Diplodia seriata Isolated from Declining Olive Trees in Salento (Apulia, Italy): Pathogenicity Trials Give a Glimpse That It Is More Virulent to Drought-Stressed Olive Trees and in a Warmth-Conditioned Environment.

The fungi Botryosphaeriaceae are involved in olive declines in both the world hemispheres and in all continents where this species is cultivated. In Salento (Apulia, Italy), the Botryosphaeriaceae Neofusicoccum mediterraneum and N. stellenboschiana have been reported as the agents of a branch and twig dieback that overlaps with olive quick decline syndrome caused by Xylella fastidiosa subsp. pauca. In this study, we report the finding of Diplodia seriata, another Botryosphaeriaceae species, in Salento in Xylella fastidiosa-infected olive trees affected by symptoms of branch and twig dieback. Given that its presence was also reported in olive in the Americas and in Europe (Croatia) with different degrees of virulence, we were prompted to assess its role in the Apulian decline. We identified representative isolates based on morphological features and a multilocus phylogeny. In vitro tests showed that the optimum growth temperature of the isolates is around 25-30 °C, and that they are highly thermo-tolerant. In pathogenicity trials conducted over eleven months, D. seriata expressed a very low virulence. Nonetheless, when we imposed severe water stress before the inoculation, D. seriata significatively necrotized bark and wood in a time frame of 35 days. Moreover, the symptoms which resulted were much more severe in the trial performed in summer compared with that in autumn. In osmolyte-supplemented media with a water potential from -1 to -3 Mpa, the isolates increased or maintained their growth rate compared with non-supplemented media, and they also grew, albeit to a lesser extent, on media with a water potential as low as -7 Mpa. This suggests that olives with a low water potential, namely those subjected to drought, may offer a suitable environment for the fungus' development. The analysis of the meteorological parameters, temperatures and rainfall, in Salento in the timeframe 1989-2023, showed that this area is subjected to a progressive increase of temperature and drought during the summer. Thus, overall, D. seriata has to be considered a contributor to the manifestation of branch and twig dieback of olive in Salento. Coherently with the spiral decline concept of trees, our results suggest that heat and drought act as predisposing/inciting factors facilitating D. seriata as a contributor. The fact that several adverse factors, biotic and abiotic, are simultaneously burdening olive trees in Salento offers a cue to discuss the possible complex nature of the olive decline in Salento.

Xylella fastidiosa subsp. pauca, Neofusicoccum spp. and the Decline of Olive Trees in Salento (Apulia, Italy): Comparison of Symptoms, Possible Interactions, Certainties and Doubts.

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.

Identification and Characterization of Neofusicoccum stellenboschiana in Branch and Twig Dieback-Affected Olive Trees in Italy and Comparative Pathogenicity with N. mediterraneum.

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.

Interlaboratory Performance of a Real-Time PCR Method for Detection of Ceratocystisplatani, the Agent of Canker Stain of Platanus spp.

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.

Neofusicoccum mediterraneum Is Involved in a Twig and Branch Dieback of Olive Trees Observed in Salento (Apulia, Italy).

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.

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

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 had been buried during planting. The bark was rotted and the xylem was discoloured. A fungal morphotype was strictly associated with symptomatic plants and identified as Dematophora (ex Rosellinia) necatrix. Pathogenicity tests on cvs Favolosa, Leccino and Ogliarola demonstrated that D. necatrix was the causal agent of the disease. Our investigations revealed that infections occurring during autumn and winter greatly favour the disease. By applying a marcottage to the inoculation point, we accelerated the course of the disease and mimicked the lethal outcome observed in the field. In in vitro tests, seven systemic (potential) fungicides strongly inhibited D. necatrix. Dentamet, Al-phosphite and Thiophanate methyl were selected to be tested in planta with a curative and preventive modality. Only Thiophanate methyl, in preventive modality, fully protected the plants from disease progression throughout the observation period. An additional fungal species was strictly associated with both diseased and apparently healthy plants. Morphological and molecular features identified the fungus as Emmia lacerata, a polypore species within the Irpicaceae, which is the agent of white rot on dead woody substrates. To our knowledge, this is the first time that E. lacerata has been reported in Italy and worldwide on olive trees. Inoculation of ?Favolosa' trees revealed that it colonizes the xylem without causing visible alterations. The possible role of E. lacerata in the olive tree-D. necatrix pathosystem is discussed. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10658-022-02458-1.

Kinase domain-targeted isolation of defense-related receptor-like kinases (RLK/Pelle) in Platanus×acerifolia: phylogenetic and structural analysis.

