Conventional PCR and Real-time Quantitative PCR Detection of Helminthosporium Solani in Soil and on Potato Tubers

Silver scurf is an economically important blemish disease of potato caused by the fungus Helminthosporium solani. Two sets of PCR primers, Hs1F1/Hs2R1 (outer) and Hs1NF1/Hs2NR1 (nested) were designed to unique sequences of the nuclear ribosomal internal transcribed spacer (ITS1 and ITS2) regions of H. solani. Nested PCR was used to increase the specificity and sensitivity of single round PCR. Each primer set amplified a single product of 447 bp and 371 bp respectively, with DNA from 71 European and North American isolates of H. solani, and the specificity of primers was confirmed by the absence of amplified product with DNA from other fungal and bacterial plant pathogens. A simple and rapid procedure for direct extraction of DNA from soils and potato tubers was modified and developed to yield DNA of a purity and quality suitable for PCR within 3 h. The sensitivity of PCR for the specific detection of H. solani in seeded soils was determined to be 1.5 spores g^–1 of soil. H. solani was also detected by PCR in naturally infested soil and from peel and peel extract from infected and apparently healthy tubers. Specific primers and a TaqMan fluorogenic probe were designed using the original primer sequences to perform real-time quantitative (TaqMan) PCR. The same levels of sensitivity for specific detection of H. solani in soil and tubers were obtained during first round mboxTaqMan-based PCR as with conventional nested PCR and gel electrophoresis. This rapid and quantitative PCR assay allows an accurate estimation of tuber and soil contamination by H. solani, thus providing a tool to study the ecology of the organism and to serve as a crucial component for disease risk assessments.     

A Combination of Baiting and Different PCR Formats, Including Measurement of Real-Time Quantitative Fluorescence, For the Detection of Phytophthora fragariae in Strawberry Plants

Phytophthora fragariae, the cause of strawberry red stele disease, is a quarantine pathogen in Europe. Detecting low levels of infection requires sensitive and specific methods. In the past, Dutch and English inspection services have used bait plants to test strawberry propagation stocks destined for export. Increasingly though, PCR is being incorporated into these testing procedures in an effort to increase sensitivity and speed. Various combinations of baiting and PCR assays were compared with existing testing procedures. Water and root samples from the bait test were screened by nested PCR and the PCR amplicon was detected by several methods, including fluorescent labelled probes (TaqMan and Molecular Beacon). PCR amplification was monitored in real-time and semi-quantitative detection was possible. Because PCR reactions are sensitive to inhibitors present in extracted DNA samples, an internal control containing the primer sequences specific for P. fragariae was developed to avoid false negatives.     

Real-time detection of <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis> and <Emphasis Type="Italic">P. citrophthora</Emphasis>in citrus roots and soil

Two primers, specific for Phytophthora nicotianae (Pn6) and P. citrophthora (Pc2B), were modified to obtain Scorpion primers for real-time identification and detection of both pathogens in citrus nursery soils and roots. Multiplex PCR with dual-labelled fluorogenic probes allowed concurrent identification of both species ofPhytophthora among 150 fungal isolates, including 14 species of Phytophthora. Using P. nicotianaespecific primers a delayed and lower fluorescence increase was also obtained from P. cactorumDNA. However, in separate real-time amplifications, the aspecific increase of fluorescence from P. cactorum was avoided by increasing the annealing temperature. In multiplex PCR, with a series of 10-fold DNA dilutions, the detection limit was 10 pg l^-1 for P. nicotianaeand 100 pg l^–1 for P. citrophthora, whereas in separate reaction DNA up to 1 pg l^-1 was detected for both pathogens.Simple and rapid procedures for direct DNA extraction from soil and roots were utilised to yield DNA whose purity and quality was suitable for PCR assays. By combining these protocols with a double amplification (nested Scorpion-PCR) using primers Ph2-ITS4 amplifying DNA from the main Phytophthora species (first round) and PnB5-Pn6 Scorpion and Pc2B Scorpion-Pc7 (second round), it was possible to achieve real-time detection of P. nicotianaeand P. citrophthora from roots and soil. The degree of sensitivity was similar to that of traditional detection methods based on the use of selective media. The analyses of artificially and naturally infested soil showed a high and significant correlation between the concentration of pathogen propagules and the real-time PCR cycle threshold.     

