Didier Mugniery

Resistance Quantitative Trait Loci Originating from Solanum sparsipilum Act Independently on the Sex Ratio of Globodera pallida and Together for Developing a Necrotic Reaction

Plant resistance to nematodes is related to the ability of the host to reduce the development of nematode juveniles into females. Resistance to the potato cyst nematode (PCN) Globodera pallida, originating from the wild species Solanum sparsipilum, was dissected by a quantitative trait loci (QTL) approach. Two QTL explained 89% of the phenotypic variation. The QTL GpaV(s)spl on chromosome V displayed the major effect on the cyst number (coefficient of determination [R2] = 76.6%). It restricted G. pallida development to 16.2% of juveniles, 81.5% of males, and 2.3% of females. The QTL GpaXI(s)spl on chromosome XI displayed a lower effect on the cyst number (R2 = 12.7%). It restricted G. pallida development to 13.8% of juveniles, 35.4% of males, and 50.8% of females. Clones carrying both QTL restricted the nematode development to 58.1% juveniles, 41.1% of males, and 0.8% of females. We demonstrated that potato clones carrying both QTL showed a strong necrotic reaction in roots infected by nematodes, while no such reaction was observed in clones carrying a single QTL. This result underlines the importance to introgress together GpaV(s)spl and GpaXI(s)spl into potato cultivars, in order to reduce the density of this quarantine pest in soil and to decrease the risk of selecting overcoming G. pallida subpopulations.

Molecular approaches to the taxonomic position of Peruvian potato cyst nematodes and gene pool similarities in indigenous and imported populations of Globodera

Peruvian potato cyst nematode populations were analysed to assess both their inter- and intraspecific similarities. ITS–RFLP and two satellite DNA sequences were used as taxonomic tools. Both techniques have confirmed that the Peruvian populations have as their closest relatives the European Globodera pallida, despite the detection of clear differences that prevents us from assigning these South American populations unambiguously to any Globodera species. A more precise study of the variability of these Peruvian populations was investigated and they were compared with the imported European populations using protein (2-DGE) and DNA (RAPD) datasets. The clear distinction between the Peruvian and the European populations was confirmed and, inside each group, no correlation was found between the pathotype classification and the observed clustering of the populations. Surprisingly, while RAPDs revealed a higher variability in the Peruvian group than in the European one, some characteristic proteins were found by 2-DGE in some European populations, whereas it was impossible to find any in the Peruvian populations. It is concluded that the primary founders of the European populations may have an origin other than that of the Peruvian populations involved in this study.

Screening tuber-bearingSolanum spp. for resistance toGlobodera rostochiensis Ro1 Woll. andG. pallida Pa2/3 stone

SummaryAccessions of tuber-bearingSolanum spp. related toS. tuberosum subsp.tuberosum were obtained from the German-Dutch collection (Braunschweig, Germany) and the Inter-regional potato collection (Sturgeon Bay, USA). They were screened for resistance toG. rostochiensis Rol andG. pallida Pa2/3. Among 1567 clones from 52 accessions, 135 clones (23 accessions) were resistant toG. rostochiensis. They mainly representedS. andigena, gourlayi, spegazzinii andvernei. Among 1689 clones (74 accessions), 105 clones (32 accessions) were resistant toG. pallida. They representedS. gourlayi, spegazzinii, sparsipilum andvernei. About 25 clones were resistant to both species.

Variability of the ITS rDNA and identification of Nacobbus aberrans (Thorne, 1935) Thorne & Allen, 1944 (Nematoda: Pratylenchidae) by rDNA amplification

In order to identify the false root-knot nematode, Nacobbus aberrans, a nematode of quarantine importance, investigations were undertaken at the molecular level. Study of the ITS rDNA region among six South American populations showed an extremely high polymorphism. This polymorphism is due to point mutation insertions-deletions, the importance of which is increased by the presence of a degenerate microsatellite in this region. The usefulness of the ITS rDNA is therefore questionable when designing a species-specific identification tool. A study based on partial sequences of the 18S gene was carried out. A conserved part of the 18S gene, with a low level of variation when compared to Pratylenchus spp. and Meloidogyne spp., was found in all the N. aberrans populations tested. The primer set designed for this part of the 18S gene allows the amplification of a 295 bp fragment in all the N. aberrans populations tested. No amplification was obtained with other species belonging to Pratylenchidae, Heteroderidae or Hoplolaimidae. This PCR-based identification tool is effective on all N. aberrans migratory stages. From this study it is concluded that this N. aberrans specific primer set may provide a very useful tool for the identification of South American N. aberrans populations and thereby assist diagnosticians in implementing N. aberrans quarantine regulations.

Resistance to the root-knot nematode Meloidogyne fallax in Solanum sparsipilum: analysis of the mechanisms.

