Globodera Tabacum Research Articles

A 2‐year, multi‐county survey of plant‐parasitic nematodes in North Carolina flue‐cured tobacco

AbstractNorth Carolina is the leading producer of flue‐cured tobacco (Nicotiana tabacum L.) in the United States. Production in the state is threatened by numerous plant‐parasitic nematodes, including Meloidogyne spp. (root‐knot), Globodera tabacum (tobacco cyst), Pratylenchus spp. (lesion), Rotylenchulus reniformis (reniform), and Tylenchorhynchus spp. (stunt) nematodes. The invasive Meloidogyne enterolobii is also a major threat as it can infect tobacco varieties carrying the Rk gene, which conditions resistance to other Meloidogyne species. To gain an updated understanding of which nematode taxa are present in tobacco production and investigate distribution of M. enterolobii in North Carolina, a 2‐year survey was conducted. From 2021 to 2022, 188 fields were sampled across 24 tobacco‐growing counties. Samples were assayed for identification and quantification of nematodes at the genus level; where Meloidogyne spp. was identified, molecular testing was used to determine the species. Data were also collected on the previous crop and tobacco variety grown. In both years, lesion, root‐knot, dagger, and stunt nematodes were found in the highest incidence among samples. In 2022, M. enterolobii was identified in a single sample each from Granville and Pitt counties; this is the first report of M. enterolobii from Granville County. Tobacco variety NC 196 and soybean were the most common variety and previous crop grown, respectively, in both years of the survey. Tobacco variety did not have an effect on nematode population density. The previous crop had a significant effect on root‐knot and spiral nematodes in 2021, and lesion and stubby root nematodes in 2022.

PM 7/40 (5) Globodera rostochiensis and Globodera pallida

PM 7/40 (5) <i>Globodera rostochiensis</i> and <i>Globodera pallida</i>

The hatching-stimulation activity of solanoeclepin A toward the eggs of Globodera (Tylenchida: Heteroderidae) species

Plant-parasitic nematodes often interact with their host plants via chemical compounds for successful invasion. In cyst nematodes, hatching requires chemicals known as hatching factors that are released from host roots. Previous studies succeeded in identifying and synthesizing solanoeclepin A (SEA) as a host-derived hatching factor for Globodera rostochiensis (Wollenweber) Behrens and Globodera pallida (Stone) Behrens (Tylenchida: Heteroderidae). However, no published data were available about the hatching response of Globodera species, other than G. rostochiensis, toward SEA. In this study, we tested the hatching responses of G. rostochiensis, G. pallida, Globodera ellingtonae Handoo, Carta, Skantar, and Chitwood, Globodera tabacum (Lownsbery and Lownsbery) Behrens, and Globodera artemisiae (Eroshenko and Kasachenko) Behrens (Tylenchida: Heteroderidae) toward SEA by exposing eggs of each species to 0.001, 0.01, 0.1, 1, 10, and 100 ppb of SEA and observing their hatching response. SEA stimulated the hatching of Solanaceae-parasitic Globodera species (G. rostochiensis, G. pallida, G. ellingtonae, and G. tabacum). The optimal concentration of SEA for hatching was 0.01–10 ppb. However, the hatching activity of G. pallida and G. ellingtonae was lower than that of G. rostochiensis and G. tabacum. SEA showed no effects on G. artemisiae which is parasitic to Artemisiae plants.

Effect of the trap crop, Solanum sisymbriifolium, on Globodera pallida, Globodera tabacum, and Globodera ellingtonae.

