Rapid detection and quantification of viable potato cyst nematodes using qPCR in combination with propidium monoazide

Potato cyst nematodes (PCN), Globodera pallida and Globodera rostochiensis, are obligate parasites of solanaceous plants, causing severe losses in several potato growing areas throughout the world. To date, management of PCN is related to nematode population densities estimated as eggs per gram of soil, without considering the actual number of viable juveniles within the cysts. In classical nematology, the standard method to determine PCN viability is based on a staining assay, using Meldola's blue dye (MB) followed by microscopic visualization of MB‐treated nematodes. Although MB is considered to be reliable in staining embryonated juveniles within eggs and cysts, it is a time‐ and labour‐consuming assay. In the present work, a real‐time PCR (qPCR)‐based method combined with propidium monoazide (PMA), a photoreactive DNA‐intercalating dye, was developed for the quantification of viable PCN. This dye renders exposed DNA of dead cells unable to be amplified by PCR, and thus only DNA from viable/intact PCN juveniles is amplified and detected. The novelty of the present method lies in the simultaneous quantitative and qualitative estimation of viable PCN inocula using species‐specific primers and TaqMan probes. The PMA–qPCR viability method (v‐PCR) developed for the two Globodera species successfully discriminated dead from living specimens in heat‐treated samples and eggs in old and newly formed cysts. Interestingly, the detection of DNA from 34‐year‐old nematode cysts stored at room temperature was observed. In conclusion, the proposed v‐PCR method should prove to be very useful for the routine determination of PCN viability from field samples.     

Consistent action of two partially effective loci conferring resistance to Globodera pallida Pa2/3 across multiple nematode field populations

The potato cyst nematodes (PCN) Globodera rostochiensis and Globodera pallida are significant pests of potatoes worldwide. The most effective control methods are crop rotation and the deployment of resistant varieties. Complete resistance to G. rostochiensis based on a single resistance gene has successfully been integrated into many varieties. However, resistance to G. pallida has not been as successful to date, with current varieties only exhibiting partial resistance. Combining partially effective quantitative trait loci (QTLs) for resistance can increase the strength and breadth of the resistance. An additive effect on resistance has previously been demonstrated on combining two QTLs from Solanum tuberosum subsp. andigena (GpaIV^s_adg) and Solanum vernei (Gpa5). However, populations of G. pallida can be quite divergent and it was unclear whether the relative effects of the individual QTLs and the combined additive effect would be consistent across different G. pallida Pa2/3 populations. Using a mapping population segregating for both QTLs, the effect of the QTLs individually and combined was examined on four UK‐derived field populations of G. pallida pathotype Pa2/3, and the relative effects of the individual QTLs and the additive effect of the combination found to be consistent across all populations.     

Increased virulence of Globodera pallida during repeated rearing on different resistant potato cultivars explained by a simple model

Selection for virulence of Globodera pallida on potato cultivars was studied for four generations under controlled conditions. The reproduction rate (P_f/P_i) of a mixed Pa2/3 population increased by a factor of 61 during rearing on the partially resistant potato cv. Darwina compared to rearing on the susceptible cv. Irene. This was a result of selection for virulence on cv. Darwina, and achieving the Hardy–Weinberg equilibrium on cv. Irene. Increased virulence also significantly raised the reproduction rate on several other Solanum genotypes. These changes could be explained reasonably well by the monogenic inheritance of a virulence factor breaking the Grp1 locus. The virulence changes were probably mainly evoked by this gene only, inherited from S. vernei 1-3 or S. vernei 24/20. The Grp1 locus has probably provided the differential S. vernei hybrid (VTⁿ)² 62-33-3 with its resistance to the Pa2 group and not to the Pa3 group. Alternation of cultivars did not halt selection if the cultivars highly differentiated between the Pa2 and Pa3 populations. Only when alternation was with cultivars that harboured a different resistance gene against Pa3 was selection for virulence delayed. Differences in virulence levels (i.e. reproduction rates) within the nematode population determined the rate of selection, not the resistance level itself. Selection of a Pa3 population for three generations on cv. Karakter not only increased the reproduction rate on cv. Karakter itself by a factor 4.2, but also raised the reproduction on other potato genotypes. A simple monogenic model could explain these changes in virulence.     

