Elsinoë fawcettii, E. australis, and Pseudocercospora angolensis are causal agents of citrus scab and spot diseases. The three pathogens are listed as quarantine pests in many countries and are subject to phytosanitary measures to prevent their entry. Diagnosis of these diseases based on visual symptoms is problematic, as they could be confused with other citrus diseases. Isolation of E. fawcettii, E. australis, and P. angolensis from infected tissues is challenging because they grow slowly on culture media. This study developed rapid and specific detection tools for the in planta detection of these pathogens, using either conventional PCR or one-tube multiplex real-time PCR. Primers and hybridization probes were designed to target the single-copy protein-coding gene MS204 for E. fawcettii and E. australis and the translation elongation factor (Tef-1α) gene for P. angolensis. The specificity of the assays was evaluated by testing against DNA extracted from a large number of isolates (102) collected from different citrus-growing areas in the world and from other hosts. The newly described species E. citricola was not included in the specificity test due to its unavailability from the CBS collection. The detection limits of conventional PCR for the three pathogens were 100, 100, and 10 pg μl−1 gDNA per reaction for E. fawcettii, E. australis, and P. angolensis, respectively. The quadruplex qPCR was fully validated assessing the following performance criteria: sensitivity, specificity, repeatability, reproducibility, and robustness. The quadruplex real-time PCR proved to be highly sensitive, detecting as low as 243, 241, and 242 plasmidic copies (pc) μl−1 of E. fawcettii, E. australis, and P. angolensis, respectively. Sensitivity and specificity of this quadruplex assay were further confirmed using 176 naturally infected citrus samples collected from Ethiopia, Cameroon, the United States, and Australia. The quadruplex assay developed in this study is robust, cost-effective, and capable of high-throughput detection of the three targets directly from citrus samples. This new detection tool will substantially reduce the turnaround time for reliable species identification and allow rapid response and appropriate action.

Black spot symptoms were reported on vanilla plants in Reunion Island in 2011. They have repeatedly reduced annual pod production by 10% to 30%. The disease is characterized by dark spots that appear in slight depressions on flowers, pods, leaves and stems. The spots then develop into broad clearly depressed necrotic plaques. Morphological and molecular analyses, as well as pathogenicity tests, identified the fungus Colletotrichum orchidophilum (Ascomycota) as the causal agent of the disease. Inoculation tests in controlled conditions confirmed that the two C. orchidophilum strains isolated from fruit lesions are pathogenic on the leaves and fruits of Vanilla planifolia (accessions CR0001 and CR0020). However, these strains induced symptoms only when the epidermis of leaves and fruits had been punctured by a needle. In the absence of injury, no symptom appeared. Colletotrichum arxii and Fusarium proliferatum (Ascomycota) are fungal species that are also frequently isolated from black spot lesions. However, they are not pathogenic to vanilla. This is the first report of C. orchidophilum in Reunion Island. It is also the first demonstration of C. orchidophilum's pathogenicity to an orchid. Simple preventive control measures were proposed to reduce the incidence of black spot disease in vanilla plots.

Four species of the destructive forest pathogen Heterobasidion annosum sensu lato (s.l.) are present in Europe: H. annosum sensu stricto (s.s.), H. abietinum and H. parviporum are native species, while H. irregulare is a non‐native invasive species currently reported only in Italy, yet recommended for regulation throughout Europe. In this study, real‐time PCR detection tests were developed for each of the four species, which can be used simultaneously or individually thanks to probes labelled with species‐specific fluorescent dyes. The different performance criteria of each assay were evaluated, and it was determined that they were theoretically capable of detecting amounts of DNA corresponding to 311, 29 and 29 cell nuclei in H. annosum s.s., H. irregulare and H. parviporum, respectively. The specificity of each assay was assessed with a wide set of strains. Real‐time PCR tests successfully detected Heterobasidion species from 36 fruiting bodies taken from the forest, as well as from artificially inoculated or naturally infected wood samples. The multiplex real‐time PCR assays developed in this study could have practical applications both in forest management and in phytosanitary monitoring.

