On the modulation of innate immunity by plant-parasitic cyst nematodes
Postma, W.J. - \ 2013
Wageningen University. Promotor(en): Jaap Bakker, co-promotor(en): Geert Smant; Aska Goverse. - S.l. : s.n. - ISBN 9789461735560 - 154
plantenparasitaire nematoden - globodera rostochiensis - heterodera schachtii - planten - interacties - immuniteit - immuunsysteem - modulatie - receptoren - signaaltransductie - moleculaire plantenziektekunde - plant parasitic nematodes - globodera rostochiensis - heterodera schachtii - plants - interactions - immunity - immune system - modulation - receptors - signal transduction - molecular plant pathology
Plant-parasitic cyst nematodes are major agricultural pests worldwide. These obligate endoparasites invade the roots of host plants where they transform cells near the vascular cylinder into a permanent feeding site. Plants possess a multilayered innate immune system consisting of different types of extracellular and intracellular immune receptors. These enable detection of most invading nematodes and initiate immune responses that result in resistance. Many plant pathogens use effectors to overcome resistance. Here, modulation of plant innate immunity by plant-parasitic cyst nematodes was investigated. Extracellular immune receptor signaling and hormone-mediated signaling pathways were found to contain infection of susceptible Arabidopsis thalianawith Heterodera schachtii. A large family of effectors was identified in Globodera rostochiensis. One of these so-called SPRYSECs interacted with a novel CC-NB-LRR type resistance protein of a susceptible tomato without inducing resistance responses. Instead, the effector was found to suppress defense-related programmed cell death and resistance mediated by several CC-NB-LRR type resistance proteins. In addition, a secreted antimicrobial peptide was identified in G. rostochiensis. Plant-parasitic cyst nematodes thus most likely secrete effectors that protect against plant immune responses and secondary infections. The current evidence for the existence of immune modulating effectors is reviewed and directions for further research are given.
Co-evolution between Globodera rostochiensis and potato driving sequence diversity of NB-LRR resistance loci and nematode suppressors of plant immunity
Finkers-Tomczak, A.M. - \ 2011
Wageningen University. Promotor(en): Jaap Bakker, co-promotor(en): Aska Goverse; Geert Smant. - [S.l.] : S.n. - ISBN 9789085859499 - 200
solanaceae - solanum tuberosum - plaagresistentie - loci voor kwantitatief kenmerk - genen - plantenparasitaire nematoden - globodera rostochiensis - globodera pallida - co-evolutie - genetische kartering - solanaceae - solanum tuberosum - pest resistance - quantitative trait loci - genes - plant parasitic nematodes - globodera rostochiensis - globodera pallida - coevolution - genetic mapping
Sedentary plant parasitic nematodes have evolved sophisticated strategies that allow them to transform host cells in the roots of host plants into feeding structures. These complex structures enable the nematodes to complete their life cycle inside a single host plant. Feeding structure initiation and maintenance are thought to be determined by the concerted action of effectors produced by the esophageal glands of the nematodes. However, the molecular mechanisms underlying the transformation of host cells into feeding structures and the role of the effectors in this process are poorly understood. For example, it is generally thought that virulent nematodes also use effectors to protect their complex feeding structures from plant innate immune responses. However, nematode effectors suppressing plant innate immunity have not been identified to date.
The host ranges of sedentary plant parasitic nematodes can vary from very wide (e.g. root-knot nematodes) to relatively narrow, limited to single plant families (e.g. cyst nematodes). Potato cyst nematodes (PCN) are able to parasitize only Solanaceous plants. Every year, they cause substantial yield losses in potato production areas. PCN are very difficult to control by the use of cultivation methods or the application of pesticides alone. The introduction of novel nematode resistant potato cultivars to the market is therefore of great importance for potato growers all over the world. Resistances, however, can be overcome by the emergence of virulent nematode populations. The aim of this thesis is to study incompatible interactions between the potato cyst nematode Globodera rostochiensis and potato (Solanum tuberosum), by analyzing resistance loci that make the plant immune to potato cyst nematodes as well as nematode effectors that suppress plant innate immunity.
This thesis begins with an extensive review of the literature on molecular and cellular aspects of plant resistance to sedentary endoparasitic nematodes, including pre-infectional, non-host and host resistance (Chapter 2). Most research in this field has focused on host resistance to nematodes, which is determined by single (e.g.H1 and Gpa2) or multiple (e.g. GpaVSsplandGpaXISspl) resistance gene loci. Two potato cyst nematode resistance loci were studied in this thesis (i.e. Grp1 in Chapter 3 and H1 in Chapter 4) and the results of these studies are summarized below.
Grp1 locus confers broad-spectrum quantitative resistance to the potato cyst nematode species Globodera pallida and G. rostochiensis in potato. It was previously mapped to a 3 cM interval on the short arm of potato chromosome V between the markers GP21 and GP179 in a hot spot for resistance (Rouppe Van Der Voort et al., 1998). The aim of the work described in chapter 3 was to fine map the Grp1 locus. First, a diploid mapping population RHAM026, comprising 1536 genotypes was screened with the flanking markers GP21 and GP179, resulting in the identification of 61 recombinants in this region. Next, thirteen new markers were developed using the genomic sequence information available from the same region of Solanum demissum. Together with markers available from the literature, these thirteen markers were used to screen a subset of 54 recombinants. Finally, these recombinants were tested for resistance to G. pallida Pa2 and G. rostochiensis Ro5. This mapping of both resistance specificities resulted in two nearly identical LOD graphs with the highest score just north of marker TG432. We conclude that the resistances to both G. pallida and G. rostochiensis map to the same 1.08 cM interval between the markers SPUD838 and TG432. Other studies have revealed that this locus in potato harbors several gene clusters encoding classical NB-ARC-LRR resistance proteins. This finding led us to the hypothesis that the Grp1 resistance depends on one, or perhaps several tightly linked major genes.
Near-absolute resistance to G. rostochiensis pathotypes 1 and 4 is conferred by the H1 resistance locus at the distal end of chromosome V of the diploid S. tuberosum ssp. andigena genotype SH83-92-488 (SH). The H1 resistance involves a hypersensitive response in the cells surrounding the nematode feeding structure, so that it becomes isolated from the vascular tissues in the host. A high-resolution map of H1 locus was generated previously using SHxRH mapping population (Bakker et al., 2004). In chapter 4, we used markers from thismap to screen a BAC library of SH. The BAC inserts identified with the markers were used to construct a physical map covering this region in the resistant haplotype. Further sequencing of the BAC inserts, included in the physical map, revealed a genomic fragment of 341 Kb harboring a large cluster of CC-NB-ARC-LRR genes. We compared this cluster of resistance gene homologs with the sequences of the corresponding regions in the two susceptible haplotypes from the diploid genotype RH89-039-16 (S. tuberosum ssp. tuberosum/ S. phureja), spanning 700 and 319 Kb respectively. The genomic regions in all three haplotypes harbor from 17 up to 23 resistance gene homologs interspersed with numerous transposable elements, genes coding for extensin-like proteins, and an amino acid transporter. Strikingly, the three haplotypes do not reveal gene order conservation and the overall sequence homology is only confined to the coding sequences of the resistance gene homologs. These findings suggest that extensive rearrangements have shaped the H1 locus. Sequence data and marker information gained from this study will benefit future efforts to clone the H1 nematode resistance gene.
