RESUMO
Decline symptoms associated with lethal stem and branch canker stain along with root and collar rots were observed on 5- to 7-year-old roadside oriental plane trees (Platanus orientalis) in Diyarbakir, Turkey. Above-ground symptoms included leaf necrosis, leaf curling, extensive bluish or blackish staining of shoots, branches, stem bark, and wood surfaces, as well as stem cankers and exfoliation of branch bark scales. A general decline of the trees was distinctly visible from a distance. A Phytophthora/Pythium-like oomycete species with globose to ovoid, often papillate and internally proliferating sporangia was consistently isolated from the fine and coarse roots and stained branch parts and shoots. The pathogen was identified as Phytopythium litorale based on several morphological features. Partial DNA sequences of three loci, including nuclear rDNA internal transcribed spacer (ITS) and the large ribosomal subunit (LSU), and mitochondrial cytochrome c oxidase subunit II (coxII) confirmed the morphological identification. All P. litorale isolates were homothallic, developing gametangia, ornamented oogonia with elongate to lobate antheridia. Pathogenicity of P. litorale was tested by inoculation on excised shoots and by root inoculation on seedlings. P. litorale produced large lesions and blights on shoots in just 5 days and killed 100% of the seedlings in a month. This paper presents the first confirmed report of P. litorale as an important pathogen on a plant species causing branch and stem cankers, and root and collar rot, in and on P. orientalis, resulting in a rapid decline of trees and suggesting a threat to plane.
Assuntos
Corantes , Doenças das Plantas , DNA Espaçador Ribossômico , Filogenia , TurquiaRESUMO
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.
RESUMO
An effective framework for early warning and rapid response is a crucial element to prevent or mitigate the impact of biological invasions of plant pathogens, especially at ports of entry. Molecular detection of pathogens by using PCR-based methods usually requires a well-equipped laboratory. Rapid detection tools that can be applied as point-of-care diagnostics are highly desirable, especially to intercept quarantine plant pathogens such as Xylella fastidiosa, Ceratocystis platani and Phytophthora ramorum, three of the most devastating pathogens of trees and ornamental plants in Europe and North America. To this aim, in this study we developed three different loop mediated isothermal amplification (LAMP) assays able to detect each target pathogen both in DNA extracted from axenic culture and in infected plant tissues. By using the portable instrument Genie® II, the LAMP assay was able to recognize X. fastidiosa, C. platani and P. ramorum DNA within 30 min of isothermal amplification reaction, with high levels of specificity and sensitivity (up to 0.02 pg µL-1 of DNA). These new LAMP-based tools, allowing an on-site rapid detection of pathogens, are especially suited for being used at ports of entry, but they can be also profitably used to monitor and prevent the possible spread of invasive pathogens in natural ecosystems.
RESUMO
Eucalyptus trees, mostly native to Australia, are widely planted in the tropics and Southern Hemisphere for the production of wood and pulp. Worldwide surveys of diseases on these trees have yielded a large collection of Ceratocystis isolates from dying trees or from wounds on their stems. The aim of this study was to characterise these isolates and to consider their relatedness to each other. Culture appearance, morphological features and a distinctive fruity odour in all cultures were typical of species in the Ceratocystis fimbriatasensu lato (s. lat.) complex. Phylogenetic analyses of sequences for the combined ITS, ßt-1 and TEF1-α gene regions revealed a genetically diverse group of isolates residing in a single large clade, that were distinct from all other species in the C. fimbriatas. lat. complex. Based on morphology and phylogenetic inference, the Eucalyptus isolates are recognised as closely related. The South African isolates are described here as a new species, C. eucalypticola.