Climate change is predicted to alter the current pest status of Globodera pallida and G. rostochiensis in the United Kingdom

The potato cyst nematodes Globodera pallida and G. rostochiensis are economically important plant pathogens causing losses to UK potato harvests estimated at £50 m/ year. Implications of climate change on their future pest status have not been fully considered. Here, we report growth of female G. pallida and G. rostochiensis over the range 15 to 25°C. Females per plant and their fecundity declined progressively with temperatures above 17.5°C for G. pallida, whilst females per plant were optimal between 17.5 and 22.5°C for G. rostochiensis. Relative reproductive success with temperature was confirmed on two potato cultivars infected with either species at 15, 22.5 and 25°C. The reduced reproductive success of G. pallida at 22.5°C relative to 15°C was also recorded for a further seven host cultivars studied. The differences in optimal temperatures for reproductive success may relate to known differences in the altitude of their regions of origin in the Andes. Exposure of G. pallida to a diurnal temperature stress for one week during female growth significantly suppressed subsequent growth for one week at 17.5°C but had no effect on G. rostochiensis. However, after two weeks of recovery, female size was not significantly different from that for the control treatment. Future soil temperatures were simulated for medium‐ and high‐emission scenarios and combined with nematode growth data to project future implications of climate change for the two species. Increased soil temperatures associated with climate change may reduce the pest status of G. pallida but benefit G. rostochiensis especially in the southern United Kingdom. We conclude that plant breeders may be able to exploit the thermal limits of G. pallida by developing potato cultivars able to grow under future warm summer conditions. Existing widely deployed resistance to G. rostochiensis is an important characteristic to retain for new potato cultivars.     

Sequence and Characterization of Six Mitochondrial Subgenomes from <Emphasis Type="Italic">Globodera rostochiensis</Emphasis>: Multipartite Structure Is Conserved Among Close Nematode Relatives

Recently, a multipartite mitochondrial genome was characterized in the potato cyst nematode, Globodera pallida. Six subgenomic circles were detectable by PCR, while full-length genomes were not. We investigate here whether this subgenomic organization occurs in a close relative of G. pallida. We amplified and sequenced one entire mitochondrial subgenome from the cyst-forming nematode, Globodera rostochiensis. Comparison of the noncoding region of this subgenome with those reported previously for G. pallida facilitated the design of amplification primers for a range of subgenomes from G. rostochiensis. We then randomly sequenced five subgenomic fragments, each representative of a unique subgenome. This study indicates that the multipartite structure reported for G. pallida is conserved in G. rostochiensis. A comparison of subgenomic organization between these two Globodera species indicates a considerable degree of overlap between them. Indeed, we identify two subgenomes with an organization identical with that reported for G. pallida. However, other subgenomes are unique to G. rostochiensis, although some of these have blocks of genes comparable to those in G. pallida. Dot-plot comparisons of pairs of subgenomes from G. rostochiensis indicate that the different subgenomes share fragments with high sequence identity. We interpret this as evidence that recombination is operating in the mitochondria of G. rostochiensis. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users. Reviewing Editor: Dr. Rafael Zardoya     

Effects of a mixed-isolate mycorrhizal inoculum on the potato – potato cyst nematode interaction

Inoculation of microplants of potato cv. Golden Wonder with Vaminoc, a mycorrhizal inoculum of three arbuscular mycorrhizal fungi (Glomus spp.), resulted in an increase in in‐sand hatch of Globodera pallida, but not G. rostochiensis, within 2 weeks. By this time, mycorrhized plants also supported a larger number of feeding nematodes of both PCN species (50% higher for G. rostochiensis) than did non‐mycorrhized plants, with a higher proportion of the G. pallida population being fertilised females than for G. rostochiensis. After 12 weeks, the multiplication rate of G. rostochiensis on mycorrhized plants was significantly greater than on non‐mycorrhized plants, whereas no such difference was observed for G. pallida. The principal component of PCN multiplication affected by mycorrhization was increased cyst number per plant from 6 to 12 weeks. Over this period, there was no increase in cyst number per plant for either PCN species on non‐mycorrhized plants, whereas the value increased on mycorrhized plants for both G. rostochiensis (by almost 200%) and G. pallida (57%). Mycorrhization resulted in significant increases in the root and shoot dry weights of plants grown in the absence of PCN. Although mycorrhized plants carried a larger PCN burden than non‐mycorrhized plants when grown on PCN‐infested medium, as a result of the increased PCN multiplication rate, they produced larger root systems than did nonmycorrhized plants, suggesting increased tolerance to PCN of the mycorrhized plants, particularly to G. rostochiensis. Of morphological characters investigated in the absence of PCN, only stem height (increased) was significantly affected by mycorrhization. Colonisation by mycorrhizal fungi resulted in increased tuber yield both in the absence (significant increase) and presence (non significant) of PCN, as a result of increased tuber number per plant. These results are discussed in the light of the possible use of AMF as part of an integrated PCN management plan.     