The effect of the nematode trap crop Solanum sisymbriifolium was assessed against three Globodera spp., the potato cyst nematode Globodera pallida (in Idaho), the recently described Globodera ellingtonae (in Oregon), and the tobacco cyst nematode Globodera tabacum (in Connecticut) in field trials. At all locations the ability of S. sisymbriifolium to reduce Globodera encysted second-stage juveniles (J2) in egg densities compared to fallow was considered. For G. ellingtonae, the impact of planting and termination dates of S. sisymbriifolium on final egg densities was also evaluated; and for G. pallida, the ability of the nematode to reproduce on potato (Solanum tuberosum) after exposure to S. sisymbriifolium was determined. Encysted J2 in egg densities of all three Globodera spp. declined from 25 to 68% after trap cropping with S. sisymbriifolium. For G. pallida, S. sisymbriifolium reduced final encysted J2 in egg density by 23 to 50% compared to the fallow treatment, and significantly decreased G. pallida reproduction on potato after exposure to S. sisymbriifolium by 99 to 100% compared to the fallow treatment (P < 0.0001). For G. ellingtonae, the planting date of S. sisymbriifolium in May or June did not impact final egg densities (P = 0.32). Rather, percentage reduction in G. ellingtonae encysted J2 in egg density was most influenced by the length of time to which nematodes were exposed to S. sisymbriifolium, with 30 and 81% reduction after 6 vs 12 wk of exposure, respectively (P < 0.0001). Similar levels of nematode reduction after S. sisymbriifolium were observed for G. tabacum after 12 to 14 wk of exposure to the trap crop; G. tabacum density changes consisted of a 114% increase after susceptible tobacco, a 65% decrease after resistant tobacco, and an 88% decrease after S. sisymbriifolium compared to bare soil. In conclusion, this research demonstrates the widespread applicability of S. sisymbriifolium in reducing a diversity of Globodera spp. present in the USA.

Occurrence of the tobacco cyst nematode subspecies Globodera tabacum subsp. virginiae in France

Tobacco cyst nematode is a sexually diploid species composed of three subspecies, Globodera tabacum subsp. tabacum (Gtt), G. tabacum subsp. virginiae (Gtv) and G. tabacum subsp. solanacearum (Gts). In Europe, only Gtt have been reported to date. Here, we report from a plant in a French tobacco field, the identification of a mixture between cysts of the subspecies Gtt and cysts genetically different. The objective of the present study was to determine the subspecies status of those particular cysts. To this aim, we performed CAPS analyses on two genes and a principal component analysis on microsatellite data. The results obtained with both CAPS and microsatellite markers demonstrate for the first time the occurrence of G. tabacum subsp. virginiae in France.

Human influence on the dispersal and genetic structure of French Globodera tabacum populations

The dispersal abilities and the population genetic structure of nematodes living in soils are poorly known. In the present study, we have pursued these issues in the tobacco cyst nematode, Globodera tabacum, which is responsible of significant yield reductions. Nine microsatellites markers were used to explore the dispersal and genetic structure of 18 French G. tabacum populations. All the populations sampled belong to the “tabacum” subspecies and low level of gene flow was observed among G. tabacum populations in France. Bayesian genetic assignments revealed two distinct genetic groups that matched with the geographic limits of two agricultural cooperative societies. An important part of the genetic variability was observed between these agricultural cooperative societies and also within populations. Those results highlight the impact of the human organization of agricultural practices on the genetic structure of G. tabacum populations and complement previous results obtained on other cyst nematodes, showing the major contribution of human activities and soil transports during harvest in the passive dispersion of these organisms.

Sequence polymorphism of nematode effectors highlights molecular differences among the subspecies of the tobacco cyst nematode complex

Globodera tabacum is a little-known species complex of specialized plant–parasitic nematodes. In this study we investigated for the first time the sequence variability of five effectors: three genes coding for cell wall degrading enzymes (Pel1, Pel2 and ExpB3) and two genes coding for CLE peptides (CLE1 and CLE4) among a set of populations representing the different subspecies described. Substantial variability was observed in the sequence dataset which is consistent with the differential pathogenicity of the G. tabacum subspecies on a range of Nicotiana species. Using these sequence data, we were able (1) to show the presence of a novel CLE-like class in cyst nematodes, (2) to support the existence of most probably three and not four subspecies in this species complex, (3) to reveal that the subspecies G. tabacum tabacum presents the highest diversity and (4) to develop a PCR tool that allows a quick and reliable identification of this subspecies among the species complex.