At the start of the research described in this thesis no suppressor of plant immune response had been found in plant parasitic nematodes. In chapter 5, we report the first identification and functional characterization of a G. rostochiensis effector suppressing plant innate immune responses. The Nematode Suppressors of Immunity 1 (NSI-1) are specifically expressed in the dorsal esophageal gland of the nematodes, and their expression is upregulated in stages that feed on host cells. We identified many variants of NSI-1 in the Ro1-Mierenbos field population, and showed that this gene family is under diversifying selection. Knocking-down NSI-1 transcription by RNA interference strongly reduced the number of nematodes developing into full-grown cysts. Overexpression of four NSI-1 variants in susceptible potato plants resulted in enhanced susceptibility to nematodes. Moreover, overexpression of three other variants enhanced the susceptibility of potato plants to the fungus Verticillium dahliae. Down-regulation of the potato homologs of the Arabidopsis thaliana transcription factors WRKY22 and WRKY53 in these plants indicated that NSI-1 target immune signaling in plants. In an agroinfiltration assay in leaves of Nicotiana benthamiana several NSI-1 variants suppressed the hypersensitive response caused by the co-expression of specific resistance proteins and matching pathogen effectors (i.e. RBP-1/Gpa2 and AvrBlb2/Rpi-blb2) and by autoactive mutants of the resistance protein Mi-1.2 and an H1 resistance gene homolog RGH10-H1. Interestingly, other NSI-1 effector variant suppressed the hypersensitivity response induced by an autoactive mutant of the immune signaling protein NRC1. These findings altogether lead to the conclusion that potato cyst nematodes secrete suppressors of plant immunity, most likely to protect their feeding structures.
In the final chapter of this thesis, we discuss our most important findings within the broader context of recent developments in the field of molecular plant-microbe interactions. First, we argue that quantitative nematode resistance Grp1 is encoded by one or more NB-ARC-LRR genes located in one of the resistance gene clusters mapped to the GP21-GP179 interval on potato chromosome V. We further examine obstacles and offer possible solutions with regard to future cloning of the H1 nematode resistance gene. Lastly, we elaborate on possible functions, activities, and evolution of NSI-1 effectors as suppressors of plant innate immunity.
Molendijk, L.P.G. - \ 2010
Kennisakker.nl 2010 (2010)25 maart.
rassenproeven - resistentie van variëteiten - plantenparasitaire nematoden - globodera rostochiensis - globodera pallida - aardappelen - testen - akkerbouw - variety trials - varietal resistance - plant parasitic nematodes - globodera rostochiensis - globodera pallida - potatoes - testing - arable farming
In dit artikel wordt ingegaan op de achtergronden van AM-resistentie en de manier waarop deze resistentie wordt getoetst.
Globodera rostochiensis of G. pallida (Aardappelcysteaaltjes)
PPO Akkerbouw, Groene Ruimte en Vollegrondsgroente, - \ 2008
akkerbouw - gewasbescherming - plantenparasitaire nematoden - globodera rostochiensis - globodera pallida - nadelige gevolgen - nematodenbestrijding - arable farming - plant protection - plant parasitic nematodes - globodera rostochiensis - globodera pallida - adverse effects - nematode control
Het hoofdproductschap Akkerbouw heeft in het kader van het Actieplan Aaltjesbeheersing elf 'Rode lampen' laten ontwikkelen. Het gaat om leaflets die laboratoria vanaf 2008 per soort aaltje meesturen als een aaltjesmonster boven een bepaalde waarde uitkomt. Een extra attendering naar de akkerbouwer om maatregelen te nemen. In dit Actieplan wordt de akkerbouwer gewaarschuwd voor schadelijke aantallen Globodera rostochiensis of G. pallida (Aardappelcysteaaltjes)
Characterization of genes coding for small hypervariable peptides in Globodera rostochiensis
Bers, N.E.M. van - \ 2008
Wageningen University. Promotor(en): Jaap Bakker, co-promotor(en): Geert Smant; Aska Goverse. - [S.l.] : S.n. - ISBN 9789085049579 - 229
globodera rostochiensis - plantenparasitaire nematoden - peptiden - genen - genexpressie - solanum tuberosum - arabidopsis - nicotiana - gensplitsing - gastheer-pathogeen interacties - moleculaire interacties - globodera rostochiensis - plant parasitic nematodes - peptides - genes - gene expression - solanum tuberosum - arabidopsis - nicotiana - gene splicing - host pathogen interactions - molecular interactions
Plant parasitic nematodes secrete a cocktail of effector molecules, which are involved
in several aspects of the interaction with the host, eg. in host defense suppression, in
migration and in feeding cell formation. In this thesis, we performed the first study on
10 novel peptide genes, believed to be important for parasitism of the potato cyst
nematode, Globodera rostochiensis. Nine of the peptide genes described here belong
to the SECPEP gene family. The SECPEP genes are all expressed in the dorsal
esophageal gland, which is one of the main sites for the production of effector
molecules. This, together with the predominant expression in preparasitic and early
parasitic juvenile nematodes, makes it very likely that the SECPEPs code for effector
peptides essential for succesful infection and feeding site formation.
In chapter 2, we show that diversifying selection is a likely driver of the molecular
evolution of the SECPEPs. The sequences of the mature peptides appear to be highly
diverse, while the non)coding 3’UTR and intronic regions as well as the region coding
for the signal peptide for secretion are relatively conserved. In fact, a pairwise
comparison of the SECPEPs reveals no significant sequence similarity between family
members at all. In chapter 5 we further speculate on a possible role for RNA)editing as
a mechanism to yield hypervariability in the SECPEPs, because the sequence diversity
at the transcript level significantly exceeds that of the genomic locus. Chapter 5 further
elaborates on the analysis of trans)splicing in SECPEP1 transcripts. We show that
SECPEP1 transcripts are trans)spliced to a surprising diversity of novel spliced)leader
sequences. The first approach to unravel the role of the members of the SECPEP family
in plant parasitism, is described in chapter 4. We generated transgenic potato and
Arabidopsis plants expressing SECPEP3 while using the CaMV 35S promotor. The
phenotype associated with SECPEP3 in both potato and Arabidopsis plants includes a
reduction of root growth and an alteration of the leaf morphology. The SECPEP3
peptide harbors several sequence motifs first found in the cyclin)dependent kinase
inhibitors ICK1/KRP1, SIM and Smr1. We, therefore, suggest a role for SECPEP3 in cell
cycle alteration in nematode feeding site formation. Although the SECPEP genes show
only a low level of primary sequence similarity, all code for positively charged,
hydrophilic peptides with a C)x)G γ)core motif (chapter 2). These are characteristics
typical for host defense peptides, and in chapter 6 we investigate whether these
characteristics are also found for other peptides involved in plant)parasite interactions.
We show that a considerable number of these effector peptides share a positive
charge, hydrophilicity and C)x)G γ)core motif with the SECPEPs, and we speculate on a
role for the positive charge in peptide)ligand interaction.