Between‐site and ‐year variation in the relative susceptibility of native Scottish Pinus sylvestris populations to dothistroma needle blight

Dothistroma needle blight (DNB), caused by Dothistroma septosporum, is currently the disease causing most concern in British pine plantations. Previous artificial inoculation (AI) experiments showed that native Scottish Scots pine (Pinus sylvestris) populations vary in susceptibility to DNB. However, it is unclear if the relative susceptibility of Scots pine populations observed in these experiments can be replicated under natural conditions. It is also unknown whether relative susceptibility of Scots pine populations varies between sites or years. To answer these two questions, young Scots pine plants from six native Scottish populations (Abernethy, Allt Broighleachan, Amat, Beinn Eighe, Glen Cannich and Glen Loyne) were exposed to natural D. septosporum inoculum at two Scottish sites (Culbin and Torrs Warren) between 2012 and 2014. DNB disease incidence and severity was assessed each October. Relative susceptibilities of the Scots pine populations varied between sites and across years. In two of the three years at Torrs Warren (2012 and 2014), the relative susceptibilities of the populations were strongly positively correlated with those observed in previous AI experiments. In these years, trees from Glen Loyne and Glen Cannich were the most susceptible. Conversely, there was no correlation between the relative susceptibilities seen in any year at Culbin with those observed in AI experiments. At Culbin, Beinn Eighe was the most susceptible population. Across both sites, there was a strong positive relationship between total summer precipitation and DNB severity (R = 0·93, t = 8·2, P = 0·001).     

Competitive ability of ALS‐inhibitor herbicide‐resistant Fimbristylis miliacea

Fimbristylis miliacea, a weed in rice, has evolved resistance to acetolactate synthase (ALS) inhibitors. This study aimed to investigate the competitive abilities of ALS‐resistant (R) and ALS‐susceptible (S) F. miliacea with rice. A replacement series experiment was conducted in the glasshouse at the Federal University of Pelotas, Brazil. The proportions of rice to F. miliacea were 100:0, 75:25, 50:50, 25:75 and 0:100, with 1060 plants m^?2. The experimental units were arranged in a completely randomised design with four replications. A follow‐up study was conducted at the University of Arkansas, Fayetteville, USA, in a split‐plot design with four replications. The main plot was species mixture (rice × R, rice × S, R × S). The subplot was competition partitioning (below‐ and above‐ground, below‐ground only, above‐ground only and no interspecific competition). Leaf area, plant height and shoot dry mass were recorded. Rice was more competitive than the R or S F. miliacea. In equal proportions of rice and F. miliacea, regardless of ecotype, the relative leaf area, height and dry mass of rice were greater than that of F. miliacea. The ALS‐resistant ecotype was less competitive with rice than the S ecotype. Intraspecific competition among rice plants was stronger than rice competition with F. miliacea. Competition for below‐ground resources was the most critical aspect of interference among rice and F. miliacea. In production fields, high infestation levels of F. miliacea results in significant yield losses; thus, resistance to ALS inhibitors needs to be curtailed.     

Herbicide‐resistant weeds in the Philippines: Status and resistance mechanisms

Two major weeds in rice in the Philippines, Sphenochlea zeylanica Gaertn. and Echinochloa crus‐galli (L.) Beauv., are controlled with chemical and cultural methods. In the 1980s, after >10 years of continuous use of 2,4‐D, S. zeylanica evolved resistance to the chemical in those rice fields that had been treated with 2,4‐D once or twice every cropping season. In the 1990s, E. crus‐galli evolved resistance to butachlor and propanil in rice monocrop areas where both herbicides were used continuously for 7–9 years. Rice farmers continue to use 2,4‐D, butachlor and propanil extensively and are often unaware of herbicide resistance or the potential for cross‐resistance, its causes or its implications. In order to control herbicide‐resistant E. crus‐galli, farmers are shifting to locally available herbicides with different modes of action, such as bispyribac, an acetolactate synthase inhibitor, and cyhalofop, an acetyl coenzyme A carboxylase inhibitor. Follow‐up manual weeding or rotary weeding after herbicide spraying, a common farmers’ practice, removes the susceptible and resistant biotypes and could help to delay or prevent the evolution of resistance. Although the resistance mechanisms of both weeds are not determined yet, they could be related to enhanced degradation that is similar to the mechanisms that are shown by the resistant biotypes in other countries.     