Registration of ‘B2’ Connecticut Broadleaf Cigar‐Wrapper Tobacco Resistant to Fusarium Wilt, Tobacco Mosaic Virus, Cyst Nematodes, and Blue Mold

‘B2’ Connecticut broadleaf cigar‐wrapper tobacco (Nicotiana tabacum L.) (Reg. No. CV‐124, PI 664300) is a male‐sterile hybrid released by the Connecticut Agricultural Experiment Station and is resistant to Fusarium wilt, Tobacco mosaic virus (TMV), the tobacco cyst nematode [TCN; Globodera tabacum tabacum (Lownsbery and Lownsbery 1954) Stone 1973], and blue mold [caused by Peronospora tabacina de Bary Adam 1933 (= Peronospora hyoscyami de Bary 1863 f. sp. tabacina Skalicky 1964)]. Initial crosses were made in 1987 between VA‐81 and PD4 flue‐cured tobacco and three broadleaf inbreds resistant to Fusarium wilt [caused by Fusarium oxysporum Schlechtend.:Fr. f. sp. nicotianae (J. Johnson) W.C. Snyder &amp; H.N. Hans.] and TMV. F2 selections were backcrossed twice to broadleaf lines, inbred and selected across 12 generations using bulk modified single‐seed descent with 2% of plants (20 plants of 1000) selected most years; selection for TMV and TCN resistance was conducted in greenhouse screens with fewer plants. Lines were field selected for reduced ozone weather fleck. One F12 selection was backcrossed again to the broadleaf cultivar ‘Scantic’ and inbred via pedigree selection across 10 generations with field and greenhouse selection. Progeny testing was used to select F8 plants homozygous for resistance to TCN and for hypersensitive TMV resistance from Nicotiana glutinosa L. A superior male‐fertile inbred (MF‐2) resulting from 27 generations of selection for broadleaf characteristics with 8 cycles of selection each for Fusarium wilt, TMV and TCN resistance was identified and crossed with a male‐sterile CT broadleaf line resistant to TMV and Fusarium wilt (MS‐1) to produce the F1 male‐sterile hybrid B2.

Low cryoprotectant concentrations and fast cooling for nematode cryostorage

Cryopreservation protocols based on slow freezing or vitrification often result in cell injury due to ice formation, cell dehydration and/or toxic concentrations of cryoprotectant (CPA). In this study, we present a cryopreservation technique based on low, non-toxic concentrations of cryoprotectants (≈2–4 M) combined with a rapid cooling rate in the liquid nitrogen phase (−196 °C). Protocols for successfully cryopreserving the plant parasitic nematodes Globodera tabacum tabacum, Heterodera schachtii and Meloidogyne incognita were developed, as demonstrated by the high survival rates and reproducibility of cyst and root-knot nematode species post-cryostorage. This approach for effective cryopreservation of viable plant-parasitic nematodes was developed by inducing an “apparent vitrification” by rapid cooling of the microscopic samples in less than 2 M of cryoprotectant. The extremely thin structure (15–20 μm width, 350–400 μm length) of these nematodes, in combination with a direct and rapid exposure to LN 2, likely prevents the formation of damaging ice crystals. Moreover, this procedure results in viability of both short- and long-cryostorage samples. These techniques could potentially be used for the near-indefinite preservation of thousands of different nematode species. A cryo-nematode collection produced in our lab is available and presented here.

Chorismate mutase: an alternatively spliced parasitism gene and a diagnostic marker for three important Globodera nematode species

The chorismate mutase gene is widely distributed in both cyst and root-knot nematode species and believed to play a critical role in nematode parasitism. In this study, we cloned a new chorismate mutase gene (Gt-cm-1) from Globodera tabacum and further characterized the gene structure in both G. tabacum and G. pallida, a closely related species of G. rostochiensis. The genomic clones of chorismate mutase genes from these two species were found to contain three introns with the second intron having unusual 5’ and 3’ splice sites. A previous study revealed that the chorismate mutase gene from G. rostochiensis is subject to alternative splicing through retention of intron 2, a process that allows for the generation of multiple mRNA transcripts from a single gene. As expected, we discovered that alternative splicing of the chorismate mutase gene is a conserved event in three Globodera species, supporting an important role of alternative splicing in regulating chorismate mutase gene function in plant parasitism by these nematodes. In addition to the potential suboptimal 5’ and 3’ splice sites and the small size of intron 2, detailed sequence analysis also identified candidate cis-acting elements that might be responsible for regulating intron retention of Globodera chorismate mutase genes. Based on genomic sequence variations observed, we developed TaqMan qPCR assays that provided a highly specific and sensitive identification of each Globodera species, revealing a new application of using the chorismate mutase gene as a valuable diagnostic marker for plant-parasitic nematodes.

