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The species diversity of culturable endophytic fungi was studied in the leaves and twigs of symptomatic and asymptomatic Fraxinus excelsior trees. Endophytic mycobiota was dominated by Ascomycota species, with Pleosporales (44.17%) and Diaporthales (23.79%) endophytes being the most frequently observed in the tree samples. The number of endophytic isolates and species richness varied depending on the sampling date (May and October) and tissue location. Of the 54 species identified based on ITS sequences, 14 were classified as dominant. The most frequently isolated species were Diaporthe eres, followed by Alternaria alternata, Dothiorella gregaria, and Fraxinicola fraxini. The inhibitory effect of 41 species (75 isolates) of endophytes on the radial growth of a Hymenoscyphus fraxineus isolate was studied under in vitro conditions (dual cultures). The radial growth of H. fraxineus was the most inhibited by four endophytic fungi from twigs (Fusarium lateritium, Didymella aliena, Didymella macrostoma, and Dothiorella gregaria). The inhibitory effect of the four isolates was also studied under in planta conditions. The isolates artificially inoculated into the trunks of ash trees reduced the length of necroses formed by H. fraxineus co-inoculated in the same trunks. This effect depended on the isolate, and the inhibition was most prominent only on trunks inoculated with F. lateritium and D. aliena. Although the total length of necrotic lesions formed by the H. fraxineus infection was shorter in the ash trunks co-inoculated with the endophytes, the difference was not significant.

Bark beetles are serious forest pests in Slovakia. Their outbreaks may have significant ecological and economic impacts on spruce forests. There is a variety of natural enemies that activate themselves during population outbreaks of insects and entomopathogenic fungi belong to important antagonists with a potential to regulate populations of their hosts. In 2014–2016, species richness and prevalence of entomopathogenic fungi were evaluated during the bark beetle outbreaks in spruce forests affected by windstorms in the Tatra National Park in Slovakia. Three Beauveria species, B. bassiana, B. caledonica and B. pseudobassiana, with Metapochonia bulbillosa were identified from 271 specimens of three bark beetle species, Ips typographus, Ips amitinus and Pityogenes chalcographus. Beauveria bassiana was the dominant pathogen and infected all three bark beetle species. Phylogenetic analysis identified three phylogenetic groups of B. bassiana in the evaluated host populations. M. bulbillosa was reported for the first time from bark beetle hosts and Slovakia. The prevalence of fungal infection in natural populations of I. typographus was low, varied between 0.07 and 0.72%, and have little influence on the bark beetle abundance.

Symptoms of dry clove rot of Allium sativum produced in Slovakia were noticed during storage in 2019. In 2020, garlic bulbs of eight cultivars were analysed and fungal isolates were obtained. Morphological and molecular identification confirmed the isolation of Fusarium proliferatum from all cultivars. The mean proportion of symptomatic cloves in evaluated samples reached 78.68%, and the mean area of clove surface damaged by the disease ranged from 5.97% to 14.83% 5 weeks after the harvest. The pathogenicity of the selected F. proliferatum isolate was verified by inoculation of garlic cloves in laboratory bioassays. This is the first report of F. proliferatum causing dry rot of garlic bulbs in Slovakia.

The density of phytophagous insect pest populations is related (directly and indirectly) to several groups of factors that can be broadly divided into: abiotic, biotic and anthropogenic. Each extreme in the abiotic environment at a macro-level leads to a series of consecutive extremes in the biotic environment, which eventually results in micro-level responses in the individual organisms. The manifestation of factors acts in aggregate or in a sequence, creating a chain of processes around us. Insects very efficiently use the abundance of nutritional resources, resulting in a tremendous increase in their population density, and triggering control mechanisms through the emergence of parasitic and pathogenic infections (viruses, bacteria, fungi, microsporidia, protozoa and nematodes). The development of entomopathogenic infections in host populations is directly dependent on the characteristics of both the antagonist and the insect. It is associated with the lifestyle and life cycle of the insect, with features encoded in the mechanism of pathogen action, and limited by the pathogen’s virulence and pathogenicity.