In chapter 3 we describe the NEMPEP peptide, secreted by G. rostochiensis. NEMPEP
is also a positively charged, hydrophilic peptide with a C)x)G γ)core motif, although it is
genetically unrelated to the SECPEP gene family. During the life cycle of G.
rostochiensis, the expression pattern of NEMPEP reveals a striking regulation. NEMPEP
is highly expressed in preparasitic juveniles and in the parasitic life stages after initial
feeding cell formation. However, NEMPEP expression was hardly detectable in the
juveniles just after entering the plant root. Several disease resistance genes condition
nematode resistance at the onset of parasitism. The downregulation of NEMPEP at
exactly this timepoint could be a strategy to avoid recognition by the host’s immune
system. In planta expression of NEMPEP, as a fusion to GFP, shows that NEMPEP
accumulates in the nucleolus of tobacco cells. Potato plants transformed with
35S::NEMPEP were slow at forming roots and the internodes between the leaflets were
shortened. This, together with a reduced transformation efficiency, led us to
hypothesize a role for NEMPEP in cytokinin signaling (Chapter 3).
Currently, there are two models regarding the functional role of the SECPEPs and
NEMPEP. The first one concerns a role as an antimicrobial peptide, which could protect
the host plant against secondary infections by opportunistic microbes. As a competing
hypothesis, the high hydrophilicity of the peptides may point to a role as peptide
hormone. As such, they may be involved in redirecting cell cycle or hormonal regulation
upon feeding cell formation.
SPRYSEC effector proteins in Globodera rostochiensis
Rehman, S. - \ 2008
Wageningen University. Promotor(en): Jaap Bakker, co-promotor(en): Geert Smant; Aska Goverse. - S.l. : s.n. - ISBN 9789085048053 - 171
globodera rostochiensis - plantenparasitaire nematoden - pathogenesis-gerelateerde eiwitten - identificatie - gastheer parasiet relaties - planten - transgene planten - receptoren - verdedigingsmechanismen - co-evolutie - globodera rostochiensis - plant parasitic nematodes - pathogenesis-related proteins - identification - host parasite relationships - plants - transgenic plants - receptors - defence mechanisms - coevolution
Plant pathogens inject so-called effector molecules into the cells of a host plant to promote their growth and reproduction in these hosts. In plant parasitic nematodes, these effector molecules are produced in the salivary glands. The objective of this thesis was to identify and characterize effector molecules produced in the salivary glands of the potato cyst nematode Globodera rostochiensis. A combination of cDNA-AFLP and mining of EST-databases resulted in the identification of a large family of effectors named the SPRYSECs. The SPRYSECs essentially consist of a conserved SPRY domain preceded by a signal peptide for secretion. The SPRYSECs are injected into host cells through the oral stylet. A protein structure model of the SPRYSECs indicated that one particular surface of the proteins in the SPRYSEC family was hypervariable and seemed to undergo diversifying selection. This led us to believe that the SPRYSECs are important players in the co-evolution between plant and nematode. Transgenic potato plants overexpressing SPRYSEC-19 appeared to be two- to five-fold more susceptible to infections of nematodes, the fungus Verticillium dahliae, and tomato spotted wilt virus (TSWV). This hypersusceptibility to a range of unrelated plant-pathogens suggests that SPRYSECs somehow suppress the basal defense responses that are controlled by the plant’s innate immunity. SPRYSEC-19 was found to engage in a specific physical interaction with the Leucine Rich Repeat domain of a protein from the CC-NBS-LRR class of resistance genes. Many immune receptors in the plant’s innate immunity belong to the same class of NB-LRR proteins. The host interactor of SPRYSEC19 is most similar to members of SW5 R gene cluster that confers resistance to tospoviruses. Remarkably, plants harboring the CC-NB-LRR interactor of SPRYSEC19 are not resistant to nematodes. Therefore, we hypothesize that the nematode effector SPRYSEC-19 promotes its virulence in susceptible host plants by suppressing basal defense through its interaction with an NB-LRR immune receptor.
|Aardappel sterke larvenlokker
Hoek, J. ; Molendijk, L.P.G. - \ 2007
Boerderij 92 (2007)9. - ISSN 0006-5617 - p. 16 - 17.
aardappelen - globodera rostochiensis - lokgewassen - gewasbescherming - potatoes - globodera rostochiensis - trap crops - plant protection
Aardappelen kunnen als vanggewas worden ingezet om het aardappelcysteaaltje te bestrijden. De loof moet dan wel veertig dagen na poten worden dood gespoten om te voorkomen dat het aaltje zich kan vermeerderen
Vanggewas aardappel in de praktijk 2006
Hoek, J. ; Prins, F. - \ 2007
Lelystad : Praktijkonderzoek Plant & Omgeving, Sector AGV - 24
solanum tuberosum - aardappelen - globodera pallida - globodera rostochiensis - nematodenbestrijding - nematoda - gewasbescherming - akkerbouw - ondergewassen - ziektebestrijdende teeltmaatregelen - solanum tuberosum - potatoes - globodera pallida - globodera rostochiensis - nematode control - nematoda - plant protection - arable farming - catch crops - cultural control
PPO en Agrifirm zijn via een enquête onder een aantal telers nagegaan hoe men de teelt heeft uitgevoerd en welke teeltproblemen zich hebben voorgedaan. Daarnaast hebben telers suggesties gedaan voor verbetering van de regelgeving
NemaDecide: Een Beslissing Ondersteunend Systeem (BOS) voor aaltjes
Schomaker, C.H. ; Been, T.H. ; Molendijk, L.P.G. - \ 2006
globodera pallida - globodera rostochiensis - nematodenbestrijding - gewasbescherming - aardappelen - akkerbouw - beslissingsondersteunende systemen - globodera pallida - globodera rostochiensis - nematode control - plant protection - potatoes - arable farming - decision support systems
Ontwikkeling van NemaDecide, een Beslissing Ondersteunend Systeem (BOS) om aardappelcysteaaltjes te beheersen
|Raketblad als vanggewas voor aardappelcystenaaltjes
Hoek, J. ; Molendijk, L.P.G. ; Mheen, H.J.C.J. van der - \ 2006
Nieuwe oogst / Magazine gewas 2 (2006)3. - ISSN 1871-093X - p. 10 - 11.
