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This study investigated the antifungal activity of a number of pomegranate genotypes. Since the main compound of pomegranate extract is punicalagin, an important substance involved in antifungal and antimicrobial activity, we analyzed the contents of punicalagin (α and β) in 21 different pomegranate genotypes. Ellagic acid content, total phenolic content, acidity and pH were also determined. This work allowed us to determine which genotypes of pomegranate can be used to obtain extracts with the highest content of punicalagin, with the goal of developing a green alternative to synthetic pesticides. To improve the extraction system from pomegranate peel fruits, several different solvents were tested. All the pomegranate genotypes tested showed antifungal activity; some genotypes were able to almost completely inhibit the fungus, while others had very low inhibitory activity. Research results also showed that the use of water as a solvent for extraction is very effective, especially when it is combined with ethanol. This is very important for the practical use of the extracts since water is economical and environmentally friendly. The research showed that among the genotypes there is also great variability regarding the chemical parameters. Genotypes with a high phenolic and punicalagin content were significantly correlated with antifungal activity. All the other chemical parameters (pH, titratable acidity and ellagic acid content) were not correlated with antifungal activity. The results obtained indicate that the fruits of some pomegranate genotypes could be used to obtain extracts very rich in punicalagins and that these substances could be used as an alternative to synthetic products to control plant disease and improve the quality of the plant products, avoiding the impact of synthetic chemicals on the environment.

Ceratocystis platani is the causal agent of canker stain, the most destructive disease of Platanus spp. The selection of resistant tree genotypes should be an effective method of disease control. Although breeding programs for resistance have been developed, there is no validated protocol for an in depth evaluation of the resistance phenotype. Testing the variables to identify the conditions that fully challenge the genetic potential of the host is crucial not to overestimate labile resistant phenotypes. Here we report results of testing different inoculum doses and inoculation times -early and late spring, summer and autumn -on the response of susceptible plane genotypes. Late spring inoculation gave rise to the quickest death pattern, followed by early spring inoculation. Nevertheless Ceratocystis platani was aggressive also in the hottest period of the summer. The capacity of C. platani to initiate canker stain in the hottest period of the year underlines its capacity to be virulent across a wide range of temperatures. Although autumnal inoculation enabled fungus entry, its progression was precociously halted, as effective and stable resistance reactions were opposed by most trees. An in vitro study of mycelial growth and conidial germination confirmed the capacity of the fungus to be active or to keep its viability across a wide range of temperatures, such as in the three seasons that were taken into consideration. Thus, we hypothesize that the failure of symptom expression after autumnal inoculation might be due to a combination of a temperature-linked reduction in pathogen virulence, and a season-linked resistant reaction of the host. Overall, our data suggest that, in the context of legal sanitary measures, only the coldest and driest periods of the year should be considered for pruning and the removal of infected trees. The different inoculum doses did not condition the death pattern. Comparing germination in suspensions at 1000 and 10 000 conidia per µl, self-inhibition germination occurred, as the germination rate was inversely correlated with the conidia concentration. This is a first step in the definition of a resistance-testing protocol for an in depth evaluation of resistance to canker stain.

A high-performing detection method is essential to safeguard those countries that are still unaffected by canker stain, a devastating disease of Platanus spp. caused by Ceratocystis platani. We previously developed EvaGreen and Taqman-based Real-Time PCR to detect this pathogen, but in-depth validation is needed to guarantee users about its effectiveness and promote its utilization. In this work we present a validation study designed according to EPPO standards, focusing on the analytical and diagnostic sensitivity and specificity. We extend its technical application using SYBR Green. By performing standard curves and eight-replication-based experiments, we established the detection limit at 3 fg C. platani gDNA per PCR reaction. The repeatability and the operator-based reproducibility of the Real-Time PCR was demonstrated. Different gDNA extraction events by different operators and different gDNA extraction modalities did not affect the detection limit. The detection limit threshold cycle was earliest with SYBR Green, followed by Taqman, and EvaGreen. Spiking 6 µl DNA extractions of uninfected, necrotized wood with 3 fg C. platani gDNA confirmed the detection limit: 3 fg C. platani gDNA per PCR reaction, i.e., 0.5 fg gDNA per µl of wood extract. The assays tolerated 6 µl of necrotic C. platani-infected wood extracts without inhibition except for long-dead wood samples, while the 2 µl dose consistently allowed for successful detection. Detection of the pathogen in infected samples showed the highest diagnostic sensitivity with the SYBR Green assay. Agarose gel electrophoresis and staining was validated for visualizing amplicons, even at the detection limit. The specificity of the method was tested against 23 isolates representing the diversity of Ceratocystidaceae, and most species were not detected at 5 ng gDNA. However, some South American strains of the C. fimbriata complex were detected at doses as low as 5 fg. The method remains specific for C. platani detection as no other Ceratocystidaceae are known to colonize plane tree and the species within the geographic range of canker stain of plane tree were only detected at 500 pg or more gDNA. This work paves the way for a performance study of inter-laboratory comparisons.

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