Method to diagnose latent infection by <Emphasis Type="Italic">Glomerella cingulata</Emphasis> in strawberry plants using ethanol

Signs on strawberry leaves with latent infection by Glomerella cingulata became visible by a simple diagnostic method using ethanol immersion treatment (SDEI). Leaves treated with SDEI changed to dark brown or nearly black, and salmon-pink conidial masses were subsequently produced in the acervuli 5–10 days after incubation in moist petri dishes. The formation rate of conidial masses through SDEI was higher as the position of the leaves became lower. Conidial masses were produced more readily and abundantly when SDEI was performed at 28°C than at 22°, 25°, or 31°C. Latent infection was found to last 1–180 days. There was no difference in the time required for conidial production or in the rate of conidial formation regardless of isolate, cultivar, or leaf position. The varietal difference in resistance to strawberry anthracnose did not influence the rate of conidial mass formation after SDEI. SDEI is thus useful for detecting latent infections during the process of selecting disease-free plants.     

Simple diagnosis using ethanol immersion of strawberry plants with latent infection by Colletotrichum acutatum, Dendrophoma obscurans, and Fusarium oxysporum f. sp. fragariae

Simple diagnosis by ethanol immersion (SDEI) to detect Glomerella cingulata was used to detect three other fungi that also cause latent infection of strawberry plants. Signs on strawberry leaves with asymptomatic latent infection by Colletotrichum acutatum became visible using SDEI. Salmon-pink conidial masses were produced in the acervuli on the treated leaves 5 days after incubation at 28°C. In the case of Dendrophoma obscurans, pycnidia with amber conidial masses formed 5 days after incubation at 28°C. The pycnidia were observed mainly on the ribs, and conidial masses exuded from the ostiole. These macroscopic conidial masses were similar to those of G. cingulata and C. acutatum. When water was dripped onto a lesion caused by D. obscurans, the pycnidia exuded white filamentous conidial masses, making the distinction of D. obscurans from G. cingulata or C. acutatum. On petioles with latent infection by Fusarium oxysporum f. sp. fragariae, white aerial hyphae grew out from the vascular tissues on the cut surface 3 days after incubation at 28°C and were easily observed by eye or with a loupe. Thus, SDEI was also useful for diagnosing latent infection of strawberry plants by C. acutatum, D. obscurans, and F. oxysporum f. sp. fragariae.     

Suitability of Ten Plant Baits for the Rapid Detection of Pathogenic Pythium Species in Hydroponic Crops

Ten types of plant baits were tested in the laboratory to assess their capacity to detect pathogenic Pythium species. These were orange tree leaves, tomato leaves, pepper leaves, geranium leaves, Bermuda grass leaves, pine needles, immature carnation petals, hemp-seed cotyledons, pepper and cucumber fruits. The Pythium spp. tested were P. aphanidermatum, P. irregulare and Pythium group F from hydroponic market garden crops in the Poniente region of Almería (south-east Spain). The test consisted of observing the velocity at which five baits were colonized and the day of colonization of the first bait. Results indicated that the slowest baits to be infected were immature carnation petals and pine needles. These two, together with Bermuda grass leaves, were also the baits infected in lowest number, such that practically no further infection was produced in the baits after the fifth day of contact with the inoculated water. The other plant baits tested were equally suitable for detection of Pythium spp. over the first two days, although only orange leaves and hemp-seed cotyledons were infected on the first day.     

Purification of lily symptomless virus. Use and value of antisera against intact and pyrrolidine-degraded virus for testing lilies and tulips

