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In the context of biological control against the Asian chestnut gall wasp Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae) in Italy, the aim of this study was to survey its native parasitoids as well as the exotic Torymus sinensis Kamijo which had been introduced and released as a biological control agent. Furthermore, both parasitism and pest susceptibility of local chestnut trees were investigated. Surveys were carried out in 2014 in the Municipality of Sambuca Pistoiese (Pistoia), in a chestnut forest which included three cultivars: Nerattino, Carpinese, and Pastinese. On a total of 35 trees, selected among the three cultivars, 24 current-year shoots were randomly collected and examined in the laboratory. After the damage by the cynipid had been assessed, leaf samples were used for molecular analyses. Galls were also collected on the 35 selected trees both for dissection and to rear parasitoid specimens in the laboratory. The three cultivars were confirmed by genetic tests. The Carpinese cultivar resulted to be the most damaged, with highest percentage of attacked shoots (84.38%). Moreover it was also the cultivar with the lower parasitism rate (about 16%). From reared galls seven parasitoid species emerged, all belonging to the superfamily of Chalcidoidea. Exception made for the non-native T. sinensis, the other six were native species associated to oak gall wasps. Native parasitoids were more scarcely present, while the exotic T. sinensis amounted for the 84.27% of all emerged parasitoids. Our results confirm how chestnut susceptibility to D. kuriphilus varies depending on the cultivar. Overall, the Nerattino cultivar resulted as the best among the three examined: it was subject to a less intense attack than the other cultivars, and the resulting smaller galls were more effectively exploited by parasitoids.

Research Highlights: We investigated the negative effects of bark beetle mass trapping, especially non-target catches among the target’s natural enemies. Slot traps modified with mesh screen and escape windows were tested to improve their selectiveness. Background and Objectives: Two of the main natural enemies of bark beetles, Thanasimus formicarius (L.) and Temnoscheila caerulea (Olivier), are frequently trapped in high numbers in Ips sexdentatus (Böerner) pheromone traps, along with other saproxylic insects, this may lead to much larger pest populations in the successive 4–20 beetle generations. From 2016–2019, during I. sexdentatus mass-trapping in a pine forest of Tuscany (Italy), non-target catches were tallied. Trap modifications were evaluated to mitigate non-target catches, especially those concerning bark beetles’ natural enemies. Materials and Methods: A total of 25 bark-beetle slot traps were placed about 75 m apart in a pine stand infested by I. sexdentatus. Traps were baited with I. sexdentatus aggregation pheromone, whose main components are ipsenol, ipsdienol, and 2-methyl-3-buten-2-ol. Catches were collected every 10 days from March to December. In 2019, 13 traps were modified by applying a 6-mm mesh screen on top of the collection container and by providing three 60 mm × 8 mm escape windows immediately above the screen. These “modified traps” and their captures were considered separately from the 12 remaining “standard traps.” All bark beetle species were recorded, as well as all beetle species &gt, 8 mm. Results: Overall, target catches amounted for &lt, 10% of the total beetle catches. The most-collected species was the bark beetle Orthotomicus erosus Wollaston. Trap modification allowed the escape of larger species, resulting in the reduction of the average size of caught specimens. Even though non-target catches among predators were still high, the proportion of major predators (T. formicarius and T. caerulea) to bark beetles showed a statistically significant reduction of predator catches in modified traps, an encouraging outcome. Conclusions: Trap modifications may mitigate the problem of non-target catches during mass trapping, especially reducing catches of beetle species larger than the target. However, the key is to schedule mass trapping only during those seasons when the target adults are more active than the main predator adults, thus limiting their catches and, consequently, the negative effects on pest management and biodiversity.

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.

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).

Made available in DSpace on 2020-12-12T02:12:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-09-01 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. Department of Agricultural Food Environmental and Forestry Science and Technology (DAGRI) Plant Pathology and Entomology Section University of Florence Plant Protection Department Faculdade de Ciências Agronômicas (FCA) Universidade Estadual Paulista (UNESP) Instituto de Biociências Departamento de Genética Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP) Plant Protection Department Faculdade de Ciências Agronômicas (FCA) Universidade Estadual Paulista (UNESP) Instituto de Biociências Departamento de Genética Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP)

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.