Effect of a Chromosome Segment Marked by the Php Gene for Resistance to Phytophthora nicotianae on Reproduction of Tobacco Cyst Nematodes.

Host resistance is an important strategy for managing Globodera tabacum subsp. solanacearum and G. tabacum subsp. tabacum, important nematode pests of flue-cured tobacco (Nicotiana tabacum) in Virginia, and cigar wrapper tobacco (N. tabacum) in Connecticut and Massachusetts, respectively. Field research from 1992 to 2005 evaluated reproduction of G. tabacum subsp. solanacearum on genotypes with and without a chromosome segment from N. plumbaginifolia containing a gene (Php) that conferred resistance to race 0 of Phytophthora nicotianae (causal agent of tobacco black shank). Ratios of G. tabacum subsp. solanacearum eggs/500 cm3 soil at the end versus the beginning of experiments (Pf/Pi) were significantly lower in cultivars and breeding lines possessing the Php-containing chromosome segment from N. plumbaginifolia compared with genotypes without the segment. Numbers of vermiform G. tabacum subsp. solanacearum juveniles in roots were similar among genotypes but numbers of swollen and pyriform nematodes were significantly lower for the known G. tabacum subsp. solanacearum resistant cv. NC 567 and in genotypes possessing the Php gene compared with genotypes and cultivars without the gene. In a 2003 greenhouse test, the percentage of plants with visible G. tabacum subsp. tabacum cysts was also significantly lower for parental and progeny genotypes homozygous and heterozygous, respectively, for Php compared with similar lines without the gene. These results indicate a close linkage or association between a likely single, dominant gene (Php) for resistance to P. nicotianae and suppressed reproduction by G. tabacum subsp. solanacearum and G. tabacum subsp. tabacum. Further research to accurately elucidate the relationships among these genes could lead to significant improvements in tobacco disease control.

Multiplex real-time polymerase chain reaction for identifying potato cyst nematodes, Globodera pallida and Globodera rostochiensis, and the tobacco cyst nematode, Globodera tabacum

Because the potato cyst nematodes (PCNs), Globodera rostochiensis and Globodera pallida, and tobacco cyst nematode (TCN), Globodera tabacum, may occur in the same geographic regions, their accurate identification is essential for implementing appropriate regulatory and crop loss mitigating strategies. Molecular methods for differentiating PCNs based on the internal genomic transcribed spacer (ITS) sequences have been developed but the potential of the many DNA amplification-based test formats have not yet been fully explored and most published work has not included TCN. In our study, the two PCN species and TCN could be distinguished by melting curve analysis of ITS amplicons generated in an EvaGreen-based real-time polymerase chain reaction (PCR) test. However, a multiplex real-time Taqman PCR test, which is also based on ITS sequences, was developed in this study with primers and probes modified with locked nucleic acids and proved to be superior for identification of one or more Globodera spp. in samples containing DNA from cysts and (or) second-stage juveniles. The test was specific for the PCN and TCN species with efficiencies of 0.91, 1.02, and 0.89 for G. rostochiensis, G. pallida, and G. tabacum tabacum, respectively. The multiplex, real-time PCR test was useful for distinguishing PCNs and TCN from other nematodes as well as their identification to species level in a single assay, correctly identifying each species in a DNA template from mixed populations. Key words: potato cyst nematode, golden cyst nematode, pale cyst nematode, tobacco cyst nematode, real-time PCR, melting curve analysis, locked nucleic acid, molecular identification. Étant donné que les nématodes à kystes de la pomme de terre (NKP) Globodera pallida et G. rostochiensis ainsi que le nématode à kystes du tabac (NKT) G. tabacum peuvent être présents dans une même zone géographique, il est essentiel de pouvoir les identifier avec précision afin d'appliquer les mesures adéquates relatives aux stratégies d'atténuation en cas de pertes de cultures. Des méthodes moléculaires permettant de différencier les NKP, basées sur les séquences de l'espaceur interne transcrit (ITS), ont été développées, mais le potentiel des nombreux tests d'amplification de l'ADN n'a pas encore été complètement exploré, et la plupart des travaux qui ont été publiés sur le sujet n'ont pas encore été faits sur les NKT. Dans notre étude, les deux espèces de NKP et les NKT pouvaient être caractérisés par l'analyse de la courbe de fusion des amplicons des ITS générée par PCR en temps réel faisant appel au marqueur EvaGreen. Toutefois, une PCR multiplex en temps réel Taqman basée également sur les ITS, conçue au cours de l'étude et faisant appel à l'utilisation d'amorces et de sondes modifiées à l'aide d'acides nucléiques bloqués (LNA), s'est avérée plus efficace quant à l'identification d'une ou de plusieurs espèces de Globodera dans les échantillons contenant de l'ADN extrait de kystes ou de plantules ayant atteint le deuxième stade de croissance. Le test était spécifique des espèces de NKP et NKT à des taux d'efficacité de 0,91, 1,02 et 0,89 pour G. rostochiensis, Globodera pallida et G. tabacum, respectivement. La PCR multiplex en temps réel s'est avérée utile pour distinguer les NKP et les NKT d'autres nématodes ainsi que pour les caractériser au niveau de l'espèce en effectuant qu'un seul biotest, tout en permettant d'identifier chaque espèce à partir d'une matrice d'ADN constituée d'un mélange de populations. Mots-clés : nématode à kystes de la pomme de terre, nématode doré, nématode blanc, nématode à kystes du tabac, PCR en temps réel, analyse de la courbe de fusion, acide nucléique bloqué, identification moléculaire.