Molecular Variability and Evolution of the Pectate Lyase (<Emphasis Type="Italic">pel</Emphasis>-2) Parasitism Gene in Cyst Nematodes Parasitizing Different <Emphasis Type="Italic">Solanaceous</Emphasis> Plants

While pectate lyases are major parasitism factors in plant-parasitic nematodes, there is little information on the variability of these genes within species and their utility as pathotype or host range molecular markers. We have analysed polymorphisms of pectate lyase 2 (pel-2) gene, which degrades the unesterified polygalacturonate (pectate) of the host cell-wall, in the genus Globodera. Molecular variability of the pel-2 gene and the predicted protein was evaluated in populations of G. rostochiensis, G. pallida, G. “mexicana” and G. tabacum. Seventy eight pel-2 sequences were obtained and aligned. Point mutations were observed at 373 positions, 57% of these affect the coding part of the gene and produce 129 aa replacements. The observed polymorphism does not correlate either to the pathotypes proposed in potato cyst nematodes (PCN) or the subspecies described in tobacco cyst nematodes. The trees reveal a topology different from the admitted species topology as G. rostochiensis and G. pallida sequences are more similar to each other than to G. tabacum. Species-specific sites, potentially applicable for identification, and sites distinguishing PCN from tobacco cyst nematodes, were identified. As both G. rostochiensis and G. pallida display the same host range, but distinct from G. tabacum, which cannot parasitize potato plants, it is tempting to speculate that pel-2 genes polymorphism may be implicated in this adaptation, a view supported by the fact that no active pectate lyase 2 was found in G. “mexicana”, a close relative of G. pallida that is unable to develop on cultivated potato varieties.     

A high-resolution map of the <Emphasis Type="Italic">Grp1</Emphasis> locus on chromosome V of potato harbouring broad-spectrum resistance to the cyst nematode species <Emphasis Type="Italic">Globodera pallida</Emphasis> and <Emphasis Type="Italic">Globodera rostochiensis</Emphasis>

The Grp1 locus confers broad-spectrum resistance to the potato cyst nematode species Globodera pallida and Globodera rostochiensis and is located in the GP21-GP179 interval on the short arm of chromosome V of potato. A high-resolution map has been developed using the diploid mapping population RHAM026, comprising 1,536 genotypes. The flanking markers GP21 and GP179 have been used to screen the 1,536 genotypes for recombination events. Interval mapping of the resistances to G. pallida Pa2 and G. rostochiensis Ro5 resulted in two nearly identical LOD graphs with the highest LOD score just north of marker TG432. Detailed analysis of the 44 recombinant genotypes showed that G. pallida and G. rostochiensis resistance could not be separated and map to the same location between marker SPUD838 and TG432. It is suggested that the quantitative resistance to both nematode species at the Grp1 locus is mediated by one or more tightly linked R genes that might belong to the NBS-LRR class. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. A. Finkers-Tomczak and S. Danan contributed equally to this research.     

A DNA probe which distinguishes Globodera pallida Pa2/3 from G. rostochiensis Ro1 and other cyst-forming nematodes

Abstract

A small DNA fragment (approx. 350 base pairs) from the genome of the potato cyst nematode Globodera pallida Pa2/3 was cloned in a bacterial plasmid. When used as a probe in dot-blot DNA hybridisations against a range of nematodes, the cloned DNA bound to G. pallida Pa2/3 but not to Globodera rostochiensis Rol. The cereal cyst nematode Heterodera avenae, the clover cyst nematode Heterodera trifolii, the root knot nematodes Meloidogyne hapla and Meloidogyne incognita, and the beet cyst nematode Heterodera schactii did not cross-hybridise. This probe can detect as few as six larvae of G. pallida.     

