Production of monoclonal antibodies to potato virus YNTN strain and their use for strain differentiation

Four mouse monoclonal antibodies (MAbs) against potato virus Y (PVY) were produced. MAb 4C1 reacted with four isolates of PVY^NTN and only very weakly with one isolate of the necrotic strain of PVY (PVY^N). It did not react with other isolates of the ordinary strain of PVY tested. MAb 2C9 reacted with all isolates tested and can be used to produce a specific diagnostic kit for routine PVY detection. Other MAbs had different specificities and reacted with isolates of various strains of PVY. MAbs did not react with seven other members of the Potyvirus group including potato virus A. A MAb-based ELISA, using MAb 4C1, was devised and shown to detect PVY^NTN specifically.     

PVYNTN-NW, a novel recombinant strain of Potato virus Y predominating in potato fields in Syria

Detailed characterization of a number of isolates of PVY^SYR, a novel recombinant strain of Potato virus Y (PVY) from Syria, was conducted to elucidate their origin, assess their significance and achieve a final classification of PVY^SYR. Recombination analysis grouped isolates of PVY^SYR into three recombination patterns, SYR-I, SYR-II and SYR-III, which varied in the first 700 nucleotides of their genomes, with the second recombination pattern, SYR-II, the most frequent. PVY^SYR isolates shared highest genomic identity and close phylogenetic relationships with PVY^NTN and PVY^NW isolates from Syria, suggesting a common origin and local emergence of these isolates in Syria. All PVY^SYR isolates (total of 20) induced tobacco veinal necrosis, but reacted to a PVY^O monoclonal antibody, typical characteristics of the previously reported PVY^NW (or PVY^N:O). In potato, however, four isolates tested (one of SYR-I and three of SYR-II) induced potato tuber necrotic ringspot disease (PTNRD), which is the characteristic phenotype of PVY^NTN. Given the shared properties of SYR-I and SYR-II isolates with PVY^NTN and PVY^NW, it was decided that they represent a new recombinant strain of the PVY^N strain group, with the proposed name PVY^NTN-NW. The classification of SYR-III will be possible only after testing the phenotype in potato. The high prevalence of PVY^NTN-NW in potatoes and weeds, as well as its ability to induce PTNRD, demonstrates its importance and the necessity for its control.     

Spread of potato virus Y in potato cultivars (Solanum tuberosum L.) with different levels of sensitivity

The aim of this work was to correlate the appearance of the symptoms, multiplication and spread of virus after mechanical inoculation of potato (Solanum tuberosum L.) cultivars showing different levels of susceptibility and sensitivity to Potato virus Y^NTN (PVY^NTN). The potato cultivars used were the resistant cultivar Sante and susceptible cultivars Igor, Pentland squire and Désirée. The spread of the virus PVY^NTN in infected plants was monitored using different methods: DAS-ELISA, tissue printing, immuno-serological electron microscopy and real-time PCR. In all three susceptible cultivars, the virus was detected in the inoculated leaves 4–5 days after inoculation. From there virus spread rapidly, first into the stem, then more or less simultaneously to the upper leaves and roots. Real-time PCR was shown to be very sensitive and enabled viral RNA to be detected in non-inoculated leaves of susceptible cultivar Igor earlier than other methods. Therefore, for exact studies of plant–virus interaction, a combination of methods which detect viruses on the basis of their different properties (coat protein, morphology or RNA) should be used to monitor the spread of viruses.     

Resistance to Sphaerotheca pannosa in roses induced by 2,6-dichloroisonicotinic acid

The susceptible rose cv. Madelon and the partially resistant cv. Sonia both responded with reduced development of rose powdery mildew when they were treated with the synthetic inducer 2,6-dichloroisonicotinic acid (INA). The EC₅₀ for number of colonies cm⁻² was approximately 0.4 mg L⁻¹ in both cultivars when treated 4 days prior to inoculation. However, conspicuous differences were observed with respect to number of spores per cm². For sporulation, the EC₅₀ was 0.37 mg L⁻¹ in cv. Madelon and only 0.08 mg L⁻¹ in cv. Sonia. A comparison with the pathosystems cucumber/ Sphaerotheca fuliginea and red cabbage/ Peronospora parasitica is made and the importance of the observed phenomenon for the selection of parents in a breeding programme for (partial) resistance is discussed.     