Lily symptomless virus (LSV) was purified by clarification with chloroform, precipitation with polyethylene glycol and NaCl, and differential centrifugation. The influence of the source material and some buffers on virus yields were determined.Antisera were prepared against intact and pyrrolidine degraded LSV. It was concluded that intact and degraded LSV have very few antigenic determinants in common or none at all. The sensitivities of the micro-precipitin test and the single immunodiffusion drop test were about the same, but lower than that of electron microscopy.In the testing of lilies for LSV the most reliable results in leaves were obtained during the period from two weeks after flowering until close to the end of the growing season, and in leaves growing at a level about one-fourth of the distance from the top of the stem. In contrast to secondary infections, primary infections were detected more successfully in stored bulbs than in leaves taken from plants in the preceding growing season.In the testing of tulips, LSV was detected better in flowers than in leaves. Detection of primary infections was almost impossible. Except for those with a pink flower, experimentally infected tulips remained symptomless.Samenvatting Het symptoomloos lelievirus (LSV) were gezuiverd door klaring met chloroform, precipitatie met polyethyleenglycol en NaCl en differentieel centrifugeren. Het effect van het uitgangsmateriaal en enkele buffers op de virusopbrengst werd nagegaan.Antisera werden bereid tegen intact en met pyrrolidine afgebroken LSV. Geconcludeerd were dat intact en afgebroken LSV geen of slechts enkele antigene determinanten gemeenschappelijk hebben.De gevoeligheid van de microprecipitatietoets en de enkele immuno-diffusiedruppeltoets is ongeveer gelijk. Met het elektronenmicroscoop kunnen echter lagere virusconcentraties worden aangetoond.Het toetsen van lelies op LSV gaf de betrouwbaarste resultaten met bladeren die op ongeveer driekwart hoogte van de stengel groeien en in de periode tussen 2 weken na de bloei en vrijwel het einde van het groeiseizoen worden geplukt. Primaire infecties konden, in tegenstelling tot secundaire infecties, beter worden vastgesteld aan bolmateriaal tijdens de bewaring dan aan bladeren in het voorafgaande groeiseizoen.Bij het toetsen van tulpen werd het LSV met grotere zekerheid vastgesteld in bloemen dan in bladeren. Het vaststellen van primaire infecties was bijna niet mogelijk. Na infectie van tulpen met LSV vertoonden alleen die met een rose bloemkleur symptomen.     

Fungal and plant gene expression during synchronized infection of tomato leaves by Botrytis cinerea

An inoculation procedure was developed to obtain efficient and synchronous infection on detached tomato leaves by Botrytis cinerea. In spray-inoculated leaves incubated at 20 °C, the infection process consisted of three phases: the formation of primary necrotic lesions (until 20 hpi), a quiescent phase (20-72 hpi), and the expansion of a proportion of the primary lesions (from 72 hpi onwards), resulting in full tissue maceration. At 4 °C, the infection progressed slowly but steadily without inducing necrotic responses in the host. The actin and -tubulin genes of B. cinerea were cloned, characterized and used as probes on blots containing RNAs from leaves at various stages of the infection. The genes displayed a similar expression pattern throughout the infection and the hybridization signal reflected the amount of fungal biomass. The actin mRNA accumulated to higher levels than the -tubulin mRNA. Tomato PR protein mRNAs (chitinase, -1,3-glucanase and PR-1) were induced during the infection, albeit with different kinetics and to different levels. At 20 °C, -1,3-glucanase and PR-1 mRNAs were induced more rapidly than chitinase mRNAs. At 4 °C, mRNAs encoding extracellular -1,3-glucanase and intracellular, as well as extracellular chitinase were hardly induced.     

Characterisation of <Emphasis Type="Italic">Phoma tracheiphila</Emphasis> by RAPD-PCR,microsatellite-primed PCR and ITS rDNA sequencing and development of specific primers for <Emphasis Type="Italic">in planta</Emphasis> PCR detection

Thirty six isolates of Phoma tracheiphila from Italy, the causal agent of the mal secco disease on Citrus species, were characterised by different molecular tools in comparison with representative isolates of other phytopathogenic Phoma species. These included analysis of the distribution of RAPD and microsatellite markers and sequencing of the internal transcribed spacer (ITS) region of the nuclear rRNA genes. The results obtained with 12 RAPD primers (92 markers) and 7 microsatellite primers (56 markers) suggest that Italian isolates of P. tracheiphila are genetically homogeneous, leading to identical patterns upon amplification with all the tested primers. Accordingly, ITSI-5.8S-ITS2 sequences were highly conserved (98–100% identity along a 544-characters alignment) among all the isolates of P. tracheiphila. A neighbor-joining analysis of ITS sequences of P. tracheiphila in comparison with those of other Phoma species, as well as with alignable sequences from anamorphic and teleomorphic taxa retrieved in BLAST searches, revealed a close relationship between P. tracheiphila and Leptosphaeria congesta. A pair of P. tracheiphila-specific primers was designed on the consensus sequence (555 residues) obtained from the alignment of the newly generated P. tracheiphila ITS sequences. A PCR-based specific assay coupled to electrophoretic separation of amplicons made it possible to detect P. tracheiphila in naturally infected Citrus wood tissue collected from both symptomatic and symptomless plants. The limit of detection was 10 pg of genomic DNA and 5 fg of the ITS target sequence.     

