revealed that all investigated systemic tissues had significantly lower expression of isochorismate synthase, a key enzyme for salicylic acid production involved in basal defence and systemic acquired resistance. The systemic defence response upon migratory nematode infection was remarkably similar to fungal rice blast infection. Almost all investigated defence-related genes were up-regulated in rice shoots 3 days after root rot nematode attack, including the phenylpropanoid pathway, ethylene pathway and PR genes, but many of which were suppressed at 7 dpi. Systemic shoot tissue of RKN-infected plants showed similar attenuation of expression of almost all studied genes already at 3 dpi, with clear attenuation of the ethylene pathway and methyl jasmonate biosynthesis. These results provide an interesting starting point for further studies to elucidate how nematodes are able to suppress systemic plant defence mechanisms and the effect in multitrophic interactions.

Studies on neurolathyrism in Ethiopia: dietary habits, perception of risks and prevention.

This study describes the correlation of traditional perceptions and dietary habits with the incidence of neurolathyrism to propose preventive measures. Therefore, 118 households of South Wollo and North Gondar (Amhara Regional State, Ethiopia), of which one third had at least one neurolathyrism affected member, were interviewed. Most of the affected families in this study had one neurolathyrism victim, being predominantly male and of younger age. The incidence among youngsters (boys and girls) was significantly correlated with the consumption of green unripe seeds (eshet), confirming this as a risk factor for developing neurolathyrism. The consumption of other popular grass pea preparations was not age related. Neurolathyrism patients did not attempt any medication as most people knew that neurolathyrism is incurable, but the consumption of grass pea was abandoned after developing the disease. The minority 'Woito' tribe was virtually unaffected. They were the only people reporting to consume fish which is rich in amino acids such as methionine and using metallic kitchen utensils in addition to clay pots. This observation points to the correlation between low neurolathyrism incidence and a better balanced diet as well as metallic kitchen utensils, suggesting a new approach for neurolathyrism prevention.

Wolbachia induces strong cytoplasmic incompatibility in the predatory bug Macrolophus pygmaeus.

Macrolophus pygmaeus is a heteropteran predator that is widely used in European glasshouses for the biological control of whiteflies, aphids, thrips and spider mites. We have demonstrated that the insect is infected with the endosymbiotic bacterium Wolbachia pipientis. Several gene fragments of the endosymbiont were sequenced and subsequently used for phylogenetic analysis, revealing that it belongs to the Wolbachia supergroup B. The endosymbiont was visualized within the ovarioles using immunolocalization. Tetracycline treatments were used to cure M. pygmaeus from its infection. Although a completely cured line could not be obtained by this approach, the application of a constant antibiotic pressure over 13 generations resulted in a line with a significantly reduced Wolbachia concentration. Crosses performed with this tetracycline-treated line revealed that the endosymbiont causes severe cytoplasmic incompatibility. This is the first report of a reproductive effect induced by Wolbachia in an economically important heteropteran predator that may have vital implications for its commercial production and use in biological control.

Cross-species microsatellite amplification in Vasconcellea and related genera and their use in germplasm classification.

To generate inexpensive and efficient DNA markers for addressing a number of population genetics problems and identification of wild hybrids in Vasconcellea, we have evaluated the use of simple sequence repeat (SSR) primers previously developed for other species. A set of 103 Vasconcellea accessions and some individuals of the related genera Carica and Jacaratia were analyzed with 10 primer pairs directing amplification of chloroplast microsatellites in Nicotiana tabacum and 9 nuclear SSR primer pairs recently identified in Vasconcellea x heilbornii. Heterologous amplification of chloroplast SSRs was successful for 8 of the 10 loci, of which 6 showed polymorphism. Seven of the 9 nuclear SSR primer pairs were useful in Vasconcellea and often also in Jacaratia and Carica, all revealing polymorphism. Exclusive haplotypes for each described taxon were identified based on chloroplast microsatellite data. Clustering based on separate nuclear and chloroplast data resulted in a clear grouping per taxon, but only low resolution was obtained above species level. The codominancy of nuclear SSRs and the general high polymorphism rate of SSR markers will make them more useful in future population genetics studies and diversity assessment in conservation programs.

Patterns of genetic and morphometric diversity in baobab (Adansonia digitata) populations across different climatic zones of Benin (West Africa).