The gypsy moth is one of the most serious pests in forests and fruit tree plantations over prevailing parts of the Northern Hemisphere. This work is based on a literature review, and presents history of gypsy moth Lymantria dispar L., observed in Slovak forests within the period 1945–2020. The life cycle, hosts, natural enemies, population dynamics of pests, impact of outbreaks on forests and different management methods used in the past are discussed. Since 1945, there were nine gypsy moth outbreaks in Slovakia. Between 1945 and 2020, a total of 155,034 ha of deciduous forests were touched with varying intensity, representing an average annual damage of 2,040 ha. The strongest outbreak culminated in 2004. Totally 51,479 ha were attacked in the period of 2000–2008. We have found outbreak periods that repeat with frequency of 7.8 ±2.2 years and the average outbreak phase lasts 3.1 ±1.1 years. The period between two subsequent outbreaks seems to be more or less constant and duration of the outbreak phase seems to be gradually shortened during the study period. Several factors influencing the gypsy moth population dynamics in Slovakia are discussed. The role of biological control by using entomopathogenic fungus Entomophaga maimaiga is described.

Many small Diptera adopted at some level endophilic life style, including man-made buildings. Stables create a specific type of microhabitat, which provides shady and relatively humid conditions in combination with excess of organic matter. Unlike the parasites (mosquitos, biting midges, etc.), the commensal fauna of stables is poorly studied. Moth flies (Psychodidae) were collected in cow stable located in Šenkvice, SW Slovakia. Special traps (derived from Malaise traps) were installed along the stable internal wall and in three different heights. In total, we recorded 6325 moth flies belonging to 8 species. The flight period lasted from spring to autumn. Seasonal dynamics was strongly influenced by rainfall and mean week temperature, e.g. high temperature in mid-summer caused drop in moth flies captures. The moth flies clearly preferred the ground and moderately preferred the interior of stable.

Biogas plays an important role in reducing the amount of biowaste that is converted to methane which is usable by multiple pathways in the energy sector. Bark beetles are among the most important pests of spruce forests in Europe. Principal measures of managing bark beetle populations include trapping them with pheromone traps. Biomass of bark beetles collected by the traps constitutes a waste of forest resources that could potentially be processed in biogas plants. The aim of this study was to provide information about the trapping of bark beetles by pheromone traps in Tatra National Park during 2005-2018 and to verify the energy potential of a combination of liquid digestate and bark beetle biomass collected by pheromone traps. The bark beetle biomass is a seasonal small-volume nitrogen-rich material which could be processed at agricultural biogas plants. The biochemical methane potential batch test performed at 40 degrees C showed a high degree of digestion of milled beetles within 40 days, with a methane production of 0.242 Nm(3) per kilogram of total solids (TS). The single-stage low-solids mesophilic fed-batch anaerobic digestion of untreated beetles was carried out in a lab-scale rotary drum reactor. Within 52 days, the average organic loading rate was 1.019 kg VS m(-3)day(-)(1), where VS stands for volatile solids. The specific biogas and methane production reached 0.218 Nm(3)kg(-1) TS and 0.140 Nm(3)kg(-1) TS, respectively. The cuticles of the beetle bodies were not obviously disturbed by the fermentation process. The digestate was quite rich in nitrogen, but the other element contents did not differ too much from fertilizer made from manure or phytomass. Web of Science 44 2 179 169

Efficacy of entomopathogenic fungus Metarhizium pemphigi was evaluated against Ips typographus adults in a laboratory bioassay for the first time. A series of four conidial concentrations (2×104–2×107 conidia/ml) were used. The cumulative mortality caused by M. pemphigi varied between 75% and 100% ten days post-treatment, with LC50 value of 2.9×103 conidia/ml and LC90 value of 6.4×104 conidia/ml. The median lethal time (LT50) of treated beetles depended on conidial concentrations and ranged from 1.78 to 5.98 days. Pathogenicity of M. pemphigi was reported for the first time and the tested strain was found to be promising for further evaluation in terms of management options against the spruce bark beetle.