Organic Seed-treatment as a Substitute for Chemical Seed-treatment to Control Common Bunt of Wheat

Common bunt of wheat, caused by Tilletia laevis and T. tritici, is a major seed and soil-borne disease in West Asia and North Africa. The use of resistant cultivars and chemical seed-treatments are the current control measures used to combat this disease. The aim of this study was to investigate alternative control measures to chemical seed-treatments that are environmentally friendly to support cultivar resistance. Several organic nutrients [skimmed milk powder, hucket (local skimmed milk) and wheat flour] at a concentration of 160g per kg of seeds were used as seed-treatments on two susceptible bread and durum wheat cultivars (Bau and Sebou, respectively) to examine their effectiveness in controlling the disease. Field trials over four years showed that skimmed milk powder, hucket, and wheat flour reduced common bunt infection levels by 96%, 93% and 62%, respectively. In most cases, the effectiveness of the skimmed milk powder and hucket was equal to the chemical seed-treatment; thus, these organic nutrients offer an effective and environmentally safe alternative to chemical treatments. However, a study of their economic value as well as of their effect on seed germination, and field emergence is needed.     

Detection and Quantification of Spongospora subterranea f. sp. subterranea in Soils and on Tubers Using Specific PCR Primers

PCR-based methods were developed for the detection and quantification of the potato pathogen Spongospora subterranea f. sp. subterranea (S. subterranea) in peel, tuber washings and soil. A partial sequence was obtained for S. subterranea ribosomal DNA and specific PCR primers (Sps1 and Sps2) were chosen from the internal transcribed spacer regions. These primers amplified a 391bp product from S. subterranea DNA but did not amplify DNA from potato or a range of soil-borne microbes, including related species. Diluted S. subterranea DNA was detected at a concentration equivalent to 25×10^–5 cystosori or 1 zoospore per PCR. Amplification was detected from peel and washings of infected and apparently healthy tubers, but not from peel of Scottish classified seed potatoes or axenically micropropagated potatoes. A rapid method for extracting S. subterranea DNA from soils was developed. This yielded DNA pure enough for PCR within 3h and facilitated the detection of 1–5 cystosori per gram of soil. A PCR quantification technique was developed involving comparison of product ratios obtained after co-amplification of S. subterranea DNA along with an internal standard (competitor DNA fragment). This quantitative technique was also adapted for use in soil. PCR detection of S. subterranea in soil was considerably more sensitive than previously reported immunoassays and was quicker and easier than conventional bait plant bioassays. Such an assay could be useful for developing disease risk assessments for field soils and seed potato stocks and for future studies on the ecology and control of S. subterranea.     

Influence of Inoculum Density of Races 0 and 5 of Fusarium oxysporum f. sp. ciceris on Development of Fusarium Wilt in Chickpea Cultivars

Artificial inoculation experiments were carried out at 25°C to determine the effects of inoculum density of Fusarium oxysporum f.sp. ciceris races 0 (Foc-0) and 5 (Foc-5) and susceptibility of chickpea cultivars P-2245 and PV-61 on development of Fusarium wilt. Foc-5 proved much more virulent than Foc-0. Increasing the inoculum density of F. oxysporum f.sp. ciceris caused an exponential reduction in disease incubation period and a monomolecular increase of disease incidence and the area under the disease intensity progress curve. The extent of these effects was highest in the most conducive P-2245/Foc-5 combination and decreased in the less susceptible PV-61 and for the less virulent Foc-0, in that order. For P-2245/Foc-5, the highest disease intensity was attained with 6 chlamydospores g^–1 of soil, the lowest inoculum density in the study. One thousand chlamydospores g^–1 of soil of the same race were needed to attain a comparable disease intensity in PV-61. Twenty thousand chlamydospores g^–1 of soil of Foc-0 were required for maximum disease intensity in P-2245.The disease intensity curves were adequately described by the Gompertz model. Using this model, a response surface for disease intensity was developed, in which the model parameters are expressed as a function of both time from inoculation and inoculum density. This response surface confirmed that the final amount of disease intensity increases in a monomolecular relationship with increasing inoculum density and showed that the relative rate of disease progress increases exponentially with increasing inoculum density of the pathogen.     