Nematode quantitative resistance conferred by the pepper genetic background presents additive effects and is stable against different isolates of Meloidogyne incognita

Root‐knot nematodes (RKNs), Meloidogyne spp., are a major disease problem in solanaceous crops worldwide, including pepper (Capsicum spp.). Genetic control provides an economically and environmentally sustainable protection alternative to soil fumigants. In pepper, resistance to the main RKN species (M. incognita, M. javanica and M. arenaria) is conferred by the major genes (R genes) Me1, Me3 and N. However, RKNs are able to develop virulence, thus endangering the efficiency of R genes. Quantitative resistance (QR) against Meloidogyne spp. is expected to provide an alternative to R genes, or to be combined with R genes, to increase the resistance efficiency and durability in pepper. In order to explore the ability of QR to protect pepper against RKNs, five pepper inbred lines, differing in their QR level, were tested directly, or after combination with the Me1 and Me3 genes, for their resistance to a panel of M. arenaria, M. javanica and M. incognita isolates. The M. arenaria and M. javanica isolates showed low pathogenicity to pepper, unlike the M. incognita isolates. The QR, controlled by the pepper genetic background, displayed a high resistance level with a broad spectrum of action, protecting pepper against Me3‐virulent as well as avirulent M. incognita isolates. The QR was also expressed when combined with the Me1 and Me3 genes, but presented additive genetic effects so that heterozygous F₁ hybrids proved less resistant than homozygous inbred lines. The discovery of this QR is expected to provide promising applications for preserving the efficiency and durability of nematode resistance.     

High‐resolution melting analysis for rapid detection and characterization of Botrytis cinerea phenotypes resistant to fenhexamid and boscalid

A novel, high‐resolution melting (HRM) analysis was developed to detect single nucleotide polymorphisms (SNPs) associated with resistance to fenhexamid (hydroxyanilides) and boscalid (succinate dehydrogenase inhibitors) in Botrytis cinerea isolates. Thirty‐six single‐spore isolates arising from 13 phenotypes were selected and tested for fungicide sensitivity. Germ tube elongation assays showed two distinct sensitivity levels for each fungicide. Sequencing revealed that resistance to fenhexamid was due to a nucleotide change in the erg27 gene, resulting in an amino acid replacement of phenylalanine (F) with serine (S) or valine (V) at position 412 of the protein, whereas in isolates resistant to boscalid, a nucleotide change in the sdhB gene resulted in the replacement of histidine (H) with arginine (R) or tyrosine (Y) at position 272 of the respective protein. In each case, melting curve analysis generated three distinct profiles corresponding to the presence of each nucleotide in the targeted areas. HRM analysis successfully detected and differentiated the substitutions associated with resistance to both fungicides. In vitro bioassays, direct sequencing and high‐resolution melting analysis showed a 100% correlation with detection of resistance. The results demonstrate the utility of HRM analysis as a potential molecular tool for routine detection of fungicide resistance using known polymorphic genes of B. cinerea populations.     