solanum sisymbriifolium - globodera rostochiensis - globodera pallida - nematiciden bevattende planten - nematodenbestrijding - nematodenlarven - plagenbestrijding - onkruidbestrijding - grondsterilisatie - aardappelen - solanum tuberosum - solanum sisymbriifolium - globodera rostochiensis - globodera pallida - nematicidal plants - nematode control - nematode larvae - pest control - weed control - soil sterilization - potatoes - solanum tuberosum
Aardappelcysteaaltjes zijn te bestrijden door natte grondontsmetting. Eén andere bestrijdingsmaatregel, die door de PD (Plantenziektenkundige Dienst) ook wordt erkend, is de teelt van een 'vanggewas'. Raketblad is zo'n vanggewas en een verwant van aardappelen, afkomstig uit Zuid-Amerka. Uit onderzoek van Wageningen Universiteit bleek dat raketblad in staat is om de larven van de aardappelcysteaaltjes uit rust te laten komen. Omdat raketblad resistent is tegen aardappelcysteaaltjes, kunnen de larven zich niet voeden en sterven af
Dissecting host plant manipulation by cyst and root-knot nematodes
Karczmarek, A. - \ 2006
Wageningen University. Promotor(en): Jaap Bakker, co-promotor(en): Aska Goverse; Hans Helder. - Wageningen : Wageningen Universiteit - ISBN 9789085044642 - 120
globodera rostochiensis - heterodera schachtii - meloidogyne incognita - plantenparasitaire nematoden - waardplanten - plantengroeiregulatoren - auxinen - interacties - reuzencellen - celbiologie - gastheer parasiet relaties - globodera rostochiensis - heterodera schachtii - meloidogyne incognita - plant parasitic nematodes - host plants - plant growth regulators - auxins - interactions - giant cells - cellular biology - host parasite relationships
Cyst ( Globodera spp. and Heterodera spp.) and root-knot nematodes ( Meloidogyne spp.), one of the most damaging crop pests, are a perfect example of highly adapted, sophisticated root parasites. These nematodes induces specialized feeding structures (cyst nematodes-syncytia, root-knot nematode-giant cells) within the host root and benefits from guaranteed continues supply of food and water from their host plant and protection within the cyst or gall for their progeny. This dissertation aims at elucidation of the molecular mechanisms controlling the induction and maintenance of those feeding structures. This knowledge is indispensable to make novel advances in constructing durable host plant resistance.A number of different observations suggested a primary role of the phytohormone auxin in the feeding cell development. However, it has never been directly proven. Therefore, we have used the DR5 promoter element fused to gusA reporter gene to visualize spatial and temporal changes in auxin distribution, during nematode infection in Arabidopsis thaliana roots infected with cyst or root-knot nematodes. DR5 promoter element is considered to be a specific indicator for auxin and is activation points at an increase of the perceived auxin concentration. For both, cyst and root-knot nematode species, strong GUS expression was observed at the very onset of parasitism, already at 18 hours post inoculation. This high expression level was maintained until 3-5 days post inoculation and then gradually was reduced. Semi-thin sections of infected roots were investigated microscopically, revealing the presence of GUS inside the initial cells, and in case of cyst nematodes, in cells to-be incorporated into syncytium. This implies that expansion of syncytium is marked by the regions with high gusA expression. Activation of DR5 promoter element can be explained by: an accumulation or an increased sensitivity to auxin. However, based on formerly gathered data, a local accumulation seems to be the more probable explanation. Moreover, the feeding structures of cyst and root-knot nematodes arise by distance mechanisms, hence the highly similar DR5 activation patterns in both feeding structures, is remarkable. This promptthe conclusion that notwithstanding the highly dissimilar structure and ontogeny, both nematodes interfere with similar signal transduction pathways in root morphogenesis.Apart from phytohormones, small peptide signalling plays a very important role in diverse aspects of plant growth and development, and such a molecule(s) could be potentially involved in the establishment of nematode feeding cell. Early nodulin ( ENOD ) genes have been defined as genes expressed in legumes during nodule formation. Recent studies, however, revealed a number of homologues of ENOD40 s in non-legume plants. In both plants types, ENOD40 expression associates with the development of the vascular tissue. Feeding site development by root-knot and cyst nematodes is accompanied by the distortion of the vascular tissue, therefore a local increase in ENOD40 expression could be anticipated. This holds truth in interaction between legume Medicago truncatula and root-knot nematodes. However, in non-legume tomato infected with cyst and root-knot nematodes, no change in ENOD40 transcript levels could be detected using RT-PCR. In addition, activation of the Sl-ENOD40 promoter was only observed in infected regions showing endogenous ENOD40 expression. Similar results were obtained for a legume ENOD40 promoter (soybean) in a non-legumes background ( Arabidopsis ), and - contrary to M. truncatula - over-expression of Gm-ENOD40 in Arabidopsis had no effect on nematode development. Hence, ENOD40 expression in tomato is basically unaffected by the induction of feeding sites by root-knot and cyst nematodes. This unexpected difference in nematode-induced ENOD40 is apparently plant background-dependent, and suggests the existence of a legume-specific factor that boosts the ENOD40 expression in legume hosts-nematode interaction.One of the most striking characteristic of the cyst nematode induced feeding structure (syncytium) is the extensive cell wall dissolution, as the syncytium expands towards the vascular bundle and along the xylem vessels. Endo-1,4-ß-glucanases (cellulases) reside among plant enzymes that are involved in this progressive cell wall dissolution.RT-PCR experiments in tomato revealed that the potato cyst nematode induces the expression of two out of the eight described cellulases, precisely, Sl-cel7 and Sl-cel8 . In situ hybridization and immunodetection studies showed that both cellulases are expressed inside and adjacent to nematode-induced syncytia. Transgenic potato plants carryinghpRNA-silencing constructs for cel7 and cel8 were infected with cyst nematode which resulted in a significant arrest of cyst nematodes development.Reduced development of juveniles into females (up to 60%) and increase in the fraction of females without eggs (up to 89%) was observed. This indicates that the recruitment of specific plant cellulases by potato cyst nematodes is essential for their development, and demonstrates how detailed knowledge of compatible plant-parasite relationships can lead to host plant resistance.The genomic sequence of potato, cel7 and cel8a was cloned and characterised, in order to enable the RNA interference experiments in the natural host of potato cyst nematodes. The analysis of sequences suggests the presence of multiple isoforms of cel8 in potato. That corresponding proteins consist solely of a catalytic core domain (St-CEL7) or of a catalytic domain linked to a carbohydrate binding module (CBM) (St-CEL8a). Only recently, the CBM domain of Sl-CEL8a was shown to belong to CBM family 2 and on the basis of shared features between CBM domains in Sl-CEL8 and St-CEL8a it can be concluded that St-CEL8a harbours a cellulose binding domain as well. Therefore, those findings imply that the potato cellulases recruited by the potato cyst nematode are able to hydrolyse both xyloglucan (CEL7) and crystalline cellulose (CEL8), and apart from cloning novel potato cellulases genes, provides an explanation why nematodes recruit exactly these two members of the cellulase family. Moreover, potato cellulase sequences were used to mine EST databases and this information was used for the generation of a phylogenetic tree of the Solanaceous endo-β-1,4-D-glucanase gene family. In this dissertation we take an effort to understand better the intimae relationship between the obligatory plant parasites, cyst and root-knot nematodes and host plants. We show that such a detailed knowledge can be beneficial in better understanding of plant physiology and can be used as a highly specific and bio-safe approach for pathogen management.