Molecular analysis of resistance mechanisms to Orobanche cumana in sunflower

Resistance to the dicotyledenous parasite Orobanche cumana in sunflower is characterized by a low number of parasitic attachments and a confinement of the parasite in host tissues leading to its necrosis. To help understand what determines such resistance mechanisms, molecular, biochemical and histological approaches were employed before (early response) and after (late response) attachment of the broomrape parasite to susceptible (2603) and resistant (LR1) sunflower genotypes. The expression patterns of 11 defence-related genes known to be involved in different metabolic pathways (phenylpropanoids, jasmonate, ethylene) and/or in resistance mechanisms against microorganisms were investigated. RT-PCR and cDNA blot experiments revealed that the resistant genotype exhibited a stronger overall defence response against O. cumana than the susceptible one, involving marker genes of the jasmonate (JA) and salicylic acid (SA) pathways. Among them, the SA-responsive gene, def. (defensin), appeared to be characteristic of LR1 sunflower resistance. However, no JA accumulation and similar SA contents (250–300 ng g⁻¹ FW) were measured by GC/MS in both genotypes, parasitized or not. In addition, three cDNAs, isolated by a suppression-subtractive hybridization, were shown to be strongly induced only in the resistant genotype 8 days post-inoculation, when the first O. cumana attachments occurred. These genes, putatively encoding a methionine synthase, a glutathione S-transferase and a quinone oxidoreductase, might be involved in detoxification of reactive oxygen species, suggesting the occurrence of an oxidative burst during the incompatible interaction. Finally, host cell-wall modifications leading to parasite-confinement were correlated with more intense callose depositions in the resistant genotype, concomitant with over-expression of the callose synthase cDNA HaGSL1 .     

Development of Globodera rostochiensis pathotype Ro1 in resistant and susceptible cultivars of potato1

Studies on the development of Globodera rostochiensis in potato cultivars with different levels of resistance have shown that both males and females of the nematode complete their whole cycle of development in susceptible cv. Svitanok Kyivskii in 61 days. In resistant cv. Vodograi, only males complete their development. Development of females is interrupted at the third larval instar, and mature females do not appear.     

Histological investigation of stripe rust (Puccinia striiformis f.sp. tritici) development in resistant and susceptible wheat cultivars

The wheat cultivar Kariega expresses complete adult plant resistance against stripe rust, whereas cv. Avocet S is susceptible. Using confocal laser scanning microscopy, initial fungal penetration into flag leaves was identical in both cultivars, with directional germ-tube growth towards stomata that were penetrated without the formation of an appressorium, followed by differentiation of a substomatal vesicle, infection hyphae, haustorial mother cells and haustoria. During the following 4 days, further fungal development occurred more quickly in the resistant than in the susceptible cultivar. However, by 7 days postinoculation (dpi) the situation changed, with exponential growth of the pathogen occurring only in the susceptible line. Induced cellular lignification, a typical defence reaction of cereals, was observed at 4 dpi in the resistant cultivar, and 2 days later lignified tissue completely surrounded the fungal colonies. In the susceptible cultivar, isolated lignified host cells occurred at 6 dpi, and long, unbranched fungal hyphae outgrowing the resistance reaction were observed.     

