Molecular Identification of a New Member of the Clover Proliferation Phytoplasma Group (16SrVI) Associated with Centaurea Solstitialis Virescence in Italy

In the United States, yellow starthistle (Centaurea solstitialis) is an annual invasive weed with Mediterranean origins. Malformed plants displaying witches' broom, fasciations, abortion of buds and flower virescence symptoms were observed in central Italy. Attempts to transmit the causal agent from the natural yellow starthistle host to periwinkle by grafting, resulted in typical symptoms of a phytoplasma, i.e. yellowing and shortening of internodes. The detection of phytoplasmas was obtained from both symptomatic yellow starthistle and periwinkle by the specific amplification of their 16S-23S rRNA genes. PCR amplification of extracted DNA from symptomatic plant samples gave a product of expected size. Asymptomatic plants did not give positive results. An amplicon obtained by direct PCR with universal primers P1/P7 was cloned and sequenced. The homology search using CLUSTALW program showed more than 99% similarity with Illinois elm yellows (ILEY) phytoplasma from Illinois (United States) and 97% with Brinjal little leaf (BLL) phytoplasma from India. Digestion of the nested-PCR products with restriction enzymes led to restriction fragment length polymorphism patterns referable to those described for phytoplasmas belonging to the clover proliferation (16S-VI) group. Since this is a previously undescribed disease, the name Centaurea solstitialis virescence has been tentatively assigned to it. This is a new phytoplasma with closest relationships to ILEY and BLL, but distinguishable from them on the basis of 16S rDNA homology, the different associated plant hosts and their geographical origin.     

Root and stem rot,and wilting of olive tree caused by Dematophora necatrix and associated with Emmia lacerata in Central Italy

European Journal of Plant Pathology - Lethal wilting was observed on young olive trees cv Favolosa in a grove in central Italy. White mycelial strands wrapped the basal portion of the stems that...     

Development of Real-Time PCR for in wood-detection of Ceratocystis platani, the agent of canker stain of Platanus spp.

Ceratocystis platani is a quarantine fungal pathogen agent of canker stain, a destructive disease affecting Platanus. Despite its diagnosis being critical for disease control, there is still no effective diagnostic tool as all known mycological and biological detection assays are problematical. In this study we developed highly effective Real-Time PCR methods based on the use of an intercalating dye, EvaGreen, and on a Taqman probe. We designed primers and probe on the internal transcribed spacer (ITS) region of rDNA, and used them for the amplification and detection of a 95?bp C. platani amplicon. Inference of standard curves revealed that both Real-Time procedures have similar and high values of amplification efficiency when applied to a range of templates, e.g. genomic fungal DNA and DNA extracted from diseased wood. The methods were sensitive with a detection limit of 10?fg???l^?1? C. platani genomic DNA. They were specific as they did not yield any detection signal when applied to non-target fungal taxa colonizers of Platanus wood. Reliability was demonstrated through the positive detection of a collection of C. platani isolates and of wood samples collected from naturally infected trees. Robustness was positively verified through detection on artificially infected trees, which were tested at different times after death, up to 27?months. Generating a standard curve with a target-amplicon-containing plasmid enabled an absolute quantification and a comparison between the discoloured wood of resistant and susceptible genotypes. The importance of the methods for studies on pathogen epidemiology and host resistance is also discussed.