First Report of Fusarium Wilt of Flue-Cured Tobacco Caused by Fusarium oxysporum f. sp. batatas in Spain.

Tobacco (Nicotiana tabaccum L.) is among the most important crops in La Vera Region (Cáceres, west-central Spain) where, since 2001, flue-cured tobacco plants showing typical symptoms of a vascular disease associated to the cyst nematode (Globodera tabacum) complex have been frequently observed (2). Symptoms observed were wilting, yellowing, and drying of the leaves, usually on one side of the plant; stunted and distorted leaves with curved midribs due to the unequal growth; and brown vascular discoloration of stems and midribs. Several diseased plants were collected during the summers of 2003 and 2004, and F. oxysporum was consistently isolated from sections of necrotic midribs. To confirm the pathogenicity of F. oxysporum isolates, plants of flue-cured tobacco (cv. Ct-681) were inoculated. Since Fusarium wilt of tobacco may be caused by F. oxysporum f. sp. vasinfectum or F. oxysporum f. sp. batatas (1), plants of cotton (cv. Acala) and sweet potato (cvs. Nemagold and Nancy Hall) were also inoculated to determine the forma specialis designation. Twenty plants per cultivar and isolate were inoculated with six isolates at the two to four true-leaf stage by pouring into the substrate 200 ml of a conidial suspension (1 × 105 CFU/ml). After inoculation, plants were incubated in a growth chamber at 28°C (day) and 24°C (night) with a 16-h photoperiod. All six F. oxysporum isolates were pathogenic to tobacco and sweet potato, but there were differences among isolates in time from inoculation to appearance of first disease symptoms (7 to 15 days after inoculation in tobacco and sweet potato) and also in disease severity 30 days after inoculation (60 to 100% wilt or mortality in tobacco and 50 to 100% in sweet potato). F. oxysporum was reisolated from stems of inoculated plants. No disease symptom was observed in cotton plants 60 days after inoculation, and F. oxysporum was not reisolated from them. Results of inoculation on the differential hosts indicated that disease symptoms in tobacco were caused by F. oxysporum f. sp. batatas. To our knowledge, this is the first report of F. oxysporum f. sp. batatas causing disease in flue-cured tobacco in Spain.

First Report of Globodera tabacum Infecting Tobacco Plants in Quebec, Canada.

The tobacco cyst nematode Globodera tabacum (Lownsbery & Lowns-bery) Behrens is a damaging soilborne pathogen of shade and broadleaf tobacco in the United States (2). Nematode infection of roots can cause dramatic stunting, yield loss, and reduce leaf quality. G. tabacum is considered a complex of three cyst nematodes not distinguishable morphologically including G. tabacum tabacum, G. tabacum virginiae, and G. tabacum solanacearum. During 1998, infested flue-cured tobacco plants were harvested in the Lanaudière Region near Montreal, Quebec. Cysts recovered from roots and soil were identified by morphological and morphometric analysis of mature cysts and unhatched juveniles. Although multiple interceptions on nursery stock entering Quebec from infested nurseries in Hartford County, Connecticut from 1974 to 1986 have been recorded (1), to our knowledge, this is the first record of G. tabacum established in Canadian soil. On the basis of historic nematode infestations in this county, it could be inferred that it was G. tabacum tabacum, but further analysis is necessary for definitive subspecies determination. The infested tobacco plants were stunted in the field when compared with noninfested plants. Recently, the production in that area has been abandoned. Because cysts may survive for years in absence of host and also because potato, also a host of G. tabacum, is being introduced on these tobacco farms, it is believed that the distribution of this nematode could expand in the future in this part of the country.