Germination of chlamydospores of Fusarium oxysporum f. sp. pisi race 1 in the rhizosphere,and penetration of the pathogen into roots of a susceptible and a resistant pea cultivar

Germination of chlamydospores ofFusarium oxysporum f. sp.pisi race 1 in the rhizosphere of pea seedlings and red clover seedlings grown in natural soil heavily infested with the pathogen, was highest in percentage along the actively growing parts of the roots. At these sites, exudation of ninhydrinpositive substances and reducing sugars was most intense with seedlings grown in vitro.No significant difference in the percentage of germinating chlamydospores ofFusarium oxysporum f. sp.pisi race 1 were observed in the rhizosphere soil and on the root surface of homologous parts of roots of seedlings and mature plants of a susceptible Rondo and a resistant Rovar pea cultivar grown in natural soil heavily infested with the pathogen. Differences in the growth of mycelium of the pathogen on the root surface, or in the attachment of the mycelium to the root surface of both cultivars were not observed. Epidermis and cortex cells of roots of both cultivars reacted on penetration by the pathogen by producing a cellulose thickening of the cell wall, which later became infiltrated with a ligning-like material. A selective effect on the activities of the pathogen in the rhizosphere, on the root surface and in the epidermis and cortex in relation to resistance thus could not be demonstrated. Formation of new chlamydospores from germ tubes of germinating chlamydospores was frequently observed in the rhizosphere of the susceptible and resistant pea cultivar and in the rhizosphere of red clover seedlings.     

Occurrence and etiology of death of young olive trees in southern Spain

New plantations of olive tree in southern Spain are being severely affected by wilt or dieback and death, which has been locally called Drying Syndrome. To determine the etiology of this problem, a study was carried out in samples of affected young trees collected during a seven year period (1989–1995), and in two field surveys in 1994–95 and 1996. Besides some insect damage and agronomic problems, the Drying Syndrome was associated with Verticillium wilt, winter frost and root rot fungi. Although Drying Syndrome can be distinguished from Verticillium wilt, the latter was included in this study, since, frequently, Verticillium wilt symptoms were unspecific and Verticillium dahliae could not be always isolated in the diagnostic work that preceded this study. Early winter frost caused a vascular necrosis and wilt of the young olive trees. This unusual and severe damage was related with the lack of frost hardiness due to warm temperatures during the previous autumn. Root rot fungi were very frequent in the samples of diseased olive trees of field or nursery origin, and they were the main cause of Drying Syndrome in the second field survey, when a heavy rainfall level occurred during winter. Pathogenicity tests showed that five fungal species (Cylindrocarpon destructans, Phytophthora megasperma, P. palmivora, Pythium irregulare and Sclerotium rolfsii) were pathogenic to olive trees and reproduced symptoms of Drying syndrome in rooted cuttings of cultivar Picual. Other fungal species associated with root rot of olive trees in the field or in the nurseries, including Fusarium acuminatum, F. eumartii, F. oxysporum, F. solani, Macrophomina phaseolina and Rhizoctonia solani, were weakly or not pathogenic. Pathogenicity of P. megasperma, P. palmivora and P. irregulare depended on soil water content, since isolates tested caused extensive root rot and sudden plant death only when the soil was continuously waterlogged. The high frequency of P. megasperma in waterlogged field soils and its pathogenicity dependence on soil water content suggest that this pathogen may play an important role in the well known sensitivity of young olive trees to root asphyxiation.     