Diverse communities of bacterial endophytes inhabit plant tissues, and these bacteria play important roles for plant growth and health. Cherry laurel (

Endophytic fungus, usually treated as an entomopathogenic agent, can provide drought stress tolerance to red oak seedlings. Its effect is realized through root growth stimulation. Endophytic fungi are known to be active in the plant environmental stress alleviation. In this work, we proved the ability of endophytic Beauveria bassiana, which is almost exclusively treated as an entomopathogenic fungus, in mitigating drought stress in dehydrated red oak (Quercus rubra L.) seedlings. Despite small differences between experiments, in general, leaf relative water content and stomatal conductance were less reduced (ca. 50% and 15%, respectively), free proline did not accumulate over the control level, carbon isotope 13C discrimination/water use efficiency was not affected and root growth was stimulated at a similar plant transpiration area in the endophyte-colonised individuals. Regression analysis revealed that the root growth stimulation provided by the fungus under drought had the most important effect on their water status.

The health of 34 different Fraxinus taxa in association with the pathogenic fungus Hymenoscyphus fraxineus was assessed in four Slovak arboreta. Averaged across all arboreta, nearly one-quarter (24.9%) of all evaluated trees showed ash dieback symptoms. The damage was most serious on the common ash F. excelsior, a native species. The percentage of dead trees did not exceed 2% for all evaluated trees. Generally, ash trees of all ages were affected, though the intensity of the damage varied among the sites. The identity of H. fraxineus was confirmed by conventional PCR targeting the internal transcribed spacer (ITS) sequences of the nuclear ribosomal DNA, as well as the 18S gene/ITS-2 region of the rDNA operon. In Slovakia, the pathogen has expanded its host range from native species not only to their ornamental cultivars, but also to introduced North American (F. cinerea, F. latifolia, F. pennsylvanica, F. quadrangulata) and Asian (F. bungeana, F. chinensis ssp. rhynchophylla, F. mandshurica) ash species. H. fraxineus was also observed on the previous year&rsquo, s leaf petioles of the native European species F. ornus, considered a weakly susceptible host. In Slovak arboreta, H. fraxineus was found on 23 Fraxinus taxa, 21 of them represent first records for the country. F. bungeana is recorded as a new host species of H. fraxineus.

Ips typographus is a serious pest for forestry in Eurasia. Effective control is difficult due to its cryptic habits and insect pathogenic microorganisms, including entomopathogenic fungi that are believed to be a promising alternative to the traditional control measures of this pest. In 2018, diversity of entomopathogenic fungi of the genus Beauveria was studied in populations of I. typographus in the Vitosha National Park, Bulgaria. Two species, B. bassiana and B. caledonica, were identified and 33 in vitro strains were obtained. Phylogenetic positions of the strains were evaluated according to phylogenetic inferences based on ITS and TEF-1α. Pathogenicity of the strains against bark beetles was tested in laboratory. All strains were pathogenic, although there was some variability in the efficacy of B. bassiana strains. Virulence of the five most pathogenic strains (four B. bassiana strains and one B. caledonica strain) was compared with the commercial mycoinsecticide Boverol® and highly-virulent B. bassiana strain ARSEF 12957 isolated from I. typographus in Slovakia. The strain from Boverol® was least virulent and the Slovak strain ARSEF 12957 was more efficient than the Bulgarian strains, but the difference was not significant. The laboratory experiments suggest that the Bulgarian strains have a potential for the control of bark beetle adults.