Assessment of partial resistance of potato to, and pathotype and virulence differences in, potato cyst nematodes

Results are reported for several international collaborative experiments which examined methods of assessing degrees of partial resistance in potato and virulence in potato cyst nematode (PCN, Globodera pallida). It was demonstrated that absolute rates of multiplication can be extremely variable on both susceptible and partially resistant clones, even when the same population and test procedures are employed. It was therefore concluded that, on clones with quantitatively inherited resistance (i.e. partially resistant), absolute rates of multiplication cannot be used to separate pathotypes. Expression of these rates as percentages of those on the susceptible controls reduced the absolute differences between tests, but the values obtained were still too variable for statutory use. However, whatever the environment or nematode population used, it was observed that the resistance of the test clones and virulence of the nematode populations were generally ranked in a similar order. The main exceptions to this were: (1) a Petri-dish test where cv. Vantage was less resistant than in canisters or pots and (2) a pot test where cv. Darwina tended to be more interactive with environmental factors than the other test clones. It was also observed that with some populations of PCN in pots the susceptible cv. Bintje was a less good host than cv. Désirée. On the basis of these results it is suggested that certain partially resistant clones should be used as internal references in statutory, recommended list and breeders' assessment tests against which the resistance of the test clones are compared. For international comparability it is necessary that the different centres conducting such tests use the same reference clones and nematode populations, and similar test methods.     

Characterization of aryl acylamidase activity from propanil-resistant barnyardgrass (Echinochloa crus-galli [L.] Beauv.)

The enzyme, aryl acylamidase, was characterized in propanil-susceptible and propanil-resistant barnyardgrass with respect to kinetic parameters, the effects of inhibitors, and the levels of activity in dark- and light-grown tissues. The enzyme reaction in the resistant tissue preparation proceeded linearly with time over a 5 h time course, while activity in the susceptible tissue preparation was 2- to 4-fold lower and the activity tended to decrease after 2 h. The apparent K_m values were 62.1 mmol L⁻¹ and 3.1 mmol L⁻¹ for the enzyme activity in the susceptible and resistant tissue preparations, respectively. Two herbicides (anilofos and piperophos), previously shown to synergize propanil injury against the resistant biotype, were found to be potent inhibitors of the in vitro aryl acylamidase activity.     

Reaction of three potato cultivars to potato virus S and alteration of miRNAs and mRNA targets in the resistant genotypes

Reactions of three Polish potato cultivars to potato virus S (PVS) were investigated at 22°C. Cultivars Tajfun and Tonacja exhibited partial resistance with systemic infection detected in some inoculated plants; cultivar Bryza was susceptible to PVS with systemic infection detected in all inoculated plants. The virus was not detectable by ELISA at 23 days postinoculation (dpi) but was detected after 40 dpi. Infection rate and viral accumulation were significantly lower in Tonacja and Tajfun than in Bryza, but no statistically significant difference between Tajfun and Tonacja was detected. Both susceptible and resistant genotypes displayed various, either common or cultivar-specific, symptoms. Delayed systemic infection at 56 dpi was observed in some cases in Tonacja and Tajfun. Resistance-related alteration of a set of miRNAs and mRNA targets in the tested cultivars in response to PVS at 22°C exhibited inter- and intracultivar variability. The majority of tested genes were altered only in the partially resistant Tajfun and Tonacja but not in the susceptible Bryza. Enhanced expression of AGO1-2, DCL1, stu-miR482 and its target Gpa2 was observed in Tonacja and plants of Tajfun in which PVS was detected, with the highest induction of Gpa2 in Tajfun (30.2-fold). However, their expression remained unchanged or decreased in plants of Tajfun in which PVS was undetected. Increased expression of stu-miR168a and stu-miR172e was observed in Tonacja and the PVS-undetected plants of Tajfun, respectively, but not in the PVS-detected plants of Tajfun. This is the first report on cultivar-specific alteration of miRNA in a potato–PVS resistance interaction.     

