Search

Environmental changes are occurring on a global scale, but their effects are most pronounced in climate change hotspot zones, such as the Mediterranean basin. Within this area Italy, extending from its southern coasts in the core of the Mediterranean Sea to its northernmost pre-Alpine and Alpine regions, is characterized by a variety of climatic conditions and vegetation types. Surveys conducted in 2018–2022 in forest formations of Central-Northern Italy revealed that the enhanced warming trend and irregular distribution of precipitations are strongly impacting the health of Fraxinus species, with some pathogenic fungi and oomycetes being important contributing factors to the decline of the three main ash species growing there: common ash (Fraxinus excelsior), flowering ash (Fraxinus ornus), and narrow-leaved ash (Fraxinus angustifolia). Isolation from symptomatic plant material collected countrywide under different site conditions and pathogenicity tests revealed a complex phytopathological framework, with several pathogenic species in addition to Hymenoscyphus fraxineus involved with a prominent role in the ash dieback etiology. Key microbial taxa included the fungal and oomycete pathogens Botryosphaeria dothidea, Diplodia fraxini, Diplodia subglobosa, Phytophthora acerina, and Phytophthora plurivora. The disease impact was higher on sites where ash trees grew under environmental stress (i.e., areas characterized by mild dry winters, hot summers with intense and prolonged drought) and exhibited reduced vigor, also as a consequence of anthropogenic interference (i.e., silvicultural management and fires). The identified causative agents are emerging pathogens that thrive under warmer conditions, their impact in the investigated areas being prevalent compared to H. fraxineus, which appears to be restricted on the Italian peninsula to the cooler and wetter valleys of the Alps and Central-Northern Apennines.

Gnomoniopsis castaneae is responsible for brown or chalky nut rot in sweet chestnut (Castanea sativa), causing heavy reductions in nut production. Controlling it is challenging, due to its inconspicuous infections, erratic colonization of host tissues and endophytic lifestyle. Fungicides are not applicable because they are prohibited in chestnut forests and strongly discouraged in fruit chestnut groves. Trichoderma species are safe and wide-spectrum biocontrol agents (BCAs), with a variety of beneficial effects in plant protection. This study tested selected strains of T. viride, T. harzianum and T. atroviride for their ability to suppress G. castaneae. Field experiments were conducted in four chestnut groves (two test plots plus two controls) at two sites with a different microclimate. As the size of the trees were a major drawback for uniform and effective treatments, the Trichoderma strains were delivered directly by trunk injection, using the BITE® (Blade for Infusion in TrEes) endotherapic tool. The BCA application, repeated twice in two subsequent years, significantly reduced nut rot incidence, with a more marked, presumably cumulative, effect in the second year. Our data showed the tested Trichoderma strains retain great potential for the biological control of G. castaneae in chestnut groves. The exploitation of Trichoderma spp. as biopesticides is a novelty in the forestry sector and proves the benefits of these microbes in plant disease protection.

A severe dieback of flowering ash (Fraxinus ornus L.) has been observed in north-central Italy in the last decades. Symptoms include typical sunken, light-brown cankers on the stem and branches; vascular discoloration; tip and shoot dieback; and foliage necroses. The disease was more evident at the beginning of the growing season, and more severe on young regeneration. Six Botryosphaeriaceae species were consistently isolated from symptomatic plant tissues: Botryosphaeria dothidea, Diplodia fraxini, Diplodia subglobosa, Dothiorella iberica, Dothiorella omnivora and Neofusicoccum parvum. B. dothidea and D. fraxini expressed higher aggressiveness and showed a widespread incidence, being the species most frequently associated with cankers; the other four species were less virulent and more erratic, occurring mainly on succulent branch tips and foliage. Isolates were characterized using morphological and molecular approaches (colony/conidial phenotyping and rDNA-ITS genotyping). Phylogenetic analysis provided congruent phylogenies depicting the relationships of the six taxa with the most closely related conspecifics. Pathogenicity tests on 2-year-old seedlings confirmed the higher virulence of B. dothidea and D. fraxini. Extensive, multi-year field surveys at different sites supported the hypothesis that climatic vagaries, mainly heat, water and drought stresses, impaired tree health and vigor, facilitating infection and pervasive colonization by these Botryosphaeriaceae species. Environmental stressors are thus the key factor bringing the six fungal pathogens together in a multitrophic interaction with F. ornus in a novel, lethal fashion.