The threat of root‐knot nematodes (Meloidogyne spp.) in Africa: a review

Meloidogyne species pose a significant threat to crop production in Africa due to the losses they cause in a wide range of agricultural crops. The direct and indirect damage caused by various Meloidogyne species results in delayed maturity, toppling, reduced yields and quality of crop produce, high costs of production and therefore loss of income. In addition, emergence of resistance‐breaking Meloidogyne species has partly rendered various pest management programmes already in place ineffective, therefore putting food security of the continent at risk. It is likely that more losses may be experienced in the future due to the on‐going withdrawal of nematicides. To adequately address the threat of Meloidogyne species in Africa, an accurate assessment and understanding of the species present, genetic diversity, population structure, parasitism mechanisms and how each of these factors contribute to the overall threat posed by Meloidogyne species is important. Thus, the ability to accurately characterize and identify Meloidogyne species is crucial if the threat of Meloidogyne species to crop production in Africa is to be effectively tackled. This review discusses the use of traditional versus molecular‐based identification methods of Meloidogyne species and how accurate identification using a polyphasic approach can negate the eminent threat of root knot nematodes in crop production. The potential threat to Africa posed by highly damaging and resistance‐breaking populations of ‘emerging’ Meloidogyne species is also examined.     

The importance of the infected seed tuber and soil inoculum in transmitting Potato mop‐top virus to potato plants

Potato mop‐top virus (PMTV), the cause of spraing in potato tubers, is transmitted by Spongospora subterranea, the cause of powdery scab, and by planting infected seed tubers. This study was undertaken to determine the relative importance of these sources of infection in seed potato production in Scotland. The transmission of PMTV from tested seed tubers to daughter plants was examined over 2 years and six cultivars. The development of foliar symptoms varied with year and cultivar. Infection of daughter tubers derived from PMTV‐infected seed tubers was more prevalent on plants affected by foliar symptoms than those without symptoms. The rate of transmission of PMTV from infected seed tubers to daughter tubers ranged from 18 to 54%. Transmission was affected by cultivar and by origin of seed tubers used for a cultivar, but not by a cultivar's sensitivity to PMTV infection. The incidence of PMTV in daughter tubers of cv. Cara grown from seed potatoes from one source (common origin) by more than 25 seed producers was examined over two successive generations. The incidence of PMTV in daughter tubers was not correlated with that in the seed tubers but appeared to be strongly associated with soil inoculum. The incidence of PMTV was correlated with powdery scab in those crops in which both were present. There was some evidence from soil tests conducted in 2006 using a tomato bait plant and real‐time RT‐PCR that planting PMTV‐infected seed potatoes could increase the risk of introducing the virus into land not infested by PMTV.     

Disruption of Cerevisin via Agrobacterium tumefaciens‐mediated transformation affects microsclerotia formation and virulence of Verticillium dahliae

Agrobacterium tumefaciens‐mediated transformation (ATMT) was used to obtain a large number of Verticillium dahliae (Vd991) T‐DNA insertion mutants that were randomly integrated. Insertion mutants that produced significantly fewer microsclerotia were chosen for further analysis. Mutant T0065 was identified as having the Cerevisin gene interrupted by T‐DNA, and it was named cerevisin. The cerevisin protein showed a high amino acid sequence similarity with the vacuolar protease B. The mutant strain cerevisin displayed decreased production of microsclerotia and conidia, significantly reduced growth rate and reduction in virulence compared to the wild type. Moreover, the composition of secreted proteins differed between the cerevisin mutant and the wild type. Loss of function of Cerevisin decreased secretion of proteins of low molecular weight (14–25 kDa). Upon treatment with the secreted proteins of the mutant, the degree of leaf wilting decreased, indicating that Cerevisin is implicated in production of these proteins, which are putative pathogenicity factors of V. dahliae. The results suggest that Cerevisin is involved in controlling multiple processes of development and metabolism, plays an important role in vegetative growth and microsclerotia formation and affects virulence of V. dahliae.     