High mortality was noticed in a 10-year-old stand of Pinus pinea in the Alberese area (Grosseto, central Italy, elev. 40 m, 42° 39′ 46″ N, 11° 06′ 25″ E) in July 2010. Aerial symptoms of trees included chlorosis, crown thinning, stunted growth, bark lesions at the stem base with resinous exudations, and extensive necroses of the underlying xylem tissue. Woody roots of two uprooted trees exhibited bark necroses and a high proportion of fine roots was destroyed. Soil around necrotic roots was baited using apple fruits (cv. Gala). After 1 week of incubation at 24°C, typical firm fruit rot developed and small tissue samples were transferred to clarified V8 agar (V8A) amended with 5 ml/l PARPNH and incubated at 24°C. After 7 days, stellate to rosaceous, finely lobed cottony colonies arose that were transferred to FPM medium and incubated at 24°C. Within 7 days, spherical oogonia with a smooth surface and predominantly paragynous antheridia formed; sporadic amphyginous antheridia could be observed. Colony squares (1 cm2) were then placed in filtered and sterilized pond water. After 48 h, ovoid, obpyriform, or clavate, nonpapillate, persistent sporangia with internal nested and extended proliferation were formed. Fifty oogonia and 30 sporangia were measured. The diameter of the 50 spherical oogonia varied from 33.6 to 44.9 μm (avg. 39 μm); dimensions of the 30 sporangia were 42.6 to 59.8 × 28.9 to 47.8 μm (avg. 52.95 × 38.98 μm; 1:b ratio 1.37). The isolate was identified as Phytophthora humicola W. H. Ko & Ann on the basis of colony type, size and morphology of oogonia and sporangia, average length/width ratio of sporangia, the homothallic formation of oogonia (4), and ITS rDNA sequence information (GenBank Accession No. JQ757060). A BLAST search of the ITS sequence of P. humicola isolate B33 revealed a 99% identity with the Phytophthora ITS Clade 6 species P. humicola and P. inundata (2). This latter species could be ruled out, however, since it is self-sterile, whereas our isolate B33 was self-fertile (3). A strain of P. humicola was deposited in the CBS-KNAW Fungal Biodiversity Centre, strain no. CBS129249. Pathogenicity tests were conducted on 10 one-year-old twigs of Pinus pinea. A bark portion was removed aseptically and a V8A disc (0.5 cm diam.) of P. humicola mycelium was placed on the wound. Control twigs (3) received sterile V8A discs. Inoculated and control twigs were incubated at 20°C in the dark. Clearly noticeable necrotic lesions (avg. length 2.2 × 0.68 cm) were observed after 15 days on inoculated twigs. Control twigs showed no symptoms. Reisolations on selective V8-PARPNH-agar confirmed P. humicola as the causal agent. P. humicola is mainly associated with woody horticultural crops (1, 3), while the other taxa grouped with this species in Clade 6 are mainly found in forest and riparian ecosystems (1). These aquatic Phytophthora species normally have a saprophytic lifestyle, but under favourable environmental circumstances can act as opportunistic pathogens, attacking susceptible trees and causing scattered mortality in forest stands and natural ecosystems (3). To our knowledge, this is the first report of P. humicola from a pine stand. It is supposed that the pathogen reached the stand through infected plant material or infested soil introduced into the stand. References: (1) C. M. Brasier et al. Mycol. Res. 107:277, 2003. (2) D. E. L. Cooke et al. Fungal Genet. Biol. 30:17, 2000. (3) T. Jung et al. Persoonia 26:13, 2011. (4) W. H. Ko and P. J. Ann. Mycologia 77:631, 1985.

Native parasitoids may play an important role in biological control. They may either support or hinder the effectiveness of introduced nonnative parasitoids released for pest control purposes. Results of a three-year survey (2011–2013) of the Asian chestnut gall wasp (ACGW) Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae) populations and on parasitism rates by native indigenous parasitoids (a complex of chalcidoid hymenopterans) in Italian chestnut forests are given. Changes in D. kuriphilus gall size and phenology were observed through the three years of study. A total of 13 species of native parasitoids were recorded, accounting for fluctuating parasitism rates. This variability in parasitism rates over the three years was mainly due to the effect of Torymus flavipes (Walker) (Hymenoptera: Torymidae), which in 2011 accounted for 75% of all parasitoid specimens yet decreased drastically in the following years. This strong fluctuation may be related to climatic conditions. Besides, our data verified that parasitoids do not choose host galls based on their size, though when they do parasitize smaller ones, they exploit them better. Consequently, ACGWs have higher chances of surviving parasitism if they are inside larger galls.

The present-day phytosanitary disasters caused by biological invasions are afflicting urban and peri-urban forest stands worldwide, as well as the varied services they normally provide. In Europe, we are witnessing an alarming situation due to an increasing introduction of infectious diseases and pests. The authors present an up-to-date list of alien microbial pathogens and insect pests affecting urban greening that have been accidentally imported in Italy or that are likely to be introduced. Information about the biology, epidemiology, ethology, and control of these invasive organisms is provided. For each species, the current geographical distribution, including newly-colonized areas, is also given, as well as the chronological progression of its occurrence. Particular detail is used for describing symptoms—the key diagnostic elements for appropriate and timely phytosanitary management. This paper will benefit urban forest management, which is a crucial factor in maintaining the social and ecological viability of urban green spaces, as well as ecosystem services. The importance of engaging citizens in community-based monitoring of urban greenspaces for tracking the location, abundance, and pathways of invasive pathogens and pests will also be touched upon.