Development of a TaqMan Real‐Time Polymerase Chain Reaction Assay for Detection and Quantification of Fusarium oxysporum f. sp. lycopersici in Soil

Fusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici (FOL), is an important disease of tomato. Pathogenicity and vegetative compatibility tests, although reliable, are laborious for the identification of FOL isolates and cannot efficiently quantify population densities of FOL in the soil. The objective of this study was to develop a rapid, sensitive and quantitative real‐time polymerase chain reaction (PCR) assay for detecting and quantifying FOL in soil. An inexpensive and relatively simple method for soil DNA extraction and purification was developed based on bead‐beating and a silica‐based DNA‐binding method. A TaqMan probe and PCR primers were designed using the DNA sequence of the species‐specific virulence gene SIX1, which is only present in isolates of FOL, not in isolates of other formae speciales or non‐pathogenic isolates of F. oxysporum. The real‐time PCR assay successfully amplified isolates of three races of FOL used in this study and quantified FOL DNA in soils, with a detection limit of 0.44 pg of genomic DNA of FOL in 20 μl of the real‐time PCR. A spiking test performed by adding different concentrations of conidia to soil showed a significant linear relationship between the amount of genomic DNA of FOL detected by the real‐time PCR assay and the concentration of conidia added. In addition, the real‐time PCR assay revealed a significant quadratic regression for a glasshouse experiment between disease severity and DNA concentration of FOL. The soil DNA extraction method and real‐time PCR assay developed in this study could be used to determine population densities of FOL in soil, develop threshold models to predict Fusarium wilt severity, identify high‐risk fields and measure the impact of cultural practices on FOL populations in soils.     

Morphological and Molecular Identification of Globodera pallida Associated with Potato in Idaho

The identity of a newly discovered population of pale potato cyst nematode Globodera pallida associated with potato in eastern Idaho was established by morphological and molecular methods. Morphometrics of cysts and second-stage juveniles were generally within the expected ranges for G. pallida with some variations noted. The Idaho population and paratype material from Epworth, Lincolnshire, England, both showed variations in tail shape, with bluntly rounded to finely pointed tail termini. Compared to literature values for the paratypes, second-stage juveniles of the Idaho population had a somewhat shorter mean body length, and cysts had a slightly higher mean distance from the anus to the nearest edge of the fenestra. PCR-RFLP of the rDNA ITS region, sequence-specific multiplex PCR and DNA sequence comparisons all confirmed the identity of the Idaho population as G. pallida. The ITS rDNA sequence of the Idaho isolate was identical to those from York, England, and the Netherlands. Species-specific primers that can positively identify the tobacco cyst nematode Globodera tabacum were also developed, providing a new assay for distinguishing this species from G. pallida and the golden potato cyst nematode Globodera rostochiensis.     

Laboratory rearing of potato cyst nematode; a method suitable for pathotyping and biological studies

A method is described for the laboratory rearing of potato cyst nematode, Globodera rostochiensis (Wollenweber, 1923) Mulvey & Stone, 1976 and G. pallida (Stone, 1973) Mulvey & Stone, 1976. Potato root systems were grown in 500 ml of sand in closed, clear plastic canisters. The enclosed system inhibited foliage growth and stabilised moisture balance. Root systems were sustained by the seed tubers, but supplementary nutrient was added. Potato cyst nematode eggs were inoculated into the sand after root initiation. At optimum rearing temperatures of 15–20°c the life cycle was completed within 8–12 weeks. The cyst‐to‐cyst multiplication factor was 10–20‐fold. The method has the advantage of not requiring light, and rearing canisters can be stacked in standard incubators. Its high reliability renders the method particularly suited to pathotype analysis.     

A QTL for broad-spectrum resistance to cyst nematode species (Globodera spp.) maps to a resistance gene cluster in potato

 Broad-spectrum resistance in potato to the potato cyst nematode (PCN) species Globodera rostochiensis and G. pallida is commonly regarded as a polygenically inherited trait. Yet, by use of QTL analysis and a selected set of PCN populations, resistance to both PCN species could be ascribed to the action of locus Grp1. Grp1 confers major resistance to G. rostochiensis line Ro₅-22 and G. pallida population Pa₂-D383 and partial resistance to G. pallida population Pa₃-Rookmaker. Grp1 was mapped on chromosome 5 using previously characterized AFLP markers. Cleaved amplified polymorphic sequence (CAPS) markers available for RFLP loci GP21 and GP179 revealed that Grp1 maps on a genomic region harboring other resistance factors to viral, fungal and nematodal pathogens. The present data indicate that Grp1 is a compound locus which contains multiple genes involved in PCN resistance. Received: 10 September 1997 / Accepted: 6 October 1997     