Nitrite as a factor in the decline of Fusarium oxysporum f. sp. dianthi in soil supplemented with urea or ammonium chloride

Addition of 1 g urea or NH₄Cl per kg dry soil (0.1%) reduced the population ofFusarium oxysporum f. sp.dianthi in one of two soils tested. Ammonia does not seem to be the responsible factor since it accumulated similarly in both soils upon addition of NH₄Cl or urea. Addition of Nitrapyrine in combination with 0.1% urea or NH₄Cl increased ammonia concentrations in soil but decreased the population-declining effect. Addition of nitrate in amounts corresponding to those measured after decomposition of urea in soil had no effect on population development. Addition of nitrite in amounts corresponding to those measured during decomposition of urea in soil decreased the population ofF. oxysporum f. sp.dianthi. In vitro, nitrite inhibited chlamydospore formation. Upon addition of 0.1% urea, nitrite accumulated 10 to 100 times more in the susceptible soil than in the not-susceptible soil. It is concluded that nitrite rather then ammonia is responsible for the decline effect of ammonia-generating compounds on populations ofF. oxysporum f. sp.dianthi in soil.Samenvatting Toevoeging van 0.1% ureum of 0.1% NH₄Cl verminderde de populatie vanF. oxysporum f. sp.dianthi in een van de twee getoetste gronden. Ammoniak lijkt niet verantwoordelijk voor deze afname, aangezien ammoniak in beide gronden gelijkelijk ontwikkelde na toevoeging van ureum of NH₄Cl. Toevoeging van Nitrapyrine tesamen met ureum of NH₄Cl aan de actieve grond verhoogde de concentratie ammoniak in de grond, maar verlaagde het remmend effect. Toevoeging van nitraat in hoeveelheden die overeenkoment met die welke gemeten worden na volledige omzetting van ureum had geen effect op de populatie vanF. oxysporum f. sp.dianthi. Toevoeging van nitriet in hoeveelheden die overeenkomen met die welke gemeten worden na volledige afbraak van ureum verminderde de populatie wel. In vitro remde nitriet de chlamydosporevorming vanF. oxysporum f. sp.dianthi. Toediening van 0.1% ureum aan de grond gaf een 10 tot 100 maal hogere nitrietaccumulatie in de actieve gron dan in de niet-actieve grond. Daarom wordt de conclusie getrokken, dat nitriet veeleer dan ammoniak verantwoordelijk is voor de vermindering van de populatie vanF. oxysporum f. sp.dianthi in grond waaraan ammoniak-genererende verbindingen zijn toegevoegd.     

Histology of roots of resistant and susceptible carnation cultivars from soil infested with fusarium oxysporum f.sp. dianthi

Fusarium wilt-resistant Novada and wilt-susceptible Early Sam carnations were planted in soil infested withFusarium oxysporum f.sp.dianthi, and their roots studied after five and ten weeks. Both in Novada and Early Sam, the extravascular tissue of undamaged young root parts were scarsely colonized. In roots of Novada, infected xylem vessels were usually occluded with gums and surrounded by phellem tissue. In mature parts of roots, the phellem surrounding occluded vessels often merged with the external phellem surrounding the vascular cylinder, after which the occluded vessels were shed from the roots. The phellem at the root surface appeared to be a strong barrier to fungal invasion. In roots of Early Sam carnations, as well as in a few roots of Novada carnations, the defence responses did not result in compartmentation of the fungus, and colonization and degradation of the vascular tissues followed. Diseased roots finally healthy. Shoots of Early Sam carnations, and eventually a few shoots of Novada carnations, were colonized and developed wilt symptoms.Samenvatting Resistente Novada en vatbare Early Sam anjers werden geplant in grond besmet metFusarium oxysporum f.sp.dianthi. Na vijf en tien weken werden de wortels van de planten bestudeerd. De extravasculaire delen van onbeschadigde jonge wortelgedeelten waren in beide cultivars nauwelijks gekoloniseerd. In de wortels van Novada waren geïnfecteerde houtvaten meestal verstopt door gommen en omgeven door kurkweefsel. In oudere wortelgedeelten sloot het kurkweefsel rond verstopte vaten vaak aan op het kurkweefsel aan de buitenkant van het vaatweefsel, waarna de aangetaste vaten werden uitgestoten uit de wortel. Het kurkweefsel aan het worteloppervlak leek een belangrijke barrière te vormen tegen het binnendringen van de schimmel. In de wortels van Early Sam en in enkele wortels van Novada mislukte het afweerproces, en werd het vaatweefsel door de schimmel gekoloniseerd en vervolgens afgebroken. Aangetaste wortels werden na afloop van tijd hol. De bovengrondse delen van de meeste Novada anjers werden niet gekoloniseerd en bleven gezond. De bovengrondse delen van de Early Sam anjers, en op den duur die van enkele Novada anjers, werden gekoloniseerd en verwelkten.     

