Olive trees are infected and damaged by Botryosphaeriaceae fungi in various countries. The botryosphaeriaceous fungus Neofusicoccum mediterraneum is highly aggressive and is a major concern for olive groves in Spain and California (USA), where it causes ‘branch and twig dieback’ characterized by wood discoloration, bark canker, and canopy blight. During surveys of olive groves in Apulia (southern Italy), we noticed that—in some areas—trees were heavily affected by severe branch and twig dieback. In addition, chlorosis and the appearance of red-bronze patches on the leaf preceded the wilting of the foliage, with necrotic leaves persisting on the twigs. Given the severity of the manifestation in zones also subject to olive quick decline syndrome (OQDS) caused by Xylella fastidiosa subsp. pauca, we investigated the etiology and provide indications for differentiating the symptoms from OQDS. Isolation from diseased wood samples revealed a mycete, which was morphologically and molecularly identified as N. mediterraneum. The pathogenicity tests clearly showed that this fungus is able to cause the natural symptoms. Therefore, also considering the low number of tested samples, N. mediterraneum is a potential causal agent of the observed disease. Specifically, inoculation of the twigs caused complete wilting in two to three weeks, while inoculation at the base of the stem caused severe girdling wedge-shaped cankers. The growth rate of the fungus in in vitro tests was progressively higher from 10 to 30 °C, failing to grow at higher temperatures, but keeping its viability even after prolonged exposure at 50 °C. The capacity of the isolate to produce catenulate chlamydospores, which is novel for the species, highlights the possibility of a new morphological strain within N. mediterraneum. Further investigations are ongoing to verify whether additional fungal species are involved in this symptomatology.

For about a decade, olive groves in Apulia (Southern Italy) have been progressively destroyed by Olive Quick Decline Syndrome (OQDS), a disease caused by the bacterium Xylella fastidiosa subsp. pauca (Xfp). Recently, we described an additional wilting syndrome affecting olive trees in that area. The botryosphaeriaceous fungus Neofusicoccum mediterraneum was found associated with the diseased trees, and its high virulence toward olive trees was demonstrated. Given the common features with Branch and Twig Dieback (BTD) of olive tree, occurring in Spain and California, we suggested that the observed syndrome was BTD. During our first survey, we also found a botryosphaeriaceous species other than N. mediterraneum. In the present article, we report the morphological and molecular characterization of this fungal species which we identified as Neofusicoccum stellenboschiana. In the study, we also included for comparison additional N. stellenboschiana isolates obtained from olive trees in Latium and Tuscany region (Central Italy). The occurrence of N. stellenboschiana in olive trees is reported here for the first time in the northern hemisphere. The pathogenicity and virulence were tested in nine inoculation trials, where the Apulian N. stellenboschiana isolate was compared with the isolate from Latium and with the Apulian isolate of N. mediterraneum. Both isolates of N. stellenboschiana proved pathogenic to olive trees. They caused evident bark canker and wood discolouration when inoculated at the base of the stem of two/three-year-old trees and on one-year-old twigs. However, virulence of N. stellenboschiana was significantly lower, though still remarkable, compared with N. mediterraneum in term of necrosis progression in the bark and the wood and capacity of wilting the twigs. Virulence of N. stellenboschiana and N. mediterraneum did not substantially change when inoculations were performed in spring/summer and in autumn, suggesting that these fungal species have the potential to infect and damage olive trees in all seasons. The high thermotolerance of N. stellenboschiana was also revealed with in vitro growth and survival tests. The high virulence of these Botryosphaeriaceae species highlights their contribution in BTD aetiology and the necessity to investigate right away their diffusion and, possibly, the role of additional factors other than Xfp in the general decline of olive groves in Apulia. Hence the importance of assessing the degree of overlap of BTD/Botryosphariaceae with OQDS/Xfp is discussed.

Xylella fastidiosa subsp. pauca (XFP), Neofusicoccum mediterraneum, N. stellenboschiana and other fungi have been found in olive groves of Salento (Apulia, Italy) that show symptoms of severe decline. XFP is well known to be the cause of olive quick decline syndrome (OQDS). It has also been assessed that Neofusicoccum spp. causes a distinct disease syndrome, namely, branch and twig dieback (BTD). All these phytopathogens incite severe symptoms that can compromise the viability of large canopy sectors or the whole tree. However, their specific symptoms are not easily distinguished, especially during the final stages of the disease when branches are definitively desiccated. By contrast, they can be differentiated during the initial phases of the infection when some facets of the diseases are typical, especially wood discoloration, incited solely by fungi. Here, we describe the typical symptomatological features of OQDS and BTD that can be observed in the field and that have been confirmed by Koch postulate experiments. Similar symptoms, caused by some abiotic adverse conditions and even by additional biotic factors, are also described. Thus, this review aims at: (i) raising the awareness that declining olive trees in Salento do not have to be linked a priori to XFP; (ii) defining the guidelines for a correct symptomatic diagnosis to orient proper laboratory analyses, which is crucial for the application of effective control measures. The possibility that bacterium and fungi could act as a polyspecies and in conjunction with predisposing abiotic stresses is also widely discussed.

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.

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 controlling the disease. 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 occurred, 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 in 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 the majority of the trees. An in vitro study of mycelial growth and conidial germination confirmed the capacity of the fungus to be active or to keep the viability in 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 1.000 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.