Changes in relative abundance of two potato cyst nematode species Globodera rostochiensis and G. pallida in one generation

Changes in relative abundance of the two potato cyst nematode species Globodera rostochiensis and Globodera pallida were studied during the 1983/84 season at two different population levels in small pots in the glasshouse and at a single population density on plants grown outdoors in 2 litre terylene cloth bags. In both environments G. rostochiensis was the more successful species. Although the ratio of the two species changed and G. pallida was at a lower level at the end of the experiment it was never eliminated. However, when the number of G. pallida in the mixture was small it did better than expected and demonstrated a frequency dependent response.     

Effects of Zygorrhynchus moelleri on the growth of potato and reproduction of potato cyst nematodes

Laboratory, pot and field experiments investigated the effects of the fungus Zygorrhynchus moelleri on the growth of potato and on the reproduction of the potato cyst nematodes (PCN), Globodera pallida and G rostochiensis. Preliminary laboratory tests showed that Z. moelleri growth was favoured by temperatures and pH ranges commonly present in field soils. The fungus colonised potato roots in vitro and in compost or field soil. It also stimulated in vitro root growth of three potato cultivars. In pot experiments Z. moelleri stimulated potato growth, particularly in the presence of PCN attack. In field plots infested with a mixture of G pallida and G. rostochiensis, tuber yields were not increased after application of the fungus but, in G pallida‐infested plots, yields were significantly increased after drills were inoculated with Z. moelleri. The application of Z. moelleri had no apparent effects on nematode reproduction. Factors influencing the interactions between Z. moelleri, potato and potato cyst nematodes are discussed and the potential role of the fungus as a plant growth promoter in organic potato production considered.     

Observations on the distribution of potato cyst-nematodes, Globodera rostochiensis and G. pallida on two fenland potato farms

The distribution of potato cyst-nematodes, Globodera rostochiensis and G. pallida was surveyed in two widely separated fenland potato farms in Eastern England. Evidence was obtained of the suppression of G. rostochiensis and increase of G. pallida populations when Maris Piper and other potatoes with the same gene (H₁) for resistance to G. rostochiensis are grown. Numbers of G. rostochiensis and/or G. pallida in the soil could not be related directly to recent potato cropping histories of the fields. Identification of the two species of Globodera by the presence and colour of females on the roots of Désirée, Maris Piper, Cromwell (A27/20) and A25/11 potatoes were confirmed by agarose gel isoelectric focussing of general proteins. Cromwell exhibited little or no resistance to G. pallida at Woodwalton. A25/11 was more resistant to this species at Woodwalton but was susceptible to G. rostochiensis. The results of these surveys emphasise the need for carefully planned and integrated control programmes based on the use of resistant cultivars of potato, crop rotations and appropriate nematicide use to combat the growing problem of G. pallida, especially in organic soils.     

Enterotoxigenic Escherichia coli in sewage‐impacted waters and aquatic weeds: quantitative PCR for culture‐independent enumeration

Aim: To develop quantitative PCR for culture‐independent enumeration of enterotoxigenic Escherichia coli (ETEC) in sewage‐impacted waters and aquatic weeds. Methods and Results: Two fluorescent probes (TaqMan and FRET) based on two different real‐time PCR chemistries were designed in highly conserved region of LT1 gene encoding heat labile enterotoxin. Both the assays could detect 2 CFU ml^?1 from serially diluted (two‐fold and ten‐fold) culture of reference strain (E. coli MTCC 723). FRET performed better in terms of C_T value and PCR efficiency than TaqMan. The presence of 10⁶ CFU ml^?1 of nonpathogenic E. coli reduced the detection limit two‐fold with both the probes. However, the performance for two chemistries in various environmental samples was significantly (student’s t‐test, P < 0·05) different. Conclusion: It could be inferred from this study that real‐time PCR chemistries (TaqMan and FRET) could detect very few copies of target DNA in pure cultures, but may give varied response in the presence of nonspecific DNA and natural inhibitors present in environmental sample matrices. Significance and Impact of the Study: The assays can be used for pre‐emptive monitoring of aquatic weeds (a potential nonpoint source), surface and potable waters to prevent waterborne outbreaks caused by ETEC.     