Development of specific PCR primers for identification and detection of <Emphasis Type="Italic">Phytophthora capsici</Emphasis> Leon

A PCR-based method was developed for the identification and detection of Phytophthora capsici in pepper plants. Three PCR primers (CAPFW, CAPRV1 and CAPRV2) specific for P. capsiciwere designed based on the sequence of its internal transcribed spacer regions. CAPFW/CAPRV1 amplify a 452 bp product from P. capsici DNA whereas CAPFW/CAPRV2 a 595 bp fragment; neither set amplifies DNA from pepper or several fungi pathogenic to pepper. In conventional (single-round) PCR, the limit of detection was 5 pg DNA for both primer sets, whereas in nested PCR the detection limit for both was of 0.5 fg. However, when the dilution series of target DNA were spiked with plant DNA, amplification declined two-fold in both conventional and nested PCR. The CAPFW/CAPRV2 set in conventional PCR was used to detect P. capsici DNA in inoculated plants. Detection occurred as soon as 8h post-inoculation in stem samples from infected but still symptomless plants. The method was also tested to detect fungal DNA in infected soils.     

Effects of fusaric acid on cells from tomato cultivars resistant or susceptible toFusarium oxysporum f. sp.Lycopersici

Cell suspension cultures were set up from two tomato cultivars, one resistant, (Rio grande) and one susceptible (63.5) toFusarium oxysporum f. sp.lycopersici. Growth rates of the two cell cultures were comparable. Toxicity of fusaric acid, expressed as the fresh weight loss, was analyzed: It was significant in both cases after 10 h, but toxicity was twice as high for 63.5 suspension cells. In the same way, electrolyte leakage caused by fusaric acid was three times more important for 63.5 suspension cells. Moreover, fusaric acid treatment resulted in an acidification of the extracellular medium for 63.5 suspension cells (0.4 pH unit), whereas an alkalization was observed for Rio grande suspension cells (0.2 pH unit). Preliminary experiments suggest that fusaric acid was partially metabolized by Rio grande suspension cells, however, no detoxified forms of fusaric acid were detected either in cells or in culture filtrates. For these two tomato cultivars, the differences in sensitivity to fusaric acid of cultivated cells correspond to the differences in plant susceptibility toFusarium oxysporum f. sp.lycopersici.Abbreviations BAP 6-benzylaminopurine - conductivity - 2,4-D 2,4-dichlorophenoxyacetic acid - EtOAc ethyl acetate - FA fusaric acid - resistivity     

Identification and Detection of Rosellinia Necatrix by Conventional and Real-time Scorpion-PCR

Several polymerase chain reaction (PCR) primers were designed from the internal transcribed spacer (ITS) regions of the rDNA genes of Rosellinia necatrix to develop a PCR-based identification method. Screening the primers against two isolates of R. necatrix and six other Rosellinia species resulted in the amplification of a single specific product from R. necatrix for most of the primer pairs. Two primer pairs (R2-R8 and R10-R7) confirmed their specificity when tested against 72 isolates of R. necatrix and 93 other fungi from different hosts and geographic areas. The R10 primer was modified to obtain a Scorpion primer for detecting a specific 112bp amplicon by fluorescence emitted from a fluorophore in a self-probing PCR assay. This assay specifically recognised the target sequence of R. necatrix over a large number of other fungal species. In conventional PCR, with primer pairs R2-R8 and R10-R7, 10-fold dilutions of R. necatrix DNA indicated a detection limit of 10pgul^-1 using a single set of primers and 10fgl^-1 in nested-PCR. For Scorpion-PCR, the detection limit was 1pgl^-1 and 1fgl^-1 in nested Scorpion-PCR, i.e. 10 times more sensitive than conventional PCR. A simple and rapid procedure for DNA extraction directly from soil was modified and developed to yield DNA of purity and quality suitable for PCR assays. Combining this protocol with the nested Scorpion-PCR procedure it has been possible to specifically detect R. necatrix from artificially inoculated soils in approximately 6h.     