The genotype 88S.329.15 of Solanum sparsipilum was studied in order to analyse the genetic basis and the mechanisms of its resistance to Meloidogyne fallax . In infected plants grown at 20°C, juveniles invaded the root system with a clear delay and a lower infection rate in comparison to the susceptible S. tuberosum genotype BF15 H1. No defence reaction occurred during root invasion and migration toward the vascular cylinder. The juveniles induced development of feeding sites usually composed of several giant cells, which contained condensed cytoplasm, only small vacuoles, enlarged nuclei with pronounced nucleoli and almost no endoplasmic reticulum. Abundant necrosis of surrounding parenchymatous vascular cylinder cells lead to the degeneration of the giant cells. More than 90% of the invading juveniles failed to develop. The others developed as males. The resistance inheritance was analysed on 128 F1 hybrids obtained using the susceptible line BF15 H1 as the female parent and 88S.329.15 as the male parent. Among the progenies, 68 genotypes produced a necrotic reaction to nematode infection and 60 produced no necrosis. This 1 : 1 segregation pattern suggests a monogenic control of this defence reaction. Unlike the resistant parent 88S.329.15, some M. fallax females developed in the roots of necrotically responding hybrids. There was a normal distribution of mean numbers of adult females found in the roots of these genotypes. This result suggests that the ability of the resistant genotype 88S.329.15 to suppress development of females is quantitatively inherited and likely to be controlled by more than one locus. These data indicate that the mechanism of resistance is different from the resistance to Meloidogyne incognita conferred by the Mi gene of tomato.

Characterisation of virulence in populations of Meloidogyne chitwoodi and evidence for a resistance gene in pepper Capsicum annuum L. line PM 217

Doubled haploid lines of pepper from the F1 hybrid of PM 217 × Yolo Wonder were tested for their resistance to different populations of Meloidogyne chitwoodi. PM 217 has the Me1 gene for resistance to Meloidogyne incognita, M. arenaria, M. javanica and Me2 gene for resistance to M. hispanica. With two European populations a clear segregation was observed. The necrotic reactions and the resistant: susceptible segregation of 1 : 1 suggested the occurrence of a major gene, different from but close to Me1. With another European population of M. chitwoodi and with M. fallax, no resistance was observed. Two American and two southern European M. chitwoodi populations were totally avirulent to the two pepper parents. These results demonstrate the existence of great polymorphism in M. chitwoodi populations and of a major gene in pepper controlling a specific resistance against some populations.

Application of a putative fatty-acid binding protein to discriminate serologically the two European quarantine root-knot nematodes, Meloidogyne chitwoodi and M. fallax, from other Meloidogyne species

Two major proteins, Mcf-A67 and Mcf-B66, were identified by mini two-dimensional polyacrylamide gel electrophoresis in order to distinguish the two European quarantine root-knot nematodes, Meloidogyne chitwoodi and M. fallax, from eight other species. These ‘quarantine proteinic markers’ have been microsequenced after enzymatic digestion. The internal amino acid sequences exhibit similarities to members of a family of low molecular weight intracellular lipid-binding proteins. Moreover, to explore a simple, rapid, and inexpensive way to identify the two quarantine nematodes, dot blot hybridizations were performed using an antiserum (ΣA67) produced from the longest amino-acid sequence of the protein Mcf-A67. Although several proteins stained on the M. chitwoodi and M. fallax western blot membranes, the two nematodes were easily distinguished from other root-knot nematodes, on dot blot assays with soluble proteins extracted from a single female. Because of its specificity and sensitivity, the use of the ΣA67 antiserum to improve the diagnosis of the two European quarantine root-knot nematodes is discussed.

Banning of methyl bromide for seed treatment: could Ditylenchus dipsaci again become a major threat to alfalfa production in Europe?

In Europe, the stem and bulb nematode Ditylenchus dipsaci has been listed as a quarantine pest by EPPO: without any control, it may cause complete failure of alfalfa crops. Movement of nematodes associated with seeds is considered to be the highest-risk pathway for the spread of this pest. Since the 2010 official withdrawal of methyl bromide in Europe, and in the absence of any alternative chemical, fumigation of contaminated seed batches is no longer possible, which makes the production of nematode-free alfalfa seeds difficult to achieve and leads to unmarketable seed batches. Thermotherapy is being considered as a realistic alternative strategy, but its efficiency still remains to be validated. The combination of the currently available methods (i.e. use of resistant cultivars, seed production according to a certification scheme, mechanical sieving, seed batch inspection) could significantly reduce the likelihood of seed contamination. However, it does not guarantee a total eradication of the nematode. Although it is already widely distributed all over Europe, reclassification of D. dipsaci as a regulated non-quarantine pest to reduce the possibility of further introductions and the rate of spread of this pest appears to be a risky strategy because of the lack of up-to-date documented data to evaluate damage thresholds and determine acceptable tolerance levels.

Variation in host status of Brassica spp. for isolates of the Columbia root-knot nematode, Meloidogyne chitwoodi , and potential mechanisms

Interaction of the Columbia root-knot nematode, Meloidogyne chitwoodi, with Brassica spp. was studied in pot and in Petri dish experiments for 11 nematode isolates and three F1 hybrids of cauliflower, broccoli and rapeseed. In the pot experiment, the host status of the different Brassica hybrids varied greatly (average of 4-45 egg-masses). The isolate-by-cultivar interaction effect was significant but depended only on three exceptions to the generally equal patterns for all isolates. In the Petri dish experiment, this large variation in aggressiveness was confirmed. Cauliflower, as a host for M. chitwoodi, is less favourable than rapeseed, and rapeseed is less favourable than broccoli. Several mechanisms are involved in the host-parasite interaction: limitation in penetration, in juvenile development, and in female-male ratio, each one acting with a high level of specificity to the isolates tested. No hypersensitive reaction was observed. In view of this quantitative resistance reaction, the species M. chitwoodi expresses an extremely large variability in terms of aggressiveness.