A rapid virulence assay for the Dutch elm disease fungus Ophiostoma novo‐ulmi by inoculation of apple (Malus × domestica ‘Golden Delicious’) fruits

Large‐scale virulence tests using trees or saplings are expensive, time‐consuming and require a considerable amount of space. The suitability of using ‘Golden Delicious’ apples as a rapid screen for identifying Ophiostoma novo‐ulmi transformants with reduced virulence was thus evaluated. When a collection of O. novo‐ulmi field isolates belonging to subspecies novo‐ulmi or americana was inoculated to apples, members of subsp. novo‐ulmi induced, on average, larger necrotic lesions than subsp. americana isolates. The size of the lesions on apples was not correlated with mycelial growth rate of isolates on nutrient agar. Insertional mutants from O. novo‐ulmi subsp. novo‐ulmi isolate H327 were inoculated to ‘Golden Delicious’ apples and Ulmus parvifolia × U. americana saplings in parallel experiments. Results clearly indicated that the O. novo‐ulmi transformants included several exhibiting significantly altered levels of virulence. Variability among replicates within a treatment was reduced in apple inoculation data compared to elm sapling data. Overall, the ‘Golden Delicious’ apple assay was found to be an excellent means for rapidly assessing the virulence level of O. novo‐ulmi isolates.     

Root‐knot nematodes do not affect black root rot disease severity on watermelon and bottle gourd

The effects of root‐knot nematodes on black root rot of watermelon and bottle gourd were studied using field surveys and co‐inoculation tests with Meloidogyne incognita (southern root‐knot nematode) and Diaporthe sclerotioides. The results of the field survey suggested that root‐knot nematodes had little effect on either the severity of black root rot or infection with D. sclerotioides. None of the three fields showed a significant positive correlation between disease severity and nematode gall index, with low correlation coefficients. Co‐inoculation experiments under controlled conditions found no significant effect of root‐knot nematodes on black root rot of watermelon and bottle gourd based on area under disease progress curves (AUDPC). These results were supported by the quantities of DNA of the two agents in root tissues because no significant difference was found between dual‐ and single‐inoculation treatments with M. incognita and/or D. sclerotioides. These findings suggest that root‐knot nematodes probably do not affect the infection of watermelon or bottle gourd roots by D. sclerotioides or the incidence of black root rot in these crops caused by this fungus.     

Methodology of virulence screening and race characterization of Plasmopara halstedii,and resistance evaluation in sunflower – a review

Sunflower downy mildew is a disease of high global economic impact as well as a causal agent that is extremely difficult to eradicate. During the past decades, several approaches for the determination of Plasmopara halstedii (Ph) races have been used worldwide and are discussed in this review. Procedures of isolation, cultivation and maintenance of Ph isolates, as well as the screening of sunflower for resistance, are also critically reviewed. The predominant, globally used resistance screening protocol is a ‘whole seedling immersion’ inoculation. ‘Soil drench’ inoculation allows more precise control of the number of Ph zoosporangia applied to a single sunflower seedling. A detached leaf assay has been described, but it has been used mainly for Ph subcultivation and fungicide tests. For race determination, a differential set consisting of nine sunflower genotypes has been used since 1988, coupled with a numerical triplet code for virulence phenotyping of Ph. The increasing variability in global Ph populations has demonstrated the inadequacy of the current set of differentials, and several researchers have proposed additional public lines as new differentials. Furthermore, bulk isolates may show different results in repeated tests, as Ph may contain genetically distinct zoospores within a single zoosporangium. For precise race determination, single zoosporangia or single zoospore isolates are advisable. However, due to low success of isolation, approximately 1–2%, this method cannot be applied in routine Ph race screening. Methods surveyed in this review have a broad spectrum of applications, including taxonomic studies.     

Silencing of Erwinia amylovora sy69 AHL‐quorum sensing by a Bacillus simplex AHL‐inducible aiiA gene encoding a zinc‐dependent N‐acyl‐homoserine lactonase

Quorum sensing in Gram‐negative bacteria is regulated by diffusible signal molecules called N‐acyl‐l ‐homoserine lactones (AHLs). These molecules are degraded by lactonases. In this study, six Bacillus simplex isolates were characterized and identified as a new quorum‐quenching species of Bacillus. An aiiA gene encoding an AHL‐lactonase was identified based on evidence that: (i) it showed high homology with other aiiA genes of Bacillus sp.; (ii) the deduced amino acid sequence contained two conserved regions, ¹⁰⁴SHLHFDH¹¹¹ and ¹⁶⁵TPGHTPGH¹⁷³, characteristic of the metallo‐β‐lactamase superfamily; and (iii) the protein had zinc‐dependent AHL‐degrading activity. Additionally, the expression of the aiiA gene was significantly up‐regulated by 3‐oxo‐AHL. The AHL‐lactonase inhibited multiplication of the 3‐oxo‐C6‐AHL‐producing plant pathogen Erwinia amylovora sy69 both in vitro and in planta. The results provide support for the use of the quorum‐quenching functionality of B. simplex in the integrated control of the devastating fire blight pathogen.     