Identification of Pathogenic Races 0, 1B/C, 5, And 6 Of Fusarium Oxysporum F. Sp. Ciceris With Random Amplified Polymorphic DNA (RAPD)

Ninety-nine isolates of Fusarium oxysporum f. sp. ciceris (Foc), representative of the two pathotypes (yellowing and wilt) and the eight races described (races 0, 1A, 1B/C, 2, 3, 4, 5, and 6), were used in this study. Sixty isolates were analyzed by the RAPD technique using DNA bulks for each race and 40 primers. Bands presumably specific for a DNA bulk were identified and this specificity was confirmed by further RAPD analysis of individual isolates in each DNA bulk. Primers OPI-09, OPI-18, OPF-06, OPF-10, and OPF-12 generated RAPD marker bands for races 0, 1B/C, 2, 3, 4, 5, and 6. The reliability and utility of this procedure was validated in blind trials using 39 new Foc isolates. Ten of the 39 isolates had already been typed to race by pathogenicity tests and 29 were typed both by pathogenicity and RAPD testing in this study. In these blind trials, we assigned the 39 new isolates to a race solely on the basis of their RAPD haplotype. Thus, we concluded that Foc races 0, 1B/C, 5, and 6 can be characterized by the RAPD markers. Cluster analysis of the RAPD data set resulted in three clusters of isolates within Foc. The yellowing isolates were grouped in two distinct clusters which correspond to races 0 and 1B/C. The wilt isolates constitute a third cluster that included races 1A, 2, 3, 4, 5, and 6. These results provide a means of studying the distribution of Foc races, to assist in the early detection of introduced race(s) and to facilitate the efficient deployment of available host resistance.     

Detection of Phytophthora nicotianae and P. citrophthora in Citrus Roots and Soils by Nested PCR

The polymerase chain reaction (PCR) was used for the specific detection of Phytophthora nicotianae and P. citrophthora in citrus roots and soils. Primers were based on the nucleotide sequences of the internal transcribed space regions (ITS1 and ITS2) of 16 different species of Phytophthora. Two primer pairs, Pn5B–Pn6 and Pc2B–Pc7, were designed specifically to amplify DNA from P. nicotianae and P. citrophthora, respectively. Another primer pair (Ph2–ITS4) was designed to amplify DNA from many Phytophthora species. All primer pairs were assessed for specificity and absence of cross-reactivity, using DNA from 118 isolates of Phytophthora and 82 of other common soil fungi. In conventional PCR, with a 10-fold dilution series of template DNA, the limit of detection was of 1pgl^–1 DNA for all the primer pairs (Ph2–ITS4, Pn5B–Pn6, and Pc2B–Pc7). In nested PCR, with primers Ph2–ITS4 in the first round, the detection limit was of 1fgl^–1 for both the primer sets (Pn5B–Pn6 and Pc2B–Pc7). Simple, inexpensive and rapid procedures for direct extraction of DNA from soil and roots were developed. The method yielded DNA of a purity and quality suitable for PCR within 2–3h. DNA extracted from soil and roots was amplified by nested PCR utilizing primers Ph2–ITS4 in the first round. In the second round the primer pairs Pn5B–Pn6 and Pc2B–Pc7 were utilized to detect P. nicotianae and P. citrophthora, respectively. Comparison between the molecular method and pathogen isolation by means of a selective medium did not show any significant differences in sensitivity.     