Thousand cankers disease (TCD) is a new deadly disease in walnut trees (Juglans spp.), which is plaguing commercial plantations, natural groves, and ornamental black walnut trees (Juglans nigra) in their native and invasion areas in the US and, more recently, in artificial plantations and amenity trees in the newly-invaded areas in Europe (Italy). This insect/fungus complex arises from the intense trophic activity of the bark beetle vector Pityophthorus juglandis in the phloem of Juglans spp. and the subsequent development of multiple Geosmithia morbida cankers around beetles’ entry/exit holes. After an analysis of the main biological and ecological traits of both members of this insect/fungus complex, this review explores the options available for TCD prevention and management. Special focus is given to those diagnostic tools developed for disease detection, surveillance, and monitoring, as well as to existing phytosanitary regulations, protocols, and measures that comply with TCD eradication and containment. Only integrated disease management can effectively curtail the pervasive spread of TCD, thus limiting the damage to natural ecosystems, plantations, and ornamental walnuts.

Walnut species (Juglans spp.) are multipurpose trees, widely employed in plantation forestry for high-quality timber and nut production, as well as in urban greening as ornamental plants. These species are currently threatened by the thousand cankers disease (TCD) complex, an insect–fungus association which involves the ascomycete Geosmithia morbida (GM) and its vector, the bark beetle Pityophthorus juglandis. While TCD has been studied extensively where it originated in North America, little research has been carried out in Europe, where it was more recently introduced. A key step in research to cope with this new phytosanitary emergency is the development of effective molecular detection tools. In this work, we report two accurate molecular methods for the diagnosis of GM, based on LAMP (real-time and visual) and SYBR Green qPCR, which are complimentary to and integrated with similar recently developed assays. Our protocols detected GM DNA from pure mycelium and from infected woody tissue with high accuracy, sensitivity, and specificity, without cross-reactivity to a large panel of taxonomically related species. The precision and robustness of our tests guarantee high diagnostic standards and could be used to support field diagnostic end-users in TCD monitoring and surveillance campaigns.

The ascomycete fungus Geosmithia morbida (GM) and the walnut twig beetle (WTB), Pityophthorus juglandis, constitute a recently discovered fungal–insect complex responsible for the harmful Thousand Cankers Disease (TCD) in walnut trees. Key aspects of the epidemiology and ecology of the fungus and its insect vector were investigated in a disease outbreak found in central Italy, currently the southernmost introduction area of TCD on the Old Continent. Walnut trees with symptoms of crown dieback and epicormic branching with beetle galleries were sampled for pathogen isolation, while funnel traps were used to catch beetles and obtain adult flight curves. Growth-temperature relationships were determined for the fungus; optimum growth temperature for GM was 25°C, but it thrived up to 37°C. The phloem-boring bark beetle showed a longer flight period than in northern Italy. The beetle haplotype identified in Tuscany (H1) differed from haplotypes H2 and H21 found in northern Italy, suggesting possible multiple anthropogenic introductions. The substantial high temperature tolerance shown by the two organisms suggests they will not be constrained by the high temperatures of warmer areas. As a consequence, the impact of TCD in southern Europe, where the native Juglans regia is widely cultivated for its nuts and wood, would potentially be disastrous to local economies. In fact, although J. regia is not considered as susceptible as J. nigra, it can nevertheless be attacked in conditions of high bark beetle population density, a harbinger of high fungus’ propagule pressure.