Potato cyst nematodes in England and Wales - occurrence and distribution

Potato cyst nematodes (PCN) have been known to occur in the UK for nearly a hundred years. They are the most problematic pests of potatoes and can cause severe yield losses. Previous work has shown the two species, Globodera rostochiensis and G pallida, to be distributed throughout the UK. This paper reports the results of the first structured and statistically unbiased survey undertaken to assess their occurrence and distribution in the potato growing land of England and Wales. The survey showed that PCN were present in 64% of sites sampled. Of the populations found, 67% were G pallida, 8% were G rostochiensis and 25% contained both species. The results show an increase in the incidence of PCN since previous studies were completed and confirm the perceived shift towards G pallida as the predominant species. Of the infestations found, 62% had a population density of less than 10 eggs g^?1 soil.     

A Real‐time PCR Assay to Identify Meloidogyne minor

Meloidogyne minor is a small root‐knot nematode that causes yellow patch disease in golf courses and severe quality damage in potatoes. It was described in 2004 and has been detected in The Netherlands, England, Wales, Northern Ireland, Ireland and Belgium. The nematode often appears together with M. naasi on grasses. It causes similar symptoms on potato tubers as M. chitwoodi and M. fallax, which are both quarantine organisms in Europe. An accurate identification method therefore is required. This study describes a real‐time PCR assay that enables the identification of M. minor after extraction of nematodes from soil or plant samples. Alignments of sequences of rDNA‐ITS fragments of M. minor and five other Meloidogyne species were used to design a forward primer Mminor_f299, a specific primer Mminor_r362 and the specific MGB TaqMan probe P_Mm_MGB321. PCR with this primers and probe results in an amplicon of 64 bp. The analytical specificity of the real‐time PCR assay was assessed by assaying it on six populations of M. minor and on 10 populations of six other Meloidogyne species. Only DNA from M. minor gave positive results in this assay. The assay was able to identify M. minor using DNA from a single juvenile independent from the DNA extraction method used.     

Species‐Specific PCR‐Based Assay for Identification and Detection of Phomopsis (Diaporthe) azadirachtae Causing Die‐Back Disease in Azadirachta indica

Die‐back disease caused by Phomopsis (Diaporthe) azadirachtae is the devastating disease of Azadirachta indica. Accurate identification of P. azadirachtae is always problematic due to morphological plasticity and delayed appearance of conidia. A species‐specific PCR‐based assay was developed for rapid and reliable identification of P. azadirachtae by designing a species‐specific primer‐targeting ITS region of P. azadirachtae isolates. The assay was validated with DNA isolated from different Phomopsis species and other fungal isolates. The PCR assay amplified 313‐bp product from all the isolates of P. azadirachtae and not from any other Phomopsis species or any genera indicating its specificity. The assay successfully detected the pathogen DNA in naturally and artificially infected neem seeds and twigs indicating its applicability in seed quarantine and seed health testing. The sensitivity of the assay was 100 fg when genomic DNA of all isolates was analysed. The PCR‐based assay was 92% effective in comparison with seed plating technique in detecting the pathogen. This is the first report on the development of species‐specific PCR assay for identification and detection of P. azadirachtae. Thus, PCR‐based assay developed is very specific, rapid, confirmatory and sensitive tool for detection of pathogen P. azadirachtae at early stages.     

Screening of non-tuber bearing Solanaceae for resistance to and induction of juvenile hatch of potato cyst nematodes and their potential for trap cropping

Ninety accessions of non‐tuber bearing Solanaceae were screened for (i) resistance to and (ii) stimulatory effect on juvenile hatch of potato cyst nematodes, and (iii) their growth under temperate climatic conditions. All plant species belonging to the genus Solanum tested induced hatching but this effect was most pronounced for plant species of the Solanum nigrum complex. Hatching of juveniles was hardly or not stimulated by other plant genera of the Solanaceae. Solanum sisymbriifolium combined a high hatching effect with complete resistance to both Globodera rosiochiensis and G pallida. Two S. nigrum varieties showed full resistance to G rostochiensis and a high level of resistance to G pallida. Moreover, S. sisymbriifolium and the two varieties of S. nigrum performed very well under Dutch field conditions and, therefore, they are suggested as candidate trap crops for the control of potato cyst nematodes.