The Eurasian spruce bark beetle, Ips typographus (IT), is considered the most destructive insect pest of spruce forests in the Palaearctic region. Its control is based on a range of phytosanitary measures, insecticide treatments targeting the tree trunks, and pheromone trappings. Essential oils (EOs) from aromatic plants have been recognized as ecological alternatives to synthetic pesticides. In this study, EOs from plants in the Lamiaceae family (Origanum vulgare, Thymus vulgaris, Hyssopus officinalis and Mentha × piperita) and the Apiaceae family (Pimpinella anisum and Foeniculum vulgare) were studied for insecticidal and repellent effects against IT. EOs chemical composition was analyzed with gas chromatography mass spectrometry. IT adults were exposed to different concentrations of EOs, and their effects were recorded 2–96 h post-treatment. Significant differences in biological activity were observed, and the greatest insecticidal effect was detected for O. vulgare, T. vulgaris, and P. anisum; but O. vulgare EO was significantly more toxic at all doses or exposure times. Repellency index (RI) varied among the EOs and depended on dose and exposure time. At doses of 0.077‒0.219 µl/cm2, the RI increased until 4‒6 h post-treatment; after that period, it decreased. Pimpinella anisum, O. vulgare, and T. vulgaris EOs showed significantly higher repellent effects compared to F. vulgare and H. officinalis EOs. Mentha × piperita EOs exhibited neither repellent nor insecticidal activity against IT. To our knowledge, this is the first time that EOs biological activity against IT was studied.

Two local strains of Beauveria bassiana originally isolated from naturally infected spruce bark beetles in Slovakia were tested for their virulence to Ips typographus (IT) and for their compatibility with a polymeric matrix composed of low-molecular polyethylene. Conidia could be homogenously immobilized in the low-molecular polyethylene matrix with no adverse effect on their viability and infectivity. At constant temperature (25°C), viability of immobilized conidial decreased only by 1–2% after 7 or 14 days when compared with non-formulated conidia. In field conditions, viability of conidia formulated in the matrix was even significantly higher than non-formulated conidia 35 days after their application in traps. Conidia incorporated into the polymeric matrix were infective to IT adults in laboratory bioassays. Mean values of LC50 for native conidia (0.72–2.05 × 106 conidia ml−1) and conidia immobilized in the polymeric matrix (0.64–1.03 × 105 conidia mm−2) demonstrated high virulence. The effica...

Cameraria ohridella is a major pest of horse-chestnut trees in Europe causing permanent outbreaks and severe defoliations. Entomopathogenic Beauveria species are common mortality factors of C. ohridella pupae overwintering in leaf litters. In this study, laboratory bioassays were conducted to determine the ability of local Beauveria strains to colonize horse-chestnut leaves endophytically and test their antagonistic activity against C. ohridella in colonized leaves. Two strains of Beauveria bassiana (AM_EF0111 isolated from horse-chestnut leaf tissue and AM_EP0715 isolated from a C. ohridella pupa) and one strain of B. pseudobassiana (AM_SO1015 obtained from soil) were used for in planta bioassays. The selected strains demonstrated high virulence to leaf miner pupae after a topical treatment with spore suspensions. LC50 ranged between 2.49 and 3.19 × 106 conidia ml−1. Horse-chestnut saplings were inoculated with the fungi through their direct immersion into spore suspensions (107 conidia ml−1). Endophytic colonization was successful and a mean colonization rate reached 57.44–66.08% 15 days post-inoculation. It decreased over time for all strains, but the fungi were still present in leaf tissue 54 days post-inoculation. AM_EF0111 strain was significantly more effective in colonizing saplings than AM_SO1015 and AM_EP0715. Fungus-colonized and untreated (control) saplings were infested with the leaf miner 31 days after inoculation and development of larval mines was monitored. In Beauveria-colonized saplings, damage by leaf miner was significantly lower compared to control plants. On day 23 post-infestation, the damaged leaf area of colonized leaves was 5-times smaller compared to control saplings. The negative effect on larval tunneling was significantly greater for strain AM_EP0715 than for the other two strains. Survival of leaf miners was considerably affected in colonized leaves, but mycosis was detected only in 5.41–9.23% of cadavers. No mycosed individuals were collected from control saplings. Pupae from colonized leaves were smaller in dimensions with lower weight than pupae form control plants. The same Beauveria strains recovered from leaves at the end of colonization bioassays showed no significant decrease in LC50 values.