The role of cell wall-modifying proteins in plant penetration and feeding site proliferation by the potato cyst nematode Globodera rostochiensis
Kudla, U. - \ 2006
Wageningen University. Promotor(en): Jaap Bakker, co-promotor(en): Hans Helder; Geert Smant. - Wageningen : Wageningen Universiteit - ISBN 9789085044147 - 121
globodera rostochiensis - plantenparasitaire nematoden - celwanden - eiwitten - pectaat lyase - globodera rostochiensis - plant parasitic nematodes - cell walls - proteins - pectate lyase
The main objective of this thesis was to investigate two distinct groups of proteins involved in plant cell walls modifications in the parasitism of the potato cyst nematode, Globodera rostochiensis , namely pectate lyases and expansins. Plant parasitism of potato cyst nematode proceeds through two main stages i.e. mobile and sessile. During the migratory phase, potato cyst nematode uses cell wall degrading enzymes and mechanical force of the stylet protruding from nematode's buccal cavity to disrupt cell walls in host plant tissue. In order to degrade plant cell wall components, nematodes produce a range of cell wall degrading and modifying proteins. Plant parasitic nematodes were the first animals for which it was shown that they are able to degrade cell wall polymers (i.e. cellulose microfibrils) without the help of symbiotic microorganisms. This first finding of an endoglucanase produced by G. rostochiensis turned out to be only a tip of an iceberg. To date, similar evidence is found in plant parasitic nematodes for the presence of enzymes capable of depolymerising the pectin matrix in plant cell walls (i.e. pectate lyase and exopolygalacturonase). In Chapter II, a novel putative pectate lyase gene from G. rostochiensis is described including a functional assay using Agrobacterium mediated leaf infiltration and site directed mutagenesis.In Chapters III and IV, we showed that potato cyst nematode is producing and secreting expansins, which are likely to aid the activity of cell wall degrading enzymes. Contrary to pectinolytic enzymes expansins are a relatively newly described group of proteins and there are still a lot of unresolved issues that need to be addressed with regard to their mode of action, biological functions and classification. Since their discovery in 1992, expansins were thought to be specific for land plants. In this thesis, we present the first evidence of a functional expansin produced outside of the plant kingdom. We demonstrated that invasive juveniles of G. rostochiensis secrete b-expansin, which most likely facilitates the disruption of cell walls in the root tissues of a host by making the structural cell wall polymers more accessible to cell wall degrading enzymes.The cell wall modifications occurring during feeding site formation by potato cyst nematode, described in the fourth chapter of this thesis, are part of the elaborate changes in the plant cell wall architecture during syncytium induction and maintenance. In contrast to the rigorous breakdown of cell walls and subsequent collapse of the protoplast during nematode migration, the modifications during feeding site formation aim at a subtle remodeling of the cell wall, including both cell wall dissolution and synthesis. The uniform structure of nematode induced syncytia suggests that these cell wall modifications are precisely controlled. A key role for the recruitment of plant enzymes (e.g. cellulases and pectin methyl esterases), by nematodes to modify plant cell wall structure to their advantage has been pointed at in several papers. The data presented in Chapter V shows that these cell wall modifications in a nematode-induced syncytium are mediated by enzymes and -expansins and expansin-like proteins of the host plant. This is the first report that links morphological changes in the cell walls of dicotes plants to the expression ofb- expansin genes.
|Aardappel als vanggewas voor aardappelcysteaaltjes
Beers, T.G. van - \ 2005
Kennisakker.nl 2005 (2005)15 april.
aardappelen - globodera rostochiensis - nematoda - gewasbescherming - plagenbestrijding - akkerbouw - potatoes - globodera rostochiensis - nematoda - plant protection - pest control - arable farming
In consumptieaardappelrassen is nog steeds weinig resistentie beschikbaar tegen het witte aardappelcysteaaltje, Globodera pallida. Alleen de rassen Innovator, Santé, Vechtster, Homage, Aziza, Balade en Maritiema bezitten resistentie tegen G. pallida. Er zijn dus weinig mogelijkheden om een besmetting in de hand te houden als bovengenoemde rassen niet beschikbaar zijn. Een mogelijkheid is het gebruik van aardappel als vanggewas. Mits zorgvuldig uitgevoerd, is het haalbaar om een afname in de populatiedichtheid van 80 tot 90% te halen.
Solanum sisymbriifolium (Lam.) : a trap crop for potato cyst nematodes
Timmermans, B.G.H. - \ 2005
Wageningen University. Promotor(en): Martin Kropff, co-promotor(en): Jan Vos; Tjeerd-Jan Stomph. - Wageningen : - ISBN 9789085043256 - 135
solanum sisymbriifolium - globodera pallida - globodera rostochiensis - zaadkieming - groeianalyse - wortelsystemen - uitbroeden - ziekteresistentie - phytophthora infestans - nederland - solanum sisymbriifolium - globodera pallida - globodera rostochiensis - seed germination - growth analysis - root systems - hatching - disease resistance - phytophthora infestans - netherlands
Keywords:Solanumsisymbriifolium ,Globoderapallida ,Globoderarostochiensis , germination, light use efficiency, crop growth, crop management, root length density, hatching, trap crop, tolerance,Phytophthorainfestans , resistance.Potato cyst nematodes (PCN),Globoderapallida (Stone) and G.rostochiensis (Woll.) continue to be a major pest in potato growing areas, in spite of existing control measures. Therefore,Solanumsisymbriifolium (Lam.) was introduced as a trap crop for PCN in
The identification of cell wall degrading enzymes in Globodera rostochiensis
Popeijus, H.E. - \ 2002
Wageningen University. Promotor(en): J. Bakker; A. Schots; G. Smant. - S.l. : S.n. - ISBN 9789058086914 - 86
globodera rostochiensis - plantenparasitaire nematoden - pectaat lyase - genen - genexpressie - cellulase - celwanden - complementair dna - globodera rostochiensis - plant parasitic nematodes - pectate lyase - cellulase - genes - gene expression - cell walls - complementary dna
This thesis describes the identification of cell wall degrading enzymes of the potato cyst nematode Globodera rostochiensis . A robust method using expressed sequence tags (ESTs) was applied to identify new parasitism related enzymes. One of the ESTs revealed the first pectate lyase from a metazoan origin. Another tag shared a strong identity towards a previously determined N-terminal amino acid sequence. Further analysis of corresponding cDNA sequence and the gene revealed two closely related beta-1,4-endoglucanases. Heterologous expression of the pectate lyase and both beta-1,4-endoglucanases showed that they are active enzymes towards their appropriate substrates ( e.g. polygalacturonic acid for the pectate lyase and carboxy methyl cellulose for both beta-1,4-endoglucanases respectively). The application of in situ hybridisation predict that these cell wall degrading enzymes are produced in the subventral oesophageal gland cells. Evidence is provided that nematodes use mixtures of cell wall degrading enzymes in order to penetrate and migrate in the plant root.
Molecular genetic analysis of the pathogenicity of the potato cyst nematode Globodera rostochiensis
Qin, L. - \ 2001
Wageningen University. Promotor(en): J. Bakker; J. Helder, co-promotor(en): G. Smant. - S.l. : S.n. - ISBN 9789058084521
plantenparasitaire nematoden - globodera rostochiensis - complementair dna - polymorfisme - genexpressie - pathogeniteit - moleculaire genetica - bio-informatica - plant parasitic nematodes - globodera rostochiensis - pathogenicity - complementary dna - polymorphism - gene expression - molecular genetics - bioinformatics
A new strategy to identify pathogenicity factors from the potato cyst nematode Globodera rostochiensis is developed. cDNA-AFLP technology and in situ hybridization allowed us to efficiently select putative pathogenicity factors among thousands of expressed genes. As a result, an unprecedented number of putative pathogenicity factors from this obligatory plant parasite were cloned.
A powerful bioinformatics tool named GenEST to link expression data generated from cDNA-AFLP directly to cDNA sequence data was developed. This computer program would be very useful for functional genomics studies in other systems as well.
A RanBPM (Ran-Binding Protein in Microtubule organization)-like gene family was identified. The proteins encoded by these genes are probably secreted by the nematode into plant cells and might manipulate the host cell development by changing the dynamic instability of microtubules.
A promoter region from the potato cyst nematode was shown to be functional in a distantly related nematode species Caenorhabditis elegans . This promoter could be a valuable tool to drive gene expression in transgenic plant-parasitic nematode species to assess the relative importance of putative pathogenicity factors.
A paper based from one chapter of this thesis will be submitted to Science. In view of their press embargo policy, I will not discussed the detail of this particular finding here.