BACKGROUND AND AIMS: Baobab (Adansonia digitata) is a multi-purpose tree used daily by rural African communities. The present study aimed at investigating the level of morphometric and genetic variation and spatial genetic structure within and between threatened baobab populations from the three climatic zones of Benin. METHODS: A total of 137 individuals from six populations were analysed using morphometric data as well as molecular marker data generated using the AFLP technique. KEY RESULTS: Five primer pairs resulted in a total of 217 scored bands with 78.34 % of them being polymorphic. A two-level AMOVA of 137 individuals from six baobab populations revealed 82.37 % of the total variation within populations and 17.63 % among populations (P < 0.001). Analysis of population structure with allele-frequency based F-statistics revealed a global F(ST) of 0.127 +/- 0.072 (P < 0.001). The mean gene diversity within populations (H(S)) and the average gene diversity between populations (D(ST)) were estimated at 0.309 +/- 0.000 and 0.045 +/- 0.072, respectively. Baobabs in the Sudanian and Sudan-Guinean zones of Benin were short and produced the highest yields of pulp, seeds and kernels, in contrast to the ones in the Guinean zone, which were tall and produced only a small number of fruits with a low pulp, seed and kernel productivity. A statistically significant correlation with the observed patterns of genetic diversity was observed for three morphological characteristics: height of the trees, number of branches and thickness of the capsules. CONCLUSIONS: The results indicate some degree of physical isolation of the populations collected in the different climatic zones and suggest a substantial amount of genetic structuring between the analysed populations of baobab. Sampling options of the natural populations are suggested for in or ex situ conservation.

Evidence of natural hybridization and introgression between Vasconcellea species (Caricaceae) from southern Ecuador revealed by chloroplast, mitochondrial and nuclear DNA markers.

BACKGROUND AND AIMS: Vasconcellea x heilbornii is believed to be of natural hybrid origin between V. cundinamarcensis and V. stipulata, and is often difficult to discriminate from V. stipulata on morphological grounds. The aim of this paper is to examine individuals of these three taxa and of individuals from the closely related species V. parviflora and V. weberbaueri, which all inhabit a hybrid zone in southern Ecuador. METHODS: Molecular data from mitochondrial, chloroplast and nuclear DNA from 61 individuals were analysed. KEY RESULTS: Molecular analysis confirmed occasional contemporary hybridization between V. stipulata, V. cundinamarcensis and V. x heilbornii and suggested the possible involvement of V. weberbaueri in the origin of V. x heilbornii. In addition, the molecular data indicated unidirectional introgression of the V. cundinamarcensis nuclear genome into that of V. stipulata. Several of the individuals examined with morphology similar to that of V. stipulata had genetic traces of hybridization with V. cundinamarcensis, which only seems to act as pollen donor in interspecific hybridization events. Molecular analyses also strongly suggested that most of the V. x heilbornii individuals are not F(1) hybrids but instead are progeny of repeated backcrosses with V. stipulata. CONCLUSIONS: The results of the present study point to the need for re-evaluation of natural populations of V. stipulata and V. x heilbornii. In general, this analysis demonstrates the complex patterns of genetic and morphological diversity found in natural plant hybrid zones.

Production of auxin and related compounds by the plant parasitic nematodes Heterodera schachtii and Meloidogyne incognita.

Mass spectrometric analysis revealed the presence of auxin, mainly in conjugated form, in secretions of Heterodera schachtii and Meloidogyne incognita, with or without treatment with DMT or resorcinol. M. incognita showed the highest production rates, though treatment of M. incognita with resorcinol had a negative effect on auxin production. Analysis of auxin precursor molecules in lysates of H. schachtii, M. incognita and Caenorhabditis elegans suggested that auxin is most probably a degradation product of tryptophan and that auxin may be synthesized via several intermediates, including indole-3-acetamide which is an intermediate of a pathway so far only characterized in bacteria. Furthermore, high levels of anthranilate, a degradation product of tryptophan in animals, but possibly also a precursor for auxin were detected.

Monitoring genetic diversity in tropical trees with multilocus dominant markers.

Since no universal codominant markers are currently available, dominant genetic markers, such as amplified fragment length polymorphism (AFLP), are valuable tools for assessing genetic diversity in tropical trees. However, the measurement of genetic diversity (H) with dominant markers depends on the frequency of null homozygotes (Q) and the fixation index (F) of populations. While Q can be estimated for AFLP loci, F is less accessible. Through a modelling approach, we show that the monolocus estimation of genetic diversity is strongly dependent on the value of F, but that the multilocus diversity estimate is surprisingly robust to variations in F. The robustness of the estimate is due to a mechanistic effect of compensation between negative and positive biases of H by different AFLP loci exhibiting contrasting frequency profiles of Q. The robustness was tested across contrasting theoretical frequency profiles of Q and verified for 10 neotropical species. Practical recommendations for the implementation of this analytical method are given for genetic surveys in tropical trees, where such markers are widely applied.