Pathogenicity of Fusarium solani f.sp. cucurbitae race 1 to courgette

Fusarium solani f. sp.cucurbitae race 1 causes foot rot in courgette (Cucurbita pepo). The pathogen could be distinguished fromFusarium solani from sweet pepper (Capsicum annuum) both morphologically and in its host range.In inoculation experiments all nine cultivars of the six species of Cucurbitaceae tested were susceptible. Courgette Green became diseased after inoculation with a spore suspension by root dipping or adding the suspension to the soil around the stem base or spraying the whole plant with it.Both wounded and young plants died more quickly than unwounded and older plants. With low inoculum densities the plants were affected more slowly than with high densities and the differences in susceptibility of the Cucurbitaceae tested were more pronounced.From infected courgette seeds the fungus could be reisolated until 6 months after harvest.This is the first record of this pathogen in courgettes in the Netherlands.Samenvatting Fusarium solani f. sp. cucurbitae fysio 1 is de oorzaak van een voetrot in courgette. Het pathogeen is morfologisch en door middel van een waardplantenreeks goed vanF. solani uit paprika te onderscheiden.In inoculatieproeven waren de getoetste Cucurbitaceae in meer of mindere mate gevoelig voorF. solani f. sp.cucurbitae. Bij courgette Green werden zowel gedompelde, als aangegoten, als bespoten planten door het pathogeen aangetast. Zowel verwonde als niet-verwonde planten werden aangetast als ook planten van verschillende leeftijden. Niet-verwonde en ook oudere planten stierven minder snel af dan verwonde en jongere planten. Bij lagere inoculumdichtheden werden de planten minder snel aangetast dan bij hogere dichtheden en waren de verschillen in vatbaarheid voor het pathogeen tussen de getoetste Cucurbitaceae duidelijker.Uit courgette zaad, dat met het pathogeen was besmet, kon de schimmel tot 6 maanden na zaadwinning opnieuw worden geïsoleerd.Dit is de eerste melding van dit pathogeen in courgette in Nederland.     

The β-tubulin Gene is a Useful Target for PCR-based Detection of an Albino Ophiostoma piliferum Used in Biological Control of Sapstain

The potential of Cartapip, an albino Ophiostoma piliferum, as a biocontrol agent against sapstain in logs has been tested in Germany. To detect the albino strain in field-tested wood, the usefulness of the -tubulin gene as a target region for developing PCR-based assays was evaluated with 102 strains of O. piliferum and 31 strains of other wood-inhabiting species. A partial -tubulin gene sequence of O. piliferum strains from different geographic origins was amplified by PCR and analyzed by restriction enzyme digestions and DNA sequencing. Variation in size and nucleotide sequences was found in intron regions indicating that intraspecific variation is present in the -tubulin gene. Consequently, -tubulin gene-derived PCR methods using PCR–RFLP patterns generated by HinfI and SpeI and sequence-specific primers Cat1 and Cat2, were developed and their specificity for Cartapip was accessed with field-tested logs and lumber. The -tubulin gene-based PCR methods were found to be valuable tools for rapid and reliable identification of Cartapip in field-tested logs and lumber in Germany. Specificity tests against other wood-inhabiting species and wild type O. piliferum strains from diverse nations showed that the Cat1 and Cat2 primers have potential to be used in other European countries, New Zealand, Alberta and British Columbia.     

Colonization and histopathology of susceptible and resistant carnation cultivars infected with Fusarium oxysporum f. sp. dianthi