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.     

Pathogenicity and genetic variation of Phytophthora cactorum from silver birch and strawberry

Phytophthora cactorum strains isolated from necrotic stem lesions on Betula pendula seedlings or from Fragaria ananassa plants suffering from crown rot were pathogenic to their host plants. Only isolates from birch caused clear lesions on non-wounded bark of birch. P. cactorum isolates from birch were not detrimental to strawberry. Random Amplified Polymorphic DNA (RAPD) analysis revealed variation within P. cactorum, isolates from silver birch having different banding patterns than those from strawberry. UPGMA analysis clustered isolates from silver birch and strawberry plants into separate groups. The data show that the recent outbreak in Finland of P. cactorum in birch could not be caused by the import of strawberry plants affected by crown rot.     

Acibenzolar-S-methyl Induces the Accumulation of Defense-related Enzymes in Apple and Protects from Fire Blight

Acibenzolar-S-methyl (Novartis) is a chemical inducer of systemic acquired resistance in several annual plants. The ability of this novel chemical to induce resistance was studied in a perennial plant (apple) affected by fire blight caused by Erwinia amylovora. Acibenzolar-S-methyl (100 and 200mg/l active ingredient) protected Golden Delicious seedlings, scions and trees from artificial infection when applied before inoculation. The protection of apple seedlings was similar to the protection obtained with the standard for fire blight control, plantomycin (100mg/l streptomycin sulfate), applied immediately before inoculation. The mean levels of control in scions in the greenhouse and in trees in orchards were approximately 69% and 50%, respectively. The protection of apple seedlings was constantly associated with the activation of two families of defense-related enzymes, peroxidases and -1,3-glucanases. Accumulation of both enzymes was induced locally in treated leaves and systemically, especially for -1,3-glucanases, in upper untreated leaves, and was sustained for at least 17 days. These results suggest that acibenzolar-S-methyl promotes induced systemic resistance in apple by increasing defense-related compounds. This chemical could provide a new approach of control of fire blight but its practical use needs further investigation.     

PCR-based Assays to Assess Wheat Varietal Resistance to Blotch (Septoria Tritici and Stagonospora Nodorum) and Rust (Puccinia Striiformis and Puccinia Recondita) Diseases

A multiplex Polymerase Chain Reaction (PCR) assay was developed to detect and quantify four fungal foliar pathogens in wheat. For Septoria tritici (leaf blotch) and Stagonospora nodorum (leaf and glume blotch), the -tubulin gene was used as the target region. Diagnostic targets for Puccinia striiformis (stripe or yellow rust) and P. recondita (brown rust) were obtained from PCR products amplified with -tubulin primer sequences. Final primer sets were designed and selected after being tested against several fungi, and against DNA of infected and healthy wheat leaves. For detection of the four pathogens, PCR products of different sizes were amplified simultaneously, whereas no products were generated from wheat DNA or other non-target fungi tested. The presence of each of the diseases was wheat tissue- and cultivar specific. Using real-time PCR measurements with the fluorescent dye SYBR Green I, PCR-amplified products could be quantified individually, by reference to a standard curve generated by adding known amounts of target DNA. Infection levels for each of the diseases were measured in the flag leaf of 19 cultivars at Growth Stage (GS) 60–64 in both 1998 and 1999. The infection levels for the cultivars were ranked, and showed, with a few exceptions, a good correlation with the NIAB Recommended List for winter wheat, which is based on visual assessment of symptoms. With PCR, the presence of the different pathogens was accurately diagnosed and quantification of pre-symptomatic infection levels was possible. Although sampling and DNA detection methods need further optimisation, the results show that multiplex PCR and quantitative real-time PCR assays can be used in resistance screening to measure the interaction between different pathogens and their hosts at different growth stages, and in specific tissues. This should enable an earlier identification of specific resistance mechanisms in both early-stage breeding material and field trials.