Genetic diversity within and between sulfonylurea‐resistant and susceptible populations of Schoenoplectus juncoides in Japan

To reveal the effects of herbicide selection on genetic diversity in the outcrossing weed species Schoenoplectus juncoides, six sulfonylurea‐resistant (SU‐R) and eight sulfonylurea‐susceptible (SU‐S) populations were analysed using 40 polymorphic inter‐simple sequence repeat loci. The plants were collected from three widely separated regions: the Tohoku, Kanto and Kyushu districts of Japan. Genetic diversity values (Nei's gene diversity, h) within each SU‐S population ranged from h = 0.125 to h = 0.235. The average genetic diversity within the SU‐S populations was H_S = 0.161, and the total genetic diversity was H_T = 0.271. Although the H_S of the SU‐R populations (0.051) was lower than that of the SU‐S populations, the H_T of the SU‐R populations (0.202) was comparable with that of the SU‐S populations. Most of the genetic variation was found within the region for both the SU‐S and SU‐R populations (88% of the genetic variation respectively). Two of the SU‐R populations showed relatively high genetic diversity (h = 0.117 and 0.161), which were comparable with those of the SU‐S populations. In contrast, the genetic diversity within four SU‐R populations was much lower (from h = 0 to 0.018) than in the SU‐S populations. The results suggest that selection by sulfonylurea herbicides has decreased genetic diversity within some SU‐R populations of S. juncoides. The different level of genetic diversity in the SU‐R populations is most likely due to different levels of inbreeding in the populations.     

Germination rates of weedy radish populations (Raphanus spp.) altered by crop‐wild hybridisation,not human‐mediated changes to soil moisture

Cultivated plants are known to readily hybridise with their wild relatives, sometimes forming populations with weedier life‐history strategies than their progenitors. Due to altered precipitation patterns from human‐induced global climate change, crop‐wild hybrid populations may have new and unpredictable environmental tolerances relative to parental populations, which would further challenge farming and land‐management weed control strategies. To recognise the role of seed dormancy variation in weed invasion, we compared seedbank dynamics of two cross‐type populations (wild radish, Raphanus raphanistrum, and crop‐wild hybrid radish, R. raphanistrum × R. sativus) across a soil moisture gradient. In a seed‐burial experiment, we assessed relative rates of seed germination, dormancy and seed mortality over two years across cross types (crop‐wild hybrid or wild) and watering treatments (where water was withheld, equal to annual rainfall, or double annual rainfall). Weekly population censuses in 2012 and 2013 assessed the frequency and timing of seedling emergence within a growing season. Generally, germination rates were two times higher and seed dormancy was 58% lower in hybrid versus wild populations. Surprisingly, experimental soil moisture conditions did not determine seedbank dynamics over time. Yet, seed bank dynamics changed between years, potentially related to different amounts of annual rainfall. Thus, variation in seedbank dynamics may be driven by crop‐wild hybridisation rates and, potentially, annual variation in soil moisture conditions.     