Ceratocystis paradoxa (Dade) C. Moreau is a polyphagous wound parasite causing black rot post-harvest disease in pineapple. This fungus is responsible for high losses of fruit destined for the fresh market and is a common problem in many countries (2). C. paradoxa is officially listed as a quarantine pathogen for French Guiana. In November 2013, the Plant Protection Service of French Guiana observed damage in crops of Ananas comosus var. perolera located in Corossony (4°19′10.8″ N, 52°10′17.1″ W) and Wayabo (5°01′02.3″ N, 52°36′18.7″ W) (eastern and middle part of the country). The three plants collected at each location showed a soft base rot of the stem and of young leaves, and developed a foul smell. Plant tissues were collected from the edge of the lesions, chopped in small pieces, and plated on malt extract agar supplemented with 100 ppm chloramphenicol. The plates were incubated at 25°C with a 12-h photoperiod. After 5 days, a fungal colony, first white and downy, then becoming black and velvety after 10 days, was transferred on potato dextrose agar (PDA) and incubated in the same conditions. After 7 days, the colonies produced phialides releasing cylindrical or doliform conidia that were unicellular, colorless to pale brown, in long chains (3.09 to 20.17 × 3.1 to 5.57 μm, n = 20) and oval, pyriform, brown chlamydospores (8.02 to 21.32 × 4.20 to 9.76 μm, n = 20), occurring in long chains or singly with a vertical slit, usually not very visible. Furthermore, the colonies emitted a fruity odor. On the basis of these morphological characteristics, the fungus was identified as the anamorph of C. paradoxa (Thielaviopsis paradoxa (De Seynes) Höhn.) (1). The species designation was confirmed by sequencing the ITS region of the rDNA followed by comparison with reference sequences available in GenBank. Fungal material was collected from PDA culture by scraping the mycelium with a sterile needle and transferring into 2-ml microtubes. Fungal total DNA was then extracted and the ITS region was amplified by PCR using the ITS1-ITS4 primer pair. Nucleotide sequence was determined and deposited in GenBank (KJ667047). BLAST analysis showed 100% identity with C. paradoxa. The pathogenicity of the fungus was confirmed by inoculating two pineapples with mycelium from the C. paradoxa isolate grown on PDA. The peel of fruits and the base of the crowns were wounded with a sterile scalpel blade, each at five locations. Mycelial plugs (avg. 4 mm diameter) were placed on the wounds. Inoculation sites were wrapped with Parafilm to prevent dehydration and to hold the mycelial plugs in position. Negative controls received five sterile PDA plugs. The samples were incubated at 25°C in a moist chamber with a 12-h photoperiod. Eight days after inoculation, negative controls remained symptomless, whereas characteristic soft, watery, and black rot lesions developed on the base of all the crowns that were inoculated with C. paradoxa. The pathogen was successfully re-isolated from symptomatic tissues, fulfilling Koch's postulate. To our knowledge, this is the first report of C. paradoxa on A. comosus in French Guiana, and quarantine measures have been enforced to prevent the spread of this pathogen that might also cause severe losses on other susceptible plant species that are important for the local market (e.g., banana, coconut, sugar cane). Pineapple has become a major crop in French Guiana, and is now subjected to a more intensive monitoring, which may explains why this disease was discovered recently. References: (1) T. R. Nag Raj and W. B. Kendrick. A Monograph of Chalara and Allied Genera. Wilfrid Laurier University Press, Waterloo, Ontario, 1975. (2) R. C. Ploetz et al., eds. Compendium of Tropical Fruit Diseases. American Phytopathological Society, St. Paul, MN, 1994.

No abstract

Ceratocystis platani (CP), an ascomycetous fungus, is the agent of canker stain, a lethal vascular disease of Platanus species. Ceratocystis platani has been listed as a quarantine pest (EPPO A2 list) due to extensive damage caused in Southern Europe and the Mediterranean region. As traditional diagnostic assays are ineffective, a Real-Time PCR detection method based on EvaGreen, SYBR Green, and Taqman assays was previously developed, validated in-house, and included in the official EPPO standard PM7/14 (2). Here, we describe the results of a test performance study performed by nine European laboratories for the purpose of an interlaboratory validation. Verification of the DNA extracted from biological samples guaranteed the high quality of preparations, and the stability and the homogeneity of the aliquots intended for the laboratories. All of the laboratories reproduced nearly identical standard curves with efficiencies close to 100%. Testing of blind-coded DNA extracted from wood samples revealed that all performance parameters—diagnostic sensitivity, diagnostic specificity, accuracy and reproducibility—were best fit in most cases both at the laboratory and at the assay level. The previously established limit of detection, 3 fg per PCR reaction, was also validated with similar excellent results. The high interlaboratory performance of this Real-Time PCR method confirms its value as a primary tool to safeguard C. platani-free countries by way of an accurate monitoring, and to investigate the resistance level of potentially canker stain-resistant Platanus genotypes.

Podosphaera pannosa (Wallr.:Fr.) de Bary (anamorph Oïdium leucoconium Desm.) is described as the most frequent species causing powdery mildew of members of the Rosaceae family, especially on Rosa spp. and Prunus spp. P. pannosa is reported as cosmopolitan, but its occurrence on Prunus cerasus (cherry) is limited to Hungary (3). During spring 2011, typical symptoms of powdery mildew were observed in a Prunus cerasus orchard located in La Roche de Glun (southeastern France). On average, 25% of the shoots per individual tree were affected by this disease. Although several different cultivars were grown in the orchard, cultivar Bigalise alone displayed powdery mildew symptoms. The lower surface of the leaves was covered with superficial, white, dense mycelium, whereas the upper side showed discoloration, necrosing patches, and blisters. Microscopic slides were prepared from fresh material by gently pressing a clear adhesive tape onto the lower surface covered by mycelium, which was further stained with lactic acid/methyl blue. The presence of powdery mildew was confirmed by the observation of typical microscopic features of the anamorphic stage of the fungus (2). Conidiophores were erect. Conidia (oïdia) were hyaline and keg-shaped, and developed basipetally in chains of six to eight conidia. Conidial dimensions were 17 to 29 (23) × 9 to 17 (14) μm. No cleistothecia (teleomorphic state of the fungus) were observed. Species identity was determined by sequencing the ITS region of the rDNA followed by comparison with reference sequences available on GenBank (1). Fungal material was collected from infected leaves by scraping the mycelium with a sterile needle, and was transferred into 2-ml microtubes. Fungal total DNA was then extracted using a commercial plant DNA extraction kit and the ITS region was amplified by PCR using the ITS1-ITS4 primer pair (4). Nucleotide sequence was determined and deposited in GenBank (Accession No. JN654341). Analysis of the sequence by BLAST showed 100% identity with Podosphaera pannosa. To our knowledge, this is the first report of Podosphaera pannosa on Prunus cerasus in France. This species was hitherto scarcely reported on cherry trees, and may deserve more attention in the future. References: (1) M. Gàbor et al. Eur. J. Plant Pathol. 131:135, 2011. (2) G. Grove et al. Page 12 in: Compendium of Stone Fruit Diseases. American Phytopathological Society, St Paul, MN, 1995. (3) L. Vajna et al. New Dis. Rep. 12:15, 2005. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications, 1990.