The promoter of the Arabidopsis thaliana Cel1 endo‐1,4‐β glucanase gene is differentially expressed in plant feeding cells induced by root‐knot and cyst nematodes

SUMMARY Transgenic tobacco and Arabidopsis thaliana carrying the Arabidopsis endo-1,4-beta-glucanase (EC 3.2.1.4) Cel1 promoter fused to the beta-glucuronidase (GUS) reporter gene were infected with the root-knot nematode, Meloidogyne incognita, and either the tobacco cyst nematode, Globodera tabacum (tobacco), or beet cyst nematode, Heterodera schachtii (Arabidopsis). Cel1-driven GUS expression was detected in cell elongation zones of noninfected plants and within feeding sites (giant-cells) induced in roots of both plant hosts by M. incognita. The first detectable signs of Cel1 expression within developing giant-cells occurred at the onset of giant-cell formation and continued throughout the M. incognita life cycle. UidA (Gus) transcripts were detectable within giant-cells induced in tobacco roots at 11-13 days postinoculation with M. incognita as determined by in situ mRNA hybridization. By contrast, expression of the Cel1 promoter was not detected within developing syncytia induced in tobacco or Arabidopsis roots by G. tabacum and H. schachtii, respectively, at any time point. The results demonstrate specific regulation of cell wall-degrading enzymes that may be required for cell wall modifications during feeding cell formation by sedentary endoparasitic nematodes. Differential expression of Cel1 by cyst and root-knot nematodes further supports underlying mechanistic differences in giant-cell and syncytium formation.

Inheritance of Resistance to Tobacco Cyst Nematode in Flue‐Cured Tobacco

The tobacco cyst nematode [Globodera tabacum solanacearum (Miller and Gray) Behrens] is an important pathogen affecting flue‐cured tobacco (Nicotiana tabacum L.) in Virginia and North Carolina and type 32 tobacco in Maryland. The objective of this study was to determine the mode of inheritance of resistance to G. t. solanacearum in the flue‐cured tobacco cultivars Coker 371 Gold and Kutsaga 110. Each cultivar was crossed to the susceptible cultivar K 326 and F1 progeny were backcrossed to each parent. Plants from each parent and F1, F2, BC1Ps, and BC1Pr progeny were evaluated for G. t. solanacearum resistance in the greenhouse. Six‐week‐old transplants were inoculated with 6000 G. t. solanacearum eggs from crushed cysts. Eight weeks after inoculation, a 1‐g sample of fibrous root was stained and vermiform, swollen, pyriform, and adult nematodes were counted. The number of cysts and eggs per 400 000 mm3 of soil were counted from each transplant. Generation means analyses were performed. Additive and dominance gene action play an important role in resistance to G. t. solanacearum in Coker 371 Gold and Kutsaga 110. F2 progeny data from the Coker 371 Gold cross fit a 3:1 (resistant:susceptible) segregation ratio and BC1Ps generation data fit a 1:1 segregation ratio, indicating that resistance to G. t. solanacearum is conferred by a single dominant gene. A continuous range of variation was observed among the F2 progeny for the K 326 × Kutsaga 110 cross, indicating resistance in Kutsaga 110 is quantitative. Globodera tabacum solanacearum resistance in Coker 371 Gold and Kutsaga 110 may be derived from different sources.

First Report of the Cyst Nematode (Globodera tabacum) Complex on Flue-Cured Tobacco in Spain.