Lindgren funnel traps were used to monitor Pityophthorus juglandis occurrence. Traps were placed directly on walnut trees, with the top tied to one of the lower branches (about 2m high). An 8-funnel model was used instead of a 4-funnel trap, with the specific pheromone bait positioned between the fourth and the fifth funnel. Traps were customized with a 5mm metal mesh which was placed inside the bottom funnel so that debris (mainly foliage) and larger non-target insects would not end up inside the collecting jar. Geosmithia morbida was isolated from beetle adults, larvae and necrotic woody tissue around beetle galleries. Contaminant-free colonies were subcultured in purity and identified by: a) colony phenotyping [morphology, texture and pigmentation; margin type (regular/irregular; lobed/non-lobed); mycelium compactness; surface bumpiness; growth/temperature relationships]; b) micromorphology: type, morphology and ontogeny of conidiophores, metulae and phialides; conidiogenesis; shape, dimension and pigmentation of conidia; c) DNA fingerprinting. • Our protocol was customized to prevent traps from swinging in the wind and to optimize beetle catches by transversely fixing the bottom of funnel traps to the tree trunk with wooden shafts for stability. • To enhance fungus isolation in purity, a semi-selective Potato Dextrose Agar (PDA) medium, enriched with the antibiotics Ampicillin (Policillin-N) and Rifampicin (Rifamycin), was devised to prevent contamination by Gram-positive and Gram-negative bacteria and by mycobacteria.

The cultivation of walnuts (Juglans sp.) in Europe retains high economic, social, and environmental value. The recent reporting of the Thousand Cankers Disease (TCD) fungus, Geosmithia morbida, and of its vector, Pityophthorus juglandis, in walnut trees in Italy is alarming the whole of Europe. Although Italy is at present the only foothold of the disease outside North America, given the difficulties inherent in traditional identification of both members of this beetle/fungus complex, a rapid and effective protocol for the early detection and identification of TCD organisms is an absolute priority for Europe. Here we report the development of an effective and sensitive molecular tool based on simplex/duplex qPCR assays for the rapid, accurate and highly specific detection of both the bionectriaceous fungal pathogen and its bark-beetle vector. Our assay performed excellently, detecting minute amounts of target DNA without any non-specific amplification. Detection limits from various and heterogeneous matrices were lower than other reported assays. Our molecular protocol could assist in TCD organism interception at entry points, territory monitoring for the early identification and eradication of outbreaks, delineation of quarantine areas, and tracing back TCD entry and dispersal pathways.

Forest health worldwide is impacted by many invasive alien pathogens and pests (IAPPs) that cause significant harm, with severe economic losses and environmental alterations. Destructive tree pathogens and pests have in the past devastated our forests, natural landscapes and cityscapes and still continue to represent a serious threat. The main driver of pathogen and pest invasions is human activities, above all global trade, which allows these invasive species to overstep their natural distribution ranges. While natural transport occurs according to a regular, expected colonization pattern (based on the dispersive capacity of the organism), human-mediated transport takes place on a larger, unpredictable scale. In order for a pathogen or pest species to become invasive in a new territory it must overcome distinct stages (barriers) that strongly affect the outcome of the invasion. Early detection is crucial to enabling successful eradication and containment. Although sophisticated diagnostic techniques are now available for disease and pest surveillance and monitoring, few control and mitigation options are usable in forestry; of these, biological control is one of the most frequently adopted. Since invasion by pathogens and pests is an economic and ecological problem of supranational relevance, governments should endorse all necessary preventive and corrective actions. To this end, establishing and harmonizing measures among countries is essential, both for preventing new introductions and for diminishing the eventual range expansion of IAPPs present at a local scale. Research is fundamental for: (i) developing effective and rapid diagnostic tools; (ii) investigating the epidemiology and ecology of IAPPs in newly introduced areas; and (iii) supporting policymakers in the implementation of quarantine regulations.

In this work, we rechecked, using species-specific Loop mediated isothermal AMPlification (LAMP) diagnostic assays followed by sequencing of fungal isolates at the beta-2-tubulin (tub2) gene region, a historical and never confirmed report of Dothistroma needle blight (DNB) in the introduced Monterey pine (Pinus radiata D. Don) in the mountains in the extreme tip of southern Italy. The report dates back to the mid-1970s, and predates the molecular-based taxonomic revision of the genus Dothistroma that defined the species accepted today. In the fall of 2019, symptomatic needles of Monterey pine and Corsican pine (Pinus nigra subsp. laricio (Poir.) Palib. ex Maire) were sampled in the area of the first finding. The applied diagnostic methods revealed the presence of Dothistroma septosporum (Dorogin) M. Morelet on both pine species. In this way, we: (i) confirmed the presence of the disease; (ii) clarified the taxonomic identity of the causal agent now occurring at that site; (iii) validated the species-specific LAMP diagnostic protocol we recently developed for Dothistroma for use on a portable field instrument, and (iv) showed that the pathogen now also attacks the native P. nigra subsp. laricio, a species particularly susceptible to the disease, indigenous to the mountains of Calabria, which is one of the very few areas where the species’ genetic resources are conserved. Comparative genetic analysis of the rare populations of D. septosporum found in the central Mediterranean region and in the native range of P. nigra subsp. laricio could help to clarify the history of the spread of the pathogen in southern Europe and better evaluate the risk it poses to the conservation of native pine species.