Endogenous cellulases in stylet secretions of cyst nematodes
Smant, G. - \ 1998
Agricultural University. Promotor(en): J. Bakker; A. Schots; F.J. Gommers. - S.l. : Smant - ISBN 9789054859260 - 111
plantenparasitaire nematoden - globodera rostochiensis - heterodera glycines - secreties - gastheer parasiet relaties - speeksel - cellulase - plant parasitic nematodes - globodera rostochiensis - heterodera glycines - secretions - host parasite relationships - saliva - cellulase
This thesis describes the identification ofβ-1,4-endoglucanases (cellulases) in stylet secretions of the two cyst nematodes species, Globodera rostochiensis and Heterodera glycines . A novel method was developed to raise monoclonal antibodies that were directed to subventral oesophageal gland secretions. These monoclonal antibodies were used to characterise and to immunopurify two secretory proteins. Partial sequence data from these proteins enabled the cloning of two homologous genes from each of the two cyst nematode species.
The predicted amino acid sequences revealed a high similarity with bacterial cellulases, whereas no homology was found with eukaryotic cellulases. Evidence is provided for the endogenous origin the nematode cellulases that may have been acquired from bacteria by horizontal gene transfer.
Genetic diversity of the potato cyst nematode in the Netherlands
Folkertsma, R.T. - \ 1997
Agricultural University. Promotor(en): R.W. Goldbach; F.J. Gommers; J. Bakker. - S.l. : Folkertsma - ISBN 9789054857488 - 88
plantenplagen - pratylenchus - heteroderidae - tylenchidae - solanum tuberosum - aardappelen - genetische variatie - overerving - rassen (dieren) - rassen (taxonomisch) - moleculaire genetica - plantenveredeling - ziekteresistentie - plaagresistentie - globodera pallida - globodera rostochiensis - plant pests - pratylenchus - heteroderidae - tylenchidae - solanum tuberosum - potatoes - genetic variation - inheritance - breeds - races - molecular genetics - plant breeding - disease resistance - pest resistance - globodera pallida - globodera rostochiensis
The potato cyst nematodes Globodera rostochiensis (Woll.) Skarbilovich and G. pallida (Stone) originate from the Andes region in South America and have been introduced into Western Europe since 1850. Both species are successful colonizers. Once primary founders have established vital populations, an area is rapidly colonized by secondary founding events. The mode of spread results in patchy distribution patterns. Analyses of the processes that influence the spatial variations in virulence are of major importance for the control by means of host plant resistance. The ability to unravel the mosaic distribution patterns of the two species and their pathotypes enables breeders and growers to anticipate on the dynamics of virulent populations. The aim of this thesis was to analyse the intra- and interspecific variation of G. rostochiensis and G. pallida in the Netherlands and to obtain insight in the processes that determine the spatial variations in protein, DNA and (a)virulence polymorphisms.
The molecular variation between the sibling species G.rostochiensis and G.pallida is remarkably large. The RAPD technique revealed a total of 250 DNA fragments, of which only nine DNA fragments were common to both species (Chapter 3). Similar results were obtained with the AFLP assay. A total of 1000 AFLP fragments was amplified, of which only 64 fragments could be identified in both species (Chapter 4). These results agreed with previous investigations using 2-DGE and show that morphologically nearly indistinguishable organisms can be quite distinct the molecular level.
The extensive genetic differentiation of G.rostochiensis and G . pallida offers perspectives for the development of a diagnostic assay. In addition the PCR technique enables the amplification of species specific fragments from small numbers of cysts extracted from soil samples. For example, G. rostochiensis specific DNA fragments can be amplified from single juveniles (Chapter 1).
The intraspecific variation of G.rostochiensis as revealed by RAPDs and AFLPs is relatively low (Chapter 3 and 4). The proportion of polymorphic DNA fragments among nine G. rostochiensis populations was 19% and 15.8%, respectively. Three clusters of populations were identified and each cluster could be described by one or more specific DNA fragments.
The intraspecific variation of G.pallida, as revealed with RAPDs or AFLPs, is larger in comparison with G.rostochiensis (Chapter 3 and 4). The proportion of polymorphic RAPD fragments among 17 populations was 46%. For the AFLP assay this figure was 23% polymorphic AFLP fragments among 15 populations. The majority of the populations displayed a continuous range of variations. Only a few clearly diverged clusters could be discriminated on the basis of specific DNA fragments.
Various groups have applied the RAPD technique to study the genetic divergence among conspecific populations of plant parasitic nematodes. The validity of this technique was evaluated in chapter 4 by comparison of the clustering of 36 G.pallida populations based on RAPD and 2-DGE data. Both data sets demonstrated that the majority of the G.pallida populations were not clearly differentiated from each other. The overall correlation between the distance matrices derived from both data sets was low. Careful examination of the separate dendrograms showed similarity in clustering only for clearly diverged populations or groups of populations.
Although the AFLP and RAPD technique are simple, fast and require only minute amounts of biological material, they are not suitable to resolve the subtle differences among potato cyst nematode populations. Quantitative variation in allele frequencies is often not resolved with those techniques, which is, among others, due to the virtual inability to recognize co-dominant alleles and the non-linear amplification of DNA fragments. In chapter 6 and 7 the genetic variation was studied by analysing pools of individuals with 2-DGE. The ratios between the protein quantities produced by the codominant alleles are appropriate measures for the allele frequencies. The correctness of this method has been confirmed by 2-DGE of single individuals (De Boer etal ., 1992)
bottleneck effects on the secondary founders
The intraspecific variation among potato cyst nematode populations in Europe is predominantly determined by the genetic constitution of the primary founders, directly or indirectly introduced from South America, and the effect of random genetic drift on the secondary founders. To obtain insight in these processes, 226 G.pallida populations from the Netherlands were analyzed with 2- DGE. The results strongly suggest that these populations originate from one source, or in case of multiple introductions, from a number of sources with a similar genetic makeup.
The genetic differentiation of the 226 G.pallida populations indicate that the colonization of the Netherlands has been accompanied by extensive random genetic drift. Only a limited proportion of the populations appeared to be identical. It is also shown, that the bottleneck effects differ between regions. Significant variation in population structure was observed between the three investigated areas. The genetic variation within G.pallida populations from north Friesland and the IJsselmeerpolders is in general smaller than within populations from the northeast Netherlands. A plausible explanation for this phenomena is the low multiplication rate of potato cyst nematodes due to wider crop rotation schemes in the IJsselmeerpolders and north Friesland. These factors result in a slow expansion of newly founded populations, which enhances the effect of drift.
gene-pool similarity concept
The gene-pool similarity concept rests on the hypothesis that in the absence of selection pressure by host plant resistance, degrees of similarity between populations revealed by molecular techniques are also reflected at virulence loci, including those not yet resolved. To test this concept, the genetic variation revealed by 2-DGE among 102 G.pallida populations was compared with the variation in virulence towards two resistant cultivars. This analysis showed that a decrease in genetic distance among populations is accompanied with a decrease in variation in virulence. In addition it is demonstrated that the gene pool similarity concept is also applicable to loci determining the variation in fitness among populations. The variation in Pf/Pi values among the 102 populations on Désirée is in general smaller between closely related populations than between distantly related populations.
breeding for resistance
Breeding for resistance has been dominated by trial-and-error approaches, which has stimulated the view that control by means of host plant resistance is unavoidably a short-term approach due to the 'appearance' of virulent populations. The pervasive myth that breeding for resistance against potato cyst nematodes is a lost arms race is challenged by the results of this thesis. Until recently it was assumed that the genetic variation of G.pallida in the Netherlands was too large to produce potato cultivars with broad-spectrum resistance. In this thesis it is shown that the genetic diversity introduced from the Andes region has been limited and that the variation among the Dutch G.pallida populations is mainly the result of random genetic drift. The elaborate analysis of 226 Dutch G.pallida populations offers perspectives to obtain potato cultivars with broad and durable resistance. The gene pool similarities revealed by 2-DGE can be used as guidance in testing the effectiveness of new sources of resistance.