Prohexadione-calcium induces sunflower (Helianthus annuus) resistance against the root parasitic weed Orobanche cumana

Prohexadione-calcium (PHDC) is a plant growth retardant that inhibits gibberellin biosynthesis and transiently alters the spectrum of flavonoids and their phenolic precursors. In this study, root chamber and pot experiments were conducted to evaluate the effects of PHDC on Orobanche cumana seed germination and induction of sunflower resistance to this root parasitic weed. Root chamber experiments indicated that PHDC did not interfere with the progress of Orobanche seed germination, but retarded O. cumana tubercle formation and development. In pot experiments, control of O. cumana by PHDC depended on its dose, method of application and sunflower cultivar. In sunflower cv. HA89, PHDC at 10 mg LS⁻¹ (litre soil) as two drenches or at 1 m m as two foliar sprays significantly reduced the total number and dry matter of O. cumana without influencing flower, leaf and root development. In sunflower cv. Albena, PHDC at 20 and 30 mg LS⁻¹, but not at 10 mg LS⁻¹, as two drenches significantly reduced O. cumana infection. Sunflower cv. HA89 was less susceptible to O. cumana and more inducible by PHDC than sunflower cv. Albena. The biochemical analysis of root extracts revealed a significantly higher accumulation of free phenolics in sunflower cv. HA89 after PHDC soil drench, but not in Albena. No significant lignification was observed after PHDC treatments. The results suggest that PHDC reduces O. cumana infection in sunflower by inducing host resistance, not by directly interfering with the parasite. Free phenolics play an important role in sunflower response to the parasite.     

Pathogenicity of the root-knot nematode Meloidogyne javanica on potato

Host–parasite relationships and pathogenicity of Meloidogyne javanica on potatoes (newly recorded from Malta) were studied under glasshouse and natural conditions. Potato cvs Cara and Spunta showed a typical susceptible reaction to M. javanica under natural and artificial infections, respectively. In potato tubers, M. javanica induced feeding sites that consisted of three to four hypertrophied giant cells per adult female. Infection of feeder roots by the nematode resulted in mature large galls which usually contained at least one mature female and egg mass. In both tubers and roots, feeding sites were characterized by giant cells containing granular cytoplasm and many hypertrophied nuclei. Cytoplasm in giant cells was aggregated alongside the thickened cell walls. Stelar tissues within galls appeared disorganized. The relationship between initial nematode population density ( P ) [0–64 eggs + second-stage juveniles (J2s) per cm³ soil] and growth of cv. Spunta potato seedlings was tested under glasshouse conditions. A Seinhorst model [ y = m  + (1 −  m ) z ^{( P − T )}] was fitted to fresh shoot weight and shoot height data of nematode-inoculated and control plants. Tolerance limits ( T ) for fresh shoot weight and shoot height of cv. Spunta plants infected with M. javanica were 0·50 and 0·64 eggs + J2s per cm³ soil, respectively. The m parameter in that model (i.e. the minimum possible y -values) for fresh shoot weight and shoot height were 0·60 and 0·20, respectively, at P  = 64 eggs + J2s per cm³ soil. Root galling was proportional to the initial nematode population density. Maximum nematode reproduction rate was 51·2 at a moderate initial population density ( P  = 4 eggs + J2s per cm³ soil).     

Genetic structure and pathogenicity of populations of Phytophthora infestans from organic potato crops in France, Norway, Switzerland and the United Kingdom

Genetic variation and pathogenicity of Phytophthora infestans isolates collected from organic potato crops of the susceptible cv. Bintje and the moderately resistant cv. Santé were assessed in France, Norway, and the United Kingdom in 2001 and in Switzerland in 2001 and 2002. Population structures differed considerably between the four P. infestans populations. Those from France, Switzerland and the UK were mainly clonal populations showing restricted levels of genetic diversity, whilst those from Norway were mixed A1 and A2 mating type populations with high levels of genetic diversity, suggesting periodical sexual reproduction. Isolates collected from cv. Bintje were on average more aggressive than or comparable to isolates from cv. Santé. Race complexity varied considerably between the regional P. infestans populations, with isolates from France and Switzerland showing the highest number of virulence factors. In all pathogen samples but the French, isolates collected from cv. Santé were more complex than isolates collected from cv. Bintje. No directional selection towards increased aggressiveness towards the more resistant cultivar Santé was observed. This suggests that there is no shift towards increased levels of pathogenicity in P. infestans populations following the large-scale introduction of more resistant potato varieties in organic production systems in Europe.     