The Globodera tabacum complex infects tobacco (Nicotiana tabacum L.) fields in the United States. In August 2001, plants of flue-cured tobacco cv. K326 from a field of the La Vera Region of Spain displayed a premature wilting and yellowing of foliage, but the roots looked healthy. In the laboratory under the microscope, nematode cysts were observed on the roots. At harvest in September 2001, soil and root samples were collected to identify the nematode and to quantify the population in the soil. Identification of the nematode was based on morphological characteristics of second-stage juveniles collected from cysts and perineal patterns of cysts recovered from the roots (2). Cysts were collected from roots, and second-stage juveniles were extracted from crushed cysts. The nematode population was extracted from the soil and quantified as described by Barker (1). The nematode population was identified as Globodera tabacum. Soil density of the nematode was 5,307 cysts per liter of soil, 64,286 eggs per liter of soil, and 16,071 second-stage juveniles per liter of soil. To our knowledge, this is the first report of G. tabacum complex in Spain.

The biotechnological application and limitation of IRES to deliver multiple defence genes to plant pathogens

Transgenic resistance often has enhanced efficacy when more than one transgene is expressed. Here, we explore the co-delivery of multiple discrete effectors via a single transgene using an internal ribosome entry site (IRES) sequence. As an example, we report the co-delivery of two distinct proteinase inhibitors in Nicotiana tabacum var. Xanthi from a bicistronic plant mRNA to examine resistance against plant parasitic nematodes. A cysteine proteinase inhibitor, Oc-IΔD86, is translated in a normal cap-dependent manner while translation of the serine proteinase inhibitor, CpTI, from the bicistronic mRNA is IRES-mediated. ELISAs using antibodies confirm the expression of the two inhibitors in aerial and root material and suggest that IRES-mediated expression in the roots is lower than normal cap-dependent expression. Analysis of Globodera tabacum recovered from transgenic Nicotiana expressing two discrete proteinase inhibitors revealed appreciable levels of resistance of up to 51±3%. Histochemical analysis of animals recovered from transgenic Nicotiana lines expressing Oc-IΔD86, via cap-dependent translation revealed a marked reduction in cysteine proteolytic activity in comparison to those from control untransformed plants. A less dramatic reduction was observed in similar analysis of serine proteolytic activity of animals recovered from transgenic Nicotiana lines expressing CpTI via IRES-mediated translation. The utility of using an IRES element to deliver a number of discrete anti-pathogen proteins is discussed.

Intra-species DNA polymorphism in the tobacco cyst – nematode complex (Globodera tabacum) using AFLP

Amplified fragment length polymorphism (AFLP) was used to obtain information on the within-species genetic variability of the tobacco cyst-nematode (TCN) complex. AFLP was found to be well suited to this type of study. The current classification of TCN was confirmed. Results indicate that the Globodera tabacum solanacearum group, believed to be restricted to the U.S.A., also occurs in Mexico. The within-species variability of TCN is considerable. Populations from Mexico may form a new subgroup. AFLP group-specific markers were identified for two of the TCN subgroups: Globodera tabacum tabacum and Globodera tabacum solanacearum.

Nematode-Responsive Activity of the Cauliflower Mosaic Virus 35S Promoter and Its Subdomains

Root-knot and cyst nematodes are obligate plant parasites that induce complex biotrophic feeding structures in host roots. The mechanisms by which nematodes regulate host gene expression to produce feeding sites are unknown. The cauliflower mosaic virus (CaMV) 35S promoter has been reported to be repressed strongly in the feeding sites of both root-knot and cyst nematodes. In contrast, other work has indicated that this promoter is partially active in some feeding sites. Considering the importance of the 35S promoter in biotechnology, we have defined the nematoderesponsive nature of this promoter in more detail. Transgenic tobacco harboring various 35S-uidA constructs was assayed for β-glucuronidase (GUS) activity after infection by root-knot nematodes (Meloidogyne incognita) and cyst nematodes (Globodera tabacum subsp. tabacum). The entire 35S promoter (-343 to +8) was active in giant cells induced by M. incognita and, to a lesser extent, the syncytia of G. tabacum subsp. tabacum. In the latter case, activity decreased as the feeding sites matured. Subdomains of the 35S promoter were also active in feeding sites, particularly B4 and B5 in giant cells. However, subdomain B3 was strongly down-regulated in gall tissue and syncytia. In total, 14 constructs were studied and nematode-responsive expression was always stronger and more consistent with the root-knot nematode than the cyst nematode.