BACKGROUND: Plant receptor-like kinase (RLK/Pelle) family regulates growth and developmental processes and interaction with pathogens and symbionts.Platanaceae is one of the earliest branches of Eudicots temporally located before the split which gave rise to Rosids and Asterids. Thus investigations into the RLK family in Platanus can provide information on the evolution of this gene family in the land plants.Moreover RLKs are good candidates for finding genes that are able to confer resistance to Platanus pathogens. RESULTS: Degenerate oligonucleotide primers targeting the kinase domain of stress-related RLKs were used to isolate for the first time 111 RLK gene fragments in Platanus×acerifolia. Sequences were classified as candidates of the following subfamilies: CrRLK1L, LRR XII, WAK-like, and LRR X-BRI1 group. All the structural features typical of the RLK kinase domain were identified, including the non-RD motif which marks potential pathogen recognition receptors (PRRs). The LRR XII candidates, whose counterpart in Arabidopsis and rice comprises non-RD PRRs, were mostly non-RD kinases, suggesting a group of PRRs. Region-specific signatures of a relaxed purifying selection in the LRR XII candidates were also found, which is novel for plant RLK kinase domain and further supports the role of LRR XII candidates as PRRs. As we obtained CrRLK1L candidates using primers designed on Pto of tomato, we analysed the phylogenetic relationship between CrRLK1L and Pto-like of plant species. We thus classified all non-solanaceous Pto-like genes as CrRLK1L and highlighted for the first time the close phylogenetic vicinity between CrRLK1L and Pto group. The origins of Pto from CrRLK1L is proposed as an evolutionary mechanism. CONCLUSIONS: The signatures of relaxed purifying selection highlight that a group of RLKs might have been involved in the expression of phenotypic plasticity and is thus a good candidate for investigations into pathogen resistance.Search of Pto-like genes in Platanus highlighted the close relationship between CrRLK1L and Pto group. It will be exciting to verify if sensu strictu Pto are present in taxonomic groups other than Solanaceae, in order to further clarify the evolutionary link with CrRLK1L.We obtained a first valuable resource useful for an in-depth study on stress perception systems.

Hairpin RNA-mediated silencing of Plum pox virus P1 and HC-Pro genes for efficient and predictable resistance to the virus.

We report the application of the hairpin-mediated RNA silencing technology for obtaining resistance to Plum pox virus (PPV) infection in Nicotiana benthamiana plants. Four sequences, covering the P1 and silencing suppressor HC-Pro genes of an Italian PPV M isolate, were introduced into N. benthamiana plants as two inverted repeats separated by an intron sequence under the transcriptional control of the Cauliflower Mosaic Virus 35S promoter. In a leaf disk infection assay, 38 out of 40 T0 transgenic plants were resistant to PPV infection. Eight lines, 2 for each construct, randomly selected among the 38 resistant plants were further analysed. Two hundred forty eight out of 253 T1 transgenic plants were resistant to local and systemic PPV infection. All transgenic single locus lines were completely resistant. These data indicate that the RNA silencing of PPV P1/HCPro sequences results in an efficient and predictable PPV resistance, which may be utilized in obtaining stone fruit plants resistant to the devastating Sharka disease.

Molecular and physiological characterization of Italian isolates of Pyrenochaeta lycopersici.

Corky root of tomato caused by Pyrenochaeta lycopersici is a disease of concern in Italy and for many tomato growing areas in the world. Isolates of the fungus were characterized at both the physiological and molecular level. The optimal in vitro growth temperature for all isolates was 23 degrees C. All Italian isolates of P. lycopersici showed similar RAPD and esterase banding patterns. No relevant polymorphisms were detected after enzymatic digestion of PCR-amplified ITS and IGS regions. The overall results indicate a low degree of genetic variability within a collection of 43 Italian isolates. These data are of interest in breeding programs for resistance against corky root of tomato and they provide useful information for the development of molecular diagnostic tools for the rapid identification and detection of P. lycopersici.

Tomato yellow leaf curl Sardinia virus rep-derived resistance to homologous and heterologous geminiviruses occurs by different mechanisms and is overcome if virus-mediated transgene silencing is activated.

The replication-associated protein (Rep) of geminiviruses is involved in several biological processes brought about by the presence of distinct functional domains. Recently, we have exploited the multifunctional character of the Tomato yellow leaf curl Sardinia virus (TYLCSV) Rep to develop a molecular interference strategy to impair TYLCSV infection. We showed that transgenic expression of its N-terminal 210 amino acids (Rep-210) confers resistance to the homologous virus by inhibiting viral transcription and replication. We have now used biochemical and transgenic approaches to carry out a fuller investigation of the molecular resistance mechanisms in transgenic plants expressing Rep-210. We show that Rep-210 confers resistance through two distinct molecular mechanisms, depending on the challenging virus. Resistance to the homologous virus is achieved by the ability of Rep-210 to tightly inhibit C1 gene transcription, while that to heterologous virus is due to the interacting property of the Rep-210 oligomerization domain. Furthermore, we present evidence that in Rep-210-expressing plants, the duration of resistance is related to the ability of the challenging virus to shut off transgene expression by a posttranscriptional homology-dependent gene silencing mechanism. A model of Rep-210-mediated geminivirus resistance that takes transgene- and virus-mediated mechanisms into account is proposed.