Phylogenetic analysis of the highland papayas ( Vasconcellea) and allied genera (Caricaceae) using PCR-RFLP.

The chloroplast and mitochondrial DNA diversity of 61 genotypes belonging to 18 Vasconcellea species, the so-called highland papayas, was studied by PCR-RFLP analysis of two non-coding cpDNA regions ( trnM- rbcL and trnK1- trnK2) and one non-coding mtDNA region ( nad4/1- nad4/2). This sample set was supplemented with six genotypes belonging to three other Caricaceae genera: the monotypic genus Carica, including only the cultivated papaya, and the genera Jacaratia and Cylicomorpha. Moringa ovalifolia was added as an outgroup species. The PCR-amplified cpDNA regions were digested with 18 restriction endonucleases, the mtDNA region with 11. A total of 22 point mutations and four insertion/deletions were scored in the sample. A higher level of interspecific variation was detected in the two cpDNA regions in comparison to the analysis of the mtDNA. Wagner parsimony and Neighbor-Joining analysis resulted in dendrograms with similar topologies. PCR-RFLP analysis supported the monophyly of Caricaceae, but among the 26 mutations scored, an insufficient number of markers discriminated between the different Caricaceae genera included in this study. Hence the inference of the intergeneric relationships within Caricaceae was impossible. However, some conclusions can be noted at a lower taxonomic level. The Caricaceae species were divided into two lineages. One group included only Vasconcellea spp., whereas the second included the remaining Vasconcellea spp., together with the papaya genotypes and those from the other Caricaceae genera. This may indicate a higher level of inter-fertility for the Vasconcellea species from the latter clade in interspecific crossings with papaya. The putative progenitors of the natural sterile hybrid V. x heilbornii, i.e. V. stipulata and V. cundinamarcensis, were only distantly related to V. x heilbornii. This indicates that probably none of these species was involved as the maternal progenitor in the origin of V. x heilbornii. Surprisingly, V. x heilbornii had organellar genome patterns identical with V. weberbaueri, suggesting a possible involvement of this species in the origin of V. x heilbornii. On the basis of discrepancy between morphological traits and the cpDNA profiles of some pairs of Vasconcellea species, we believe that besides V. x heilbornii, some other species have originated through interspecific hybridization. A reticulate evolution for Vasconcellea has therefore been suggested. Finally, intraspecific cpDNA variation was detected in V. microcarpa, thus providing molecular evidence for the high diversity previously indicated by morphological observations.

Molecular markers for cold tolerance and early vigour in maize (Zea mays L.).

Planting maize earlier than the current guidelines recommend, would give great contributions to ecological and sustainable agriculture. In order to plant maize earlier, maize varieties with good cold tolerance and strong early vigour are required. Therefore cold tolerance and early vigour should be important goals in modern maize breeding programmes. Both traits however have a complex, quantitative genetic background and are therefore not easily introduced into modern maize varieties. Marker assisted selection (MAS) can improve the efficiency of breeding activities. In this research project we aim to identify the molecular markers for cold tolerance and early vigour in one of our breeding populations through a QTL analysis. So far nine QTLs for cold tolerance and six QTLs for early vigour could be identified and there is even one QTL in common for the two traits under investigation. The analysis of more populations should reveal whether or not these QTLs might be useful in maize breeding programmes over the world.

The cytoskeleton in nematode feeding sites.

Sedentary edoparasitic nematodes induce specialised feeding cells in plant roots. Giant cells induced by root knot nematodes and syncytia generated by cyst nematodes in plant roots are large multinucleated cells containing a dense cytoplasm. To examine the plant cytoskeleton during feeding cell development, transcriptional activity of actin and tubulin genes and organization of the actin filaments and of the microtubules were analyzed in situ. Immunolocalizations of actins and tubulins and in vivo observation of green fluorescent protein decorated actin filaments and microtubules in nematode infected root cells revealed that major rearrangements of the cytoskeleton occur during the formation of nematode induced feeding cells.

Arabidopsis thaliana mutants with an altered susceptibility to the root-knot nematode Meloidogyne incognita.