Many biotic and abiotic factors contribute to the onset of oak decline. Among biotic agents, a variety of fungi and insects cause extensive disease and insect outbreaks in oak forests. To date, research on fungus-insect interactions in Mediterranean forest ecosystems is still scarce and fragmentary. In this study, we investigated the assemblage of endophytic mycobiota and insect pests occurring in a declining oak stand, with the aim to explore if, and to what extent, the insect species were active vectors of fungal propagules. It emerged that some known latent pathogens of the Botryosphaeriaceae family, namely Botryosphaeria dothidea, Diplodia corticola, Diplodia seriata, Dothiorella sarmentorum, and Neofusicoccum parvum were isolated at high frequency from physiologically-impaired trees. In addition, propagules of these fungi were isolated from five insects, two of which (Cerambyx welensii and Coraebus fasciatus) are main oak pests. The life-history strategies of these fungi and those of wood-boring beetles were strikingly interconnected: both the fungi and beetles exploit drought-stressed trees and both occur at high frequency during hot, dry periods. This synchronicity increased their chance of co-occurrence and, consequently, their probability of jointly leading to oak decline. If these interactions would be confirmed by future studies, they could help to better understand the extensive decline/dieback of many Mediterranean forest ecosystems.

Although mass trapping cannot be a definitive control measure, it is one of the few ones available to contain the destruction of millions of cubic metres of conifer forests perpetrated every year worldwide by bark beetles. However, using bark beetle aggregation pheromones during both monitoring and control programs may negatively affect other saproxylic insects. The aim of this study was to describe the response of both Ips sexdentatus and its saproxylic beetle associates, especially predators, to traps baited with a commercial blend of I. sexdentatus aggregation pheromone. Furthermore, the usefulness of adding pine volatiles, such as (−)-α-pinene and ethanol, to the pheromone was discussed. The commercial blend proved to be attractive to I. sexdentatus adults, both when used alone and together with pine volatiles. Pheromone attractiveness, however, was lessened by the addition of the volatiles. The pheromone blend proved to be attractive to Thanasimus formicarius, as well as to other predator species. Overall, although during our study, traps baited only with (−)-α-pinene and ethanol attracted some predator specimens, I. sexdentatus pheromone traps were more attractive. Our study confirms that calendar differences in flight activity between the bark beetle and its predators are substantial; therefore, they should be taken into account when planning control measures. According to our data, the commercial blend of I. sexdentatus pheromone seems to be the most effective, among the baits used, in catching I. sexdentatus adults, while reducing the impact on T. formicarius.

Cone and seed insects have played a key role in the decline of stone pine nut production in Italy. To evaluate the impact caused by native Palearctic and exotic insects, a greater knowledge of pest symptoms is required. During 2008–2009, first and second-year stone pine cones, as well as the seeds produced, were examined in Tuscany (Italy) to assess viability. Insect damage was characterized based on external signs on the cones and seed endosperms, and the impact of recorded insect species on nut production was evaluated. In the current study, cones attacked by anobiid beetles and Dioryctria spp. were observed, as well as asymptomatic dead cones and cones with resin drops and patches, that could not easily be related to a damaging agent. As regards the anobiid beetles, adults of Ernobius parens (Mulsant and Rey) and E. impressithorax Pic emerged from cones in laboratory rearing. A low number of cones damaged by Dioryctria spp. was recorded whereas high percentages of cones showed resin exudates. The presence of resin cannot be definitely related to a damaging agent, although the feeding activity of Leptoglossus occidentalis Heidemann could be one of the reasons. Damage by L. occidentalis was assessed by seed observation. Most of the seeds displayed tissues that had been damaged by this pest.

1 Preliminary investigations were carried out on Dryocosmus kuriphilus Yasumatsu on Castanea sativa Miller in Tuscany to assess variations in gall characteristics in coppice and high forest at two crown heights (height < 2 or 2–6 m), influence of bud size and bud position on oviposition rates and susceptibility of three cultivars. 2 Gall size may depend on various factors, including wasp population density. In the present study area, small galls (with one or two cells) were the most numerous in 2008, whereas larger galls (with more than three cells) prevailed in 2009. 3 Dryocosmus kuriphilus oviposition occurrence was influenced by both bud size and bud position. Buds with eggs tended to be larger in size compared with bud without eggs, suggesting that D. kuriphilus females prefer to lay eggs in larger buds (approximately 6 mm3) compared with smaller buds (approximately 3 mm3). The mean number of eggs per bud tended to decrease from the apical bud toward the basal bud. 4 Three C. sativa cultivars, Carpinese, Fusca and Cesurone, were examined. Fusca grafts had significantly more galls compared with Carpinese and Cesurone, whereas Cesurone grafts had more larvae per bud compared with Carpinese and Fusca. Overall, the Carpinese cultivar may be less susceptible to D. kuriphilus galling compared with the Fusca and Cesurone cultivars.