Pathology of cortical invasion by Plasmodiophora brassicae in clubroot resistant and susceptible Brassica oleracea hosts

The differences in physiological response of Brassica oleracea to infection and growth of Plasmodiophora brassicae in infected tissue were studied in clubroot resistant and susceptible hosts, grown in sand solution culture artificially inoculated with spores of P. brassicae (10⁸ spores mL⁻¹). Primary (root hair) and secondary (cortical) stages of P. brassicae occurred in both resistant and susceptible hosts. Symptoms of cortical invasion by P. brassicae in resistant and susceptible hosts included cell wall breaks, presence of vesicles or inclusion bodies within the cell walls, cell wall thickening in association with plasmodesmata and enlarged and/or disorganized host nuclei. The main difference between the resistant and susceptible host reaction was the absence of degradation of the secondary thickening and cell walls of the xylem in the resistant host. This study supports the existence of an amoeboid form of the pathogen in addition to the recognized two-phase life history of P. brassicae. Furthermore, it suggests that resistance in B. oleracea does not prevent the development of this amoeboid form. A reduced number of cell wall breakages suggests movement of the amoeboid form, may have been restricted but not prevented in the resistant host.     

Occurrence and inheritance of resistance to powdery mildew (Oidium lycopersici) in Lycopersicon species

Among 146 accessions of Lycopersicon pimpinellifolium , 132 of L. esculentum var. cerasiforme and 53 of L. peruvianum screened for resistance to powdery mildew, caused by Oidium lycopersici , a wide variation in reactions was found. Two plants of L. esculentum var. cerasiforme accession LA-1230 were resistant. One resistant symptomless plant of accession LA-1230, designated LC-95, produced homozygous resistant progenies. LC-95 was crossed with cv. Marmande (susceptible parent) and F₁, F₂ and backcrosses to the resistant and the susceptible parents were derived. These genotypes were grown in glasshouses at 23°C and 95–100% RH and inoculated with O. lycopersici . The F₁ plants were susceptible. F₂ and backcross segregations fitted the hypothesis of a single recessive gene which is here designated ol-2 .     

Sulphur accumulation after Verticillium dahliae infection of two pepper cultivars differing in degree of resistance

Elemental sulphur levels, sulphur localization in stems, and levels of sulphate, glutathione and cysteine were studied in pepper ( Capsicum annuum ) cvs Yolo Wonder (higher resistance) and Luesia (lower resistance) after inoculation with Verticillium dahliae , the cause of vascular wilt. Accumulation of elemental sulphur (S⁰) was first detected 10 days after inoculation in Yolo Wonder (mean S⁰ level 7·3  µ g g⁻¹ DW), and 15 days after inoculation in Luesia (mean S⁰ level 3·3  µ g g⁻¹ DW). The maximum level was reached 21 days after inoculation in Yolo Wonder (14·1  µ g g⁻¹ DW). In control plants, elemental sulphur was not detected. SEM-EDX (scanning electron microscopy-energy dispersive X-ray microanalysis) indicated that the sulphur was not restricted to a specific location, but was dispersed throughout the vascular tissue. Sulphate levels showed a decline at the end of the experiment in inoculated plants, possibly related to the increase in sulphur levels seen in the two cultivars. The differences in sulphate levels between the two cultivars may be due to faster sulphate breakdown in cv. Yolo Wonder.     

Diagnosis of potato virus YN: a comparison between polyclonal and monoclonal antibodies and a biological assay

In an investigation conducted over 5 years, tests of samples from the Scottish Seed Potato Classification Scheme showed that when monoclonal antibodies (McABs) were used with the enzyme-linked immunosorbent assay (ELISA), they reliably detected the tobacco veinal necrosis strain of potato virus Y (PVY^N). Polyclonal antibodies used in the first 3 years of the investigation were unreliable in differentiating PVY^N from the ordinary strain (PVY^N). It is suggested that ELISA and McABs be routinely used to diagnose PVY^N and that all samples which give negative results be further assayed biologically to identify the virus or to indicate strains of PVY^N not detectable by the McAB. Problems encountered with both assay systems are discussed.