Sedentary endoparasitic nematodes of plants are obligate parasites of roots in which they complete their sophisticated life cycle. To study the plant/root-knot nematode interaction, a screening was performed for mutants of Arabidopsis thaliana that were less susceptible to nematode infection. Ethyl methanesulfonate-mutagenized A. thaliana M2 seeds (13,000) were germinated and the seedlings were screened in vitro for nematode susceptibility. Another 5,000 plants were screened in a sand mixture under glasshouse conditions. Ten mutants that were at least a 2-fold less susceptible than the wild-type were retained and analyzed in more detail in vitro as well as in the greenhouse. All mutants with a fairly normal morphology had only a slightly, but reproducibly, decreased susceptibility to nematodes. One mutant (AMi 2) with a 17-fold lower susceptibility to nematode infection and an aberrant phenotype could be rescued by tryptophan.

AFLP analysis of genetic relationships among papaya and its wild relatives (Caricaceae) from Ecuador.

The AFLP technique was used to assess the genetic relationships among the cultivated papaya ( Carica papaya L.) and related species native to Ecuador. Genetic distances based on AFLP data were estimated for 95 accessions belonging to three genera including C. papaya, at least eight Vasconcella species and two Jacaratia species. Cluster analysis using different methods and principal co-ordinate analysis (PCO), based on the AFLP data from 496 polymorphic bands generated with five primer combinations, was performed. The resulted grouping of accessions of each species corresponds largely with their taxonomic classifications and were found to be consistent with other studies based on RAPD, isozyme and cpDNA data. The AFLP analysis supports the recent rehabilitation of the Vasconcella group as a genus; until recently Vasconcella was considered as a section within the genus Carica. Both cluster and PCO analysis clearly separated the species of the three genera and illustrated the large genetic distance between C. papaya accessions and the Vasconcella group. The specific clustering of the highly diverse group of Vasconcella x heilbornii accessions also suggests that these genotypes may be the result of bi-directional introgression events between Vasconcella stipulata and Vasconcella cundinamarcensis.

Activation of a pollenin promoter upon nematode infection.

Three glycine-rich protein genes of Arabidopsis thaliana (Atgrp-6, Atgrp-7, and Atgrp-8) that correspond to putative genes coding for pollenins (AtolnB;2, AtolnB;3, and AtolnB;4, respectively) are expressed predominantly in the anthers and, more specifically, in the tapetum layer. Tapetal cells are responsible for nutrition of developing pollen grains and show some functional similarities to nematode feeding sites (NFS) induced in plant roots by sedentary parasitic nematodes. The aim of this study was to analyze promoter activity of the Atgrp genes in NFS. Transformed Arabidopsis plants containing a promoter-ss-glucuronidase (gus) fusion of the Atgrp-7 gene were inoculated with the root-knot nematode Meloidogyne incognita and the cyst nematode Heterodera schachtii. GUS assays were performed at different time points after infection. Histochemical analysis revealed an up-regulation of Atgrp-7-gus expression 3 days after inoculation in the feeding sites of both nematodes. Maximal Atgrp-7-gus staining levels in NFS were observed 1 week after nematode infection.

Arabidopsis thaliana genes expressed in the early compatible interaction with root-knot nematodes.

In the compatible interaction between Arabidopsis thaliana and the endoparasitic nematode Meloidogyne incognita, galls are formed on the roots of the host plant. Differential display was used to identify alterations of gene expression in young A. thaliana root galls caused by M. incognita. Six genes were confirmed as plant genes by DNA gel blot hybridizations. Significant homology was found with a trypsin inhibitor, peroxidase, mitochondrial uncoupling protein, endomembrane protein, 20S proteasome alpha-subunit, and diaminopimelate decarboxylase. The cellular and temporal expression of each of the six genes was analyzed by mRNA in situ hybridizations.

Molecular characterisation of Arabidopsis lines tagged with a promoterless gus gene.

Promoter trapping has been performed through integration of a T-DNA containing a promoterless beta-glucuronidase (gus) reporter gene into the genome of Arabidopsis thaliana. A collection of T-DNA-tagged Arabidopsis lines has been produced with the vector pdeltagusBin19. Part of this collection was screened for lines with specific gus expression patterns. Here we report on the identification of two lines with gus expression in anthers and seeds. Both lines harbour complex T-DNA inserts. In one line, the integration of the T-DNA causes a male sterility phenotype and gus expression is developmentally regulated in anthers and flower bases. In the other line expression of gus is seen in the anther and seed endosperm.