Stems of the susceptible Early Sam and resistant Novada carnations were inoculated with a conidial suspension ofFusarium oxysporum f. sp.dianthi. Stem segments of either cultivar were sampled regularly and used for determination of fungal growth and for microscopical investigation.Early Sam showed typicalFusarium wilt symptoms and its stems were colonized intensively. The observed vascular browning appeared to be caused by discolouration of primary walls of infected vessels and surrounding cells. Vessels were rarely occluded with gel. Cell wall degradation led to the formation of stem cavities. Hyperplasia of xylem parenchyma was not seen.In Novada, fungal colonization remained low throughout the experiment. Macroscopic symptoms were absent except for longitudinal bursts in the stem, which appeared to be caused by hyperplasia of xylem parenchyma bordering infection. Vascular gelation occurred in the infected tissues, causing some vascular browning also. Xylem vessel regeneration was observed in the hyperplastic layer. Cavities were not formed, and wall discolouration was rare. Vascular gelation is considered part of theFusarium wilt resistance mechanism. It is followed by xylem vessel regeneration, which expresses a general plant response to vascular dysfunction rather than being part of the resistance mechanism.Although of different origin, vascular browning as such occurs in both susceptible and resistant interactions. In breeding for resistance, care should hence be taken with the current use of browning as an indication of disease.Samenvatting Anjers van de vatbare cultivar Early Sam en de resistente cultivar Novada werden geïnoculeerd met een conidiënsuspensie vanFusarium oxysporum f. sp.dianthi. Van beide cultivars werden regelmatig stengeldelen geoogst om deze microscopisch te onderzoeken en om de schimmelgroei te bepalen.Early Sam vertoonde de voor deze verwelkingsziekte kenmerkende symptomen en werd intensief gekoloniseerd. Aan het vaatweefsel waargenomen bruinkleuring bleek veroorzaakt te worden door verkleuring van de primaire wanden van geïnfecteerde vaten en de hen omringende cellen. Zelden trad er in de vaten gomvorming op. Celwandafbraak veroorzaakte de vorming van holten in de stengel. Hyperplasie van het houtparenchym werd niet waargenomen.In Novada bleef de schimmelgroei gedurende het hele experiment beperkt. Macroscopisch waren er enkel lengtescheuren in de stengel te zien, die veroorzaakt bleken te worden door hyperplasie van aan de infectie grenzend houtparenchym. In het geïnfecteerde vaatweefsel optredende gomvorming veroorzaakte ook enige bruinkleuring. In het hyperplastische weefsel werd regeneratie van houtvaten waargenomen. In de stengel werden geen holten gevormd, en verkleuring van de celwanden kwam weinig voor. De vorming van gommen in de houtvaten maakt waarschijnlijk deel uit van het resistentiemechanisme. De daarop volgende houtvatregeneratie is eerder een algemene reactie van de plant op vaatverstopping dan een deel van het resistentiemechanisme.Vaatverbruining, zij het van verschillende oorsprong, komt voor in zowel vatbare als resistente interacties. Om die reden moet men in de resistentieveredeling bij de anjer voorzichtig zijn met het gebruik van bruinkleuring als ziekteïndicatie.     

Asymmetric PCR ELISA: Increased Sensitivity and Reduced Costs for the Detection of Plant Viruses

PCR ELISA is the immunodetection of the products of a polymerase chain reaction (PCR). It is effective for detecting and differentiating plant viral nucleic acids, but as currently performed, it is laborious and expensive. The procedure has been modified and simplified by using asymmetric PCR. This eliminated the need to denature and neutralize samples prior to hybridization. It also increased the relative concentration of the target DNA species, making PCR ELISA more sensitive than TaqMan, a fluorescence-based detection method. Reducing the reaction volumes to half and the concentration of the dNTPs and the digoxigenin label by tenfold significantly reduced the costs of PCR ELISA without reducing its sensitivity. The usefulness of these modifications was demonstrated for the detection of Citrus tristeza virus and Rupestris stem pitting-associated virus. We expect that with only minor modifications asymmetric PCR ELISA could be used effectively for the detection of most nucleic acid molecules of interest.     

Distribution of the FoToml gene encoding tomatinase in formae speciales of Fusarium oxysporum and identification of a novel tomatinase from F. oxysporum f. sp. radicis-lycopersici, the causal agent of Fusarium crown and root rot of tomato

The antifungal glycoalkaloid -tomatine accumulates in tomato plants and may protect plants from fungal infection. Fusarium oxysporum f. sp. lycopersici, the causal agent of vascular wilt of tomato, produces a tomatinase (FoToml) that degrades -tomatine to the nontoxic compounds tetrasaccharide lycotetraose and tomatidine. Induction of tomatinases and the distribution of FoToml homologs were examined among 30 strains belonging to 16 formae speciales of F. oxysporum. Tomatinase activity was found in 27 strains belonging to 15 formae speciales, but FoToml homologs (>98% sequence identity) were detected in only six strains belonging to four formae speciales. To identify tomatinases other than FoToml, -tomatine-inducible proteins of another tomato pathogen F. oxysporum f. sp. radicis-lycopersici were analyzed by two-dimensional gel electrophoresis. A protein with a molecular mass of 64kDa accumulated in the -tomatine-induced culture filtrates, and the protein had tomatinase activity, degrading -tomatine to lycotetraose and tomatidine.