Interactions between the closely related potato cyst nematode species Globodera rostochiensis (Woll.) and G. pallida (Stone)
Nijs, L.J.M.F. den - \ 1992
Agricultural University. Promotor(en): L. Brussaard, co-promotor(en): C.J.H. Booij. - S.l. : Den Nijs - 75
plantenplagen - solanum tuberosum - aardappelen - pratylenchus - heteroderidae - tylenchidae - globodera rostochiensis - globodera pallida - globodera rostochiensis - globodera pallida - plant pests - solanum tuberosum - potatoes - pratylenchus - heteroderidae - tylenchidae
in integrated control of the potato cyst nematodes, Globodera rostochiensis and G. pallida , the use of resistant varieties is an important strategy, especially now the application of agrochemicals in The Netherlands is increasingly restricted. Repeated cropping of varieties with the same or similar resistance genes produces a selection pressure on nematode populations leading to an increase in virulence and a progressive loss of resistance. To prolong the efficient use of resistant varieties, alternate cropping of varieties with species specific resistance instead of totally resistant varieties might prove to be a more successful strategy. In this situation the population development of each nematode species is alternately suppressed and selection for increased virulence will be reduced at the same time. To develop rotation schemes with these objectives information is needed on the reproduction of the species on the varieties to be used, the actual species composition and distribution in the field, and the interactions between the nematode species. The latter was the subject of this thesis (chapter 1)
A method has been developed to quantify species ratios in mixed populations. The method is based on the separation of species specific thermostable proteins by SDS- PAGE. Densitometric analysis of the 17 kD protein of G. pallida and the 18 kD protein of G. rostochiensis revealed a high correlation (R=0.93) with the species ratio in the mixed samples. Within the limits of 10 to 90% of each species, one can estimate with 95% reliability the species composition with 3 to 6% deviation (chapter 2).
To determine whether interactions between the species takes place mixtures of G. rostochiensis (Ro1) and G. pallida (Pa3) were reared on susceptible (cv. Bingtje) and species specific resistant (cv. Elkana and clone ZB35-29) potato varieties at different nematode densities. The proportion of newly formed cysts belonging to each species was determined by way of SDS-PAGE and the relative population increase of each species, defined as the final population (P f ) divided by the initial population (P i ), was calculated. Comparisons of the relative population increase of each species in the single populations and in the mixtures revealed that interaction between the two species occurred. The relative population increase of G.rostochiensis was significantly decreased when G. pallida was highly dominant in the mixed populations. These effects were density independent which makes it unlikely that exploitation competition is responsible for this. It was suggested that interference competition for mates might be responsible for these interaction effects (chapter 3).
Differences in hatching behaviour of the two species in response to the host plant could interfere in the interaction. Therefore, hatching differences of G.rostochiensis and G.pallida were assessed in potato root diffusate (PRD) of cv. Bintje, cv. Elkana and clone ZB35-29. G.pallida hatched better in the PRDs than G. rostochiensis. However, the reduced relative population increase of G.rostochiensis was found at all densities indicating that G.pallida did not reach the roots sooner and occupy all available feeding sites. Therefore, it was concluded that the differences in hatching behaviour did not play any significant role in the interactions.
It was shown that the experimental test conditions strongly influenced the hatching results. The water type used in the hatching tests had a significant discriminating effect on the species; . rostochiensis had a relative hatch of 60 to 90% in demineralized and tap water, whereas the hatch of G.pallida never exceded 15%. These differences were independent of the various cyst batches that were used or the different years the tests were carried out. Silver sand percolate had an inhibiting effect on the hatching of both nematode species. The high concentration of boron may explain the latter effect (chapter 4).
An investigation was conducted to study whether both species could hybridize in pots under optimal environmental conditions and to see what consequences this might have for the next generations. Pot experiments were carried out for four years with 50% mixtures of both species and single species populations, reared on the susceptible cv. Bintje. Analysis of the cyst production, the cyst contents and the relative population increase of each species show that the two species do not interbreed. They reproduce independently and are in stable coexistence in these circumstances. The fact that hybridization did not take place confirms the idea that the observed interaction is mainly a result of interference competition (chapter 5).
The different reaction of the species to the initial hatching trigger, the consistently different behaviour in hatching and reproduction and the absence of hybridization indicate that the species are much more divergent than the term sibling species suggests. Findings on one of the two species cannot be uncritically translated to the othet species. A thorough study of the biology of the species on natural host plants in the country of origin will give a better understanding of the ecological differences.
The results were obtained under controlled conditions with optimal chances for interspecific encounters leading to interactions. Only in one of the experiments was a significant but slight interaction found. In field situations conditions are much more heterogeneous and variable causing seggregation of the species. Additionally, nematodes infest the soil as cysts ensuring a certain amount of aggregation. It is therefore unlikely that interactions occur in the field, hence both species populations will develop independently.
For control strategies, in particular for using the right resistant varieties, it is essential to know which species inhabits the soil and additionally what species composition is present. For monitoring field populations the method described here has some draw backs, a recently developed ELISA test might be of more use.
To prevent underestimation of the total population increase one ought to determine the initial density of both species separately, as nematode reproduction is dependent on its own population density before planting. Predictions of the population development based the separate species are relatively straight forward because interactions between the different potato cyst nematode species in the field are of no importance (chapter 6).
Genetics of virulence in potato cyst nematodes
Janssen, R. - \ 1990
Agricultural University. Promotor(en): A.F. van der Wal; F.J. Gommers. - S.l. : Janssen - 71
plantenplagen - pratylenchus - heteroderidae - tylenchidae - solanum tuberosum - aardappelen - heritability - genetica - overerving - globodera rostochiensis - plant pests - pratylenchus - heteroderidae - tylenchidae - solanum tuberosum - potatoes - heritability - genetics - inheritance - globodera rostochiensis
Het centrale thema van het onderzoek was de bestudering van de genetische achtergronden van virulentie en avirulentie van Globoderarostochiensis voor het H 1 resistentiegen in Solanumtuberosum ssp. andigena CPC 1673. Deze monogene resistentie is op dit moment de belangrijkste vorm van resistentie in consumptie- en fabrieksaardappelrassen in West-Europa. Net als bij andere planteparasitaire nematoden werd genetisch onderzoek aan aardappelcysteaaltjes belemmerd door problemen bij het uitvoeren van kruisingsexperimenten. Het genetisch onderzoek dat desondanks werd uitgevoerd kenmerkte zich door tijdrovende experimenten vanwege de diapauze van bijna één jaar, niet goed te controleren massa kruisingen, en het gemis aan populaties met nauwkeurig gedefinieerde niveau's van virulentie. Genetische modellen konden slechts getoetst worden aan de hand van het gedrag van veldpopulaties. Het model van Jones, gebaseerd op het gen-om-gen systeem dat voor het eerst gevonden werd bij de plant/schimmel interactie vlas/roest, beschrijft op een redelijke wijze de virulentie-ontwikkeling van veldpopulaties. In zijn theorie zijn virulente individuen voor het H 1 resistentiegen homozygoot recessief.