Inoculation of Malus genotypes with a set of Erwinia amylovora strains indicates a gene‐for‐gene relationship between the effector gene eop1 and both Malus floribunda 821 and Malus ‘Evereste’

The Gram‐negative bacterium Erwinia amylovora, causal agent of fire blight disease in pome fruit trees, encodes a type three secretion system (T3SS) that translocates effector proteins into plant cells that collectively function to suppress host defences and enable pathogenesis. Until now, there has only been limited knowledge about the interaction of effector proteins and host resistance presented in several wild Malus species. This study tested disease responses in several Malus wild species with a set of effector deletion mutant strains and several highly virulent E. amylovora strains, which are assumed to influence the host resistance response of fire blight‐resistant Malus species. The findings confirm earlier studies that deletion of the T3SS abolished virulence of the pathogen. Furthermore, a new gene‐for‐gene relationship was established between the effector protein Eop1 and the fire blight resistant ornamental apple cultivar Evereste and the wild species Malus floribunda 821. The results presented here provide new insights into the host–pathogen interactions between Malus sp. and E. amylovora.     

Occurrence and ITS diversity of wood‐associated Bursaphelenchus nematodes in Scots pine forests in Switzerland

The quarantine pathogen Bursaphelenchus xylophilus (pine wood nematode, PWN) represents a serious threat for Pinus species in Europe. To exclude its presence in Switzerland, in 2010 and 2011 a countrywide survey was conducted in 102 Pinus sylvestris stands, chosen according to whether they contained dying or dead trees or were located in areas at risk of PWN introduction. In total, 285 trees (1–5 per site) were sampled. Nematodes were extracted from wood chips using a standard procedure, and identified to species by internal transcribed spacer (ITS) sequencing. Bursaphelenchus species were present in 34% of the trees, but no B. xylophilus was identified, i.e. PWN is still not present in Switzerland. The nematodes found belonged to seven different species, with B. vallesianus the most frequent species, followed by B. sexdentati, B. mucronatus kolymensis and B. eggersi. Three other species (B. borealis, B. pinophilus, B. poligraphi) were each only present in one or two trees. Three groups of sequences could not be assigned to a species because of the lack of matching reference sequences. The species composition found in Switzerland suggests co‐existence of southern and central European Bursaphelenchus species. Intraspecific ITS variability differed considerably among the four most common species. Bursaphelenchus eggersi, B. mucronatus kolymensis and B. sexdentati had several variable sites in the ITS region, resulting in multiple ITS genotypes in each species. In contrast, all 99 B. vallesianus isolates had an identical ITS region. This could indicate a founder effect, and possibly that B. vallesianus is not native to the Alpine region.     

Virulence of ‘Dickeya solani’ and Dickeya dianthicola biovar‐1 and ‐7 strains on potato (Solanum tuberosum)

Studies were conducted to explain the relative success of ‘Dickeya solani’, a genetic clade of Dickeya biovar 3 and a blackleg‐causing organism that, after recent introduction, has spread rapidly in seed potato production in Europe to the extent that it is now more frequently detected than D. dianthicola. In vitro experiments showed that both species were motile, had comparable siderophore production and pectinolytic activity, and that there was no antagonism between them when growing. Both ‘D. solani’ and biovar 1 and biovar 7 of D. dianthicola rotted tuber tissue when inoculated at a low density of 10³ CFU mL^?1. In an agar overlay assay, D. dianthicola was susceptible to 80% of saprophytic bacteria isolated from tuber extracts, whereas ‘D. solani’ was susceptible to only 31%, suggesting that ‘D. solani’ could be a stronger competitor in the potato ecosystem. In greenhouse experiments at high temperatures (28°C), roots were more rapidly colonized by ‘D. solani’ than by biovar 1 or 7 of D. dianthicola and at 30 days after inoculation higher densities of ‘D. solani’ were found in stolons and progeny tubers. In co‐inoculated plants, fluorescent protein (GFP or DsRed)‐tagged ‘D. solani’ outcompeted D. dianthicola in plants grown from vacuum‐infiltrated tubers. In 3 years of field studies in the Netherlands with D. dianthicola and ‘D. solani’, disease incidence varied greatly annually and with strain. In summary, ‘D. solani’ possesses features which allow more efficient plant colonization than D. dianthicola at high temperatures. In temperate climates, however, tuber infections with ‘D. solani’ will not necessarily result in a higher disease incidence than infections with D. dianthicola, but latent seed infection could be more prevalent.