In Slovakia, a diversity of entomopathogenic fungi (Ascomycota, Hypocreales) associated with outbreaks of Ips typographus was studied in 81 localities and as many as 113 in vitro cultures of five entomopathogenic species were isolated from infected individuals: Beauveria bassiana (87 isolates), B. pseudobassiana (14 isolates), B. caledonica (6 isolates), Lecanicillium lecanii (4 isolates) and Isaria farinosa (2 isolates). B. pseudobassiana is recorded in natural populations of I. typographus for the first time. Biological properties of selected Beauveria isolates, including colony growth, biomass production, conidia yield and pathogenicity to I. typographus adults, were studied in a series of laboratory bioassays and much intra- and interspecific variability was detected. B. bassiana isolates produced biomass or conidia at significantly higher rate than B. pseudobassiana and B. caledonica isolates. Two B. bassiana isolates were selected as the most virulent to bark beetle adults, demonstrating a mean LC50 ranging from 0.72 to 2.05 × 106 conidia ml−1, and were qualified as promising candidates for biocontrol of I. typographus. Their virulence was significantly higher than that of the mycoinsecticides Boverol®, which was used as a reference strain in the virulence bioassays.

Species of the genus Beauveria are entomopathogenic fungi used in biological control to protect against insect pests. Identification of species is based on observation of morphological, phylogenetic and genetic differences. Currently, MALDI-TOF (Matrix Assisted Laser Desorption/Ionization- Time of Flight) mass spectrometry is often used in microbial diagnostic but identification of Beauveria species has not been tested before. The aim of this work was the possibilities of this method and to compare it with genetic analysis. Twenty strains of B. bassiana and B. pseudobassiana isolated from insect cadavers, mycoinsecticides and soil samples were used. Sequences of ITS (Internal Transcribed Spacer) region were used for genetic identification of strains. Sequences and results from protein analysis of isolates were compared with the reference strains of B. bassiana and B. pseudobassiana. Results of MALDI Biotyper softvare identification were correct for all strains. Identification was reliable with high log score (>2.1) for most of strains. Score value less than 1 was observed only in a single case - strain GHA isolated from mycoinsecticide Botanigard.The result of both analyses was also dendrogram (phylogenetic tree) which showed high degree of result consistence. MALDI-TOF identification of fungi from Beauveria genus is usable for fast and reliable identification of entomopathogenic fungi isolates acquired from environment and results can be utilized in conservation biocontrol strategies in agriculture and forest ecosystems.

The cypress aphid (Cinara cupressi) is listed among the hundred most important invasive pests in the world. In Chile, it was first detected in 2003 and currently is present throughout the country. In the course of a survey of their natural enemies in Chile, three strains of entomopathogenic fungi were isolated. The isolates were identified and tested against the aphid in laboratory experiments. Two further entomopathogenic fungi (ARSEF 5126 and 5128), formulated in the mycoinsecticides Vertalec® and Mycotal®, were used as reference strains. The three Chilean isolates were identified genomically as Lecanicillium attenuatum and were pathogenic to third-instar nymphs. The isolate ARSEF 13279 yielded the lowest overall lethal concentration (LC50), 3 × 105 conidia ml−1 at four days post-inoculation, and the shortest lethal time (LT50), 3.7 days after inoculation with 106 conidia ml−1. The results indicate that the isolates have considerable potential as microbial control agents of the invasive cypress aphid.