In de eerste drie hoofdstukken van dit proefschrift zijn de methoden beschreven waarmee binnen een acceptabele tijd genetische experimenten uitgevoerd kunnen worden. Genetische analyses van virulentie in aardappelcysteaaltjes vormen het onderwerp van de laatste twee hoofdstukken.
In het eerste hoofdstuk wordt een methode beschreven waarmede achtereenvolgende generaties aardappelcysteaaltjes gekweekt kunnen worden door de diapauze te omzeilen. Hiertoe worden de cysten gekweekt op wortels van aardappelspruiten in Petrischalen met wateragar of in grond en zorgvuldig vochtig gehouden. De larven worden uit de cyst gelokt door de cysten met een scalpel te halveren of voorzichtig door te drukken zonder de eieren te beschadigen ("crushen") en deze vervolgens te incuberen in lokstof. Op deze wijze wordt ongeveer 40% van de cysteinhoud gelokt. Deze behandeling heeft geen nadelige invloeden op de vitaliteit van de larven en de hieruit ontwikkelde mannetjes en vrouwtjes. Dit geldt zowel voor eieren uit cysten gekweekt in Petrischalen als in grond. Op deze wijze is het mogelijk drie tot vijf generaties per jaar in potten te kweken en vijf tot zes generaties in Petrischalen.
Een essentieel onderdeel voor het uitvoeren van het genetisch onderzoek was een gestandaardiseerde en nauwkeurige bepaling van de virulentie in populaties (hoofdstuk II). Hiervoor werden cysten in Petrischalen op wortels van aardappelspruiten gekweekt. Optimale resultaten werden verkregen door twee larven per wortelpunt te inoculeren. Het percentage virulente Individuen in diverse populaties werd bepaald door larven op een vatbare en een resistente cultivar te inoculeren en het aantal gevormde vrouwtjes op de resistente cultivar uit te drukken in percentages van het aantal gevormde vrouwtjes op de vatbare cultivar. Deze percentages varieerden aanzienlijk in populaties van pathotypen van zowel G.rostochiensis als G.pallida . Ter bepaling van de nauwkeurigheid van de methode werd een vergelijking gemaakt met de gangbare pathotypetoets op basis van P f /P i bepalingen. De waarden voor de variatiecoëfficient geven duidelijk aan dat de methode In Petrischalen beter geschikt is voor het bepalen van virulentieniveau's dan aan de hand van potexperimenten. Voor verder onderzoek werden de populaties Ro 1 -Mierenbos en Ro 5 -Harmerz uitgekozen met respectievelijk <0,1% en 84.6% virulente individuen voor het H 1 resistentiegen.
Het kweken van cysten onder geconditioneerde omstandigheden en de hierboven beschreven techniek maakten het mogelijk om na gecontroleerde enkelvoudige kruisingen virulente en avirulente lijnen voor het H 1 resistentiegen te selecteren (hoofdstuk III). Voor deze kruisingen werden mannetjes In grond en vrouwtjes op wortels in Petrischalen gekweekt. Kruisingen werden uitgevoerd door één mannetje te plaatsen op de gelatineuze matrix van het vrouwtje. In een groot aantal F 2 en F 3 lijnen werden de virulentieniveau's bepaald. Uit populatie Ro 1 -Mierenbos en Ro 5 -Harmerz werden respectievelijk een avirulente en een virulente lijn geselecteerd, die gebruikt werden voor een nadere genetische analyse.
Reciproke kruisingen tussen individuen van een avirulente en een virulente lijn gaven avirulente nakomelingen (F 1 ). Zelfbevruchting van de F 1 leverde in de F 2 25% virulente en 75% avirulente individuen. Deze 1:3 uitsplitsing betekent dat virulentie recessief (a) is; virulente individuen zijn homozygoot recessief (aa), en avirulente individuen homozygoot dominant (AA) of heterozygoot (Aa). Bovendien is (a)virulentie niet geslachtsgebonden (hoofdstuk IV).
Met behulp van de virulente en de avirulente lijn en de heterozygote F 1 in combinatie met een betrouwbare techniek om mannetjes te verzamelen werd het effect van heterozygotie op de vorming van mannetjes en vrouwtjes bestudeerd. Zowel de homozygote (AA) als de heterozygote (Aa) avirulente larven kunnen zich op cultivars met het H 1 gen niet tot vrouwtjes ontwikkelen maar wel tot mannetjes. Heterozygote avirulente mannetjes waren enigszins in het voordeel in vergelijking met homozygote avirulente mannetjes. Dit verschil is echter te klein om effect te kunnen hebben op populatie-genetisch niveau (hoofdstuk V).
Deze genetische analyse bewijst dat er een klassieke gen-om-gen relatie bestaat tussen (a)virulentie in G.rostochiensis en het H 1 resistentiegen en opent de weg voor het opsporen van het (a)virulentiegen en mogelijk ook het H 1 resistentiegen. Met behulp van "single copy" DNA probes kunnen RFLP's (Restriction Fragment Length Polymorphism) opgespoord worden die nauw gekoppeld zijn met het (a)virulentiegen. Hiertoe is de F 1 (Aa) van een virulente (aa) en avirulente (AA) teruggekruist met de virulente ouderlijn (aa). De T 1 is vervolgens opnieuw teruggekruist (T 2 ) met de virulente ouder om virulente lijnen te selecteren voor het H 1 resistentiegen in cultivar 'Saturna' (Figuur l). Deze kruising gaf de gewenste 1:1 uitsplitsing te zien. Er worden nu ongeveer 300 T 2 lijnen vermeerderd. Een set discriminerende probes voor de ouderlijnen is in ontwikkeling. In het geval de RFLP's niet zijn gekoppeld met (a)virulentie, zullen in de 300 T 2 lijnen de DNA-banden van beide ouderlijnen aanwezig zijn. Geldt echter koppeling dan wordt alleen het DNA-patroon van de virulente ouderlijn verkregen, tenzij er recombinatie heeft plaatsgevonden. Het percentage recombinanten geeft waardevolle informatie voor het construeren van koppelingskaarten. Ook kan de fysische afstand tussen een aantal RFLP's die nauw gekoppeld zijn met het (a)virulentiegen worden bepaald met behulp van "Pulsed Field Electrophoresis". Dit is een belangrijke stap op weg naar het isoleren en karakteriseren van dit (a)virulentiegen in het aardappelcysteaaltje, wat vervolgens kan leiden tot localisatie van het complementaire resistentiegen in de plant.
Tot slot, het hier samengevatte onderzoek betreft een viertal methodieken die ondanks hun eenvoud baanbrekend zijn geweest voor het uitvoeren van genetisch onderzoek aan aardappelcysteaaltjes: (i) het opkweken van vier tot vijf generaties aardappelcysteaaltjes per jaar, (ii) een accurate methode voor het bepalen van het aantal virulente individuen in een populatie, (iii) het kweken van cysten op aardappelwortels in Petrischalen, en (iv) het uitvoeren van gecontroleerde kruisingen tussen één vrouwtje en één mannetje. Dit veredelingswerk resulteerde in de selectie van een avirulente en een virulente lijn met behulp waarvan het genetisch bewijs werd geleverd voor het bestaan van een gen-om-gen relatie tussen (a)virulentie in G.rostochiensis en het H 1 resistentiegen.