Melampsoridium hiratsukanum is an alien rust fungus which has spread pervasively throughout several European countries following introduction into North Europe at the end of the 20th century. The authenticity of several records of the Melampsoridium species infecting alder (Alnus spp.) in the northern hemisphere is questionable, due to the misidentification and confusion that surround many of the older reports. Given this complicated taxonomic history, and since a M. hiratsukanum-like rust is strongly impacting Alnus incana stands in the Alps, probably affecting the bank protection role of this species along rivers, the unambiguous identification of this pathogen was a pressing epidemiological and ecological issue. In this study, field surveys, light (LM) and scanning electron microscopy (SEM), and molecular characterization were put together in an attempt to solve the conundrum. Field monitoring data, LM and SEM analyses of key taxonomic traits (length of ostiolar cells of uredinium, uredinio-spore shape and size, spore echinulation, number and position of germ pores) and ITS-rDNA sequence-based identification, convergently and unambiguously connected the rust that is causing the current epidemic to the non-native M. hiratsukanum. We documented the completion of the M. hiratsukanum life cycle on its two taxonomically unrelated broadleaf/conifer hosts. This is the first report of M. hiratsukanum from naturally infected Larix decidua in Europe.

The walnut twig beetle Pityophthorus juglandis is a phloem-boring bark beetle responsible, in association with the ascomycete Geosmithia morbida, for the Thousand Cankers Disease (TCD) of walnut trees. The recent finding of TCD in Europe prompted the development of effective diagnostic protocols for the early detection of members of this insect/fungus complex. Here we report the development of a highly efficient, low-cost, and rapid method for detecting the beetle, or even just its biological traces, from environmental samples: the loop-mediated isothermal amplification (LAMP) assay. The method, designed on the 28S ribosomal RNA gene, showed high specificity and sensitivity, with no cross reactivity to other bark beetles and wood-boring insects. The test was successful even with very small amounts of the target insect’s nucleic acid, with limit values of 0.64 pg/µL and 3.2 pg/µL for WTB adults and frass, respectively. A comparison of the method (both in real time and visual) with conventional PCR did not display significant differences in terms of LoD. This LAMP protocol will enable quick, low-cost, and early detection of P. juglandis in areas with new infestations and for phytosanitary inspections at vulnerable sites (e.g., seaports, airports, loading stations, storage facilities, and wood processing companies).

Successful infection by a tree pathogen depends both on species-specific traits, related to its infection biology, and on external factors, i.e. the biotic and abiotic components of the ecosystem. Among species-specific traits, virulence, biomass (propagule) production and dispersal ability all contribute to pathogen establishment and spread. Among the external factors, two crucial roles are played by an environment conducive to disease and by vector occurrence. We investigated the endophytic fungi and xylophagous beetles associated with declining oaks in a natural stand of central Italy, particularly focusing on the fungi isolated from insects’ body. This paper extends and reinterprets the outcome of a previous study, which looked into the whole mycota (pathogenic/neutral/beneficial) found on these oaks. A rich assemblage of fungi was found on declining trees on which a number of wood-boring beetles (Buprestidae and Cerambycidae) also occurred. In the laboratory two-thirds of the insects proved to convey propagules of prominent fungal pathogens (Botryosphaeriaceae, Pestalotiopsidaceae, Plectosphaerellaceae, Pleosporaceae); this confirms pathogen pervasiveness in oak stands is enhanced by their association with xylophagous insects. The outcome of this research will benefit forest manager and other stakeholder efforts to manage stands and species invasions, also in the light of climate change. Keywords: Oak decline, Endophytic fungi, Xylophagous beetles