In temperate regions of Europe, the large pine weevil, Hylobius abietis, is a major pest of coniferous forests mostly at sites where clear-felling is followed by planting of saplings. Control measures against this pest are based on silvicultural techniques, an application of physical barriers on stems of saplings and insecticide treatments. To avoid the use of insecticides, alternative measures such as biological control have been investigated. The goal of the present study was to obtain local strains of entomopathogenic fungi (Ascomycota, Hypocreales) from natural populations of H. abietis, and to investigate their efficacy against the weevil. A survey on entomopathogenic fungi was undertaken at clear-felled areas of spruce forests in northern Slovakia. Two Beauveria species, B. bassiana and B. pseudobassiana, were identified, and 22 in vitro strains were obtained. Mean prevalence of infected adults was low (2.10% &plusmn, 0.67%) and the mycosis was mostly recorded during May and June. Virulence of Beauveria strains against the weevil was tested in laboratory. B. bassiana strain AMEP20 was significantly most virulent (LC50 of 0.65 &plusmn, 0.10 &times, 108 conidia mL&minus, 1). Treatment with conidia of AMEP20 strain affected feeding damage by the weevil on bark of Scots pine twigs. Daily bark consumption by B. bassiana-treated weevils was lower than by untreated individuals and decreased with increasing conidia concentration used for the treatment. In the outdoor experiment, AMEP20 strain killed weevils that fed on spruce saplings treated with conidia suspensions. Mortality due to mycosis on weevils exposed to the conidia-treated saplings reached 30.0%&ndash, 76.5% and 55.0%&ndash, 88.2% after 32 and 46 days, respectively.

Arboreal fungal endophytes are a highly diverse group of organisms associated with asymptomatic tissues and some of them are latent pathogens. The knowledge of fungal endophytes is limited, because previous studies mainly focused on foliar fungal endophytes of a limited number of host species and locations. We assessed the fungal endophyte diversity in twigs of congeneric native and exotic tree species belonging to eleven angiosperm and gymnosperm genera in mostly temperate regions of 32 countries on six continents. We describe how host taxonomy and geographic location affect endophyte diversity and how diversity differs between native and exotic trees. More than 400 samples were collected on both hemispheres and fungal communities were assessed by high-throughput sequencing. Sampling was done in winter, because trees are mostly traded in winter, when deciduous trees lack foliage. We found around 15, 000 fungal Exact Sequence Variants (ESVs) associated with the tree genera. ESV richness varied among sampled taxa, but did not differ between native and exotic hosts. ESV richness in most of the northern hemisphere tree genera peaked at 45-50 degrees of latitude, but the pattern for southern hemisphere genera was not clear. The endophyte communities of angiosperms and gymnosperms differed ; around 30% of all ESVs were found in a single genus and less than 1% was found in all genera. Distinct geographic patterns among and within continents were found and the similarity of the endophyte communities decreased with distance between sampling locations in most genera. This study reveals fungal endophyte diversity on an unprecedented scale as a result of the wide geographic and taxonomic diversity of the samples. The geographic differences in ESVs in particular suggest that international live plant trade can facilitate the exchange of geographically isolated fungal communities, which could threaten tree-based resources in importing countries.

The large pine weevil (Hylobius abietis) is an important pest of freshly planted coniferous trees. Feeding damage to conifer seedlings can be prevented by protecting the seedlings (mechanically or chemically), or by reducing H. abietis abundance. The entomopathogenic fungus Beauveria bassiana has promise for H. abietis control. To test the effects of the B. bassiana-colonized carriers (25 mm in diameter and colonized by strain AMEP20) on H. abietis adults that move into forest clearances, we performed several experiments. These included an experiment in Petri dishes under laboratory conditions, an experiment in cages with Norway spruce seedlings under semi-natural conditions, and a field experiment. Infection by B. bassiana and mortality of H. abietis was confirmed in all three experiments. The total mortality rate in all Petri dishes with B. bassiana carriers after 14 days was 74% (80% with fresh carriers; 68% with carriers that had been treated with UV light; 82% for carriers that had been frozen; 45% for adults