Applicazioni e prospettive della Real-Time PCR in patologia forestale

The Real-Time PCR is a molecular biological technique which allow to monitor in real time the amplification of DNA fragments, during all phases of the reaction. This molecular method allow to perform two different type of DNA analyses: a qualitative analysis, aimed at detecting the DNA target in the sample, and a quantitative analysis, aimed at quantifying the DNA target during the amplification process. Real-Time PCR technologies are mainly based on the use of fluorescent dyes (intercalating molecules or oligonucleotidic probes) which are able to interact with target double strand DNA, producing detectable fluorescent light. Depending on technology used, it is possible to distinguish two main detection methods: amplicon sequence non-specific, when an intercalating dye is used, and amplicon sequence specific, when oligonucleotidic probes are utilized. The use of Real-Time PCR on diagnosis and study of phytopathogenic fungi has become popular in the recent years. In fact, the rapid identification and quantification of a pathogen achieved by using this molecular approach, also during the first phases of the infection which are often asymptomatic, has allowed to perform early diagnosis of particularly aggressive pathogens. In addition, this molecular approach favoured the understanding of mechanisms of host plant tolerance, resistance and susceptibility, through epidemiological studies. In this work, four case-studies where Real-Time PCR was used to reach different aims, were analyzed. In the first, Real-Time PCR was used to quantify airborne inoculum of the fungal pathogen Ceratocystis platani, the agent of the ¿canker stain on plane trees¿, in order to identify hazardous areas in urban environment and to estimate the minimal amount of inoculum needed to starting the infection. In the second, an early diagnostic assay based on Real-Time PCR was developed to identify the wood decay fungus Fuscoporia torulosa on asymptomatic infected trees. In the third, the disease resistance of Norway spruce clones against the forest pathogen Heterobasidion annosum was tested through the monitoring of the fungal inoculum inside the plants by using a Multiplex Real-Time PCR assay coupled with ergosterol method. In the last case-study, an epidemiological survey of the agent of ¿Sudden Oak Death¿ Phytophthora ramorum was carried out in several California forests, by using the Real-Time PCR to identify and quantify the pathogen in different substrates during the year. In conclusion, the diverse applications of the Real-Time PCR showed the great potential of this method for preventive actions, allowing to reduce or contain the spreading of aggressive pathogens, especially the invasive ones. Early diagnosis of fungal diseases by the use of this molecular technique could result in a reduction of forests losses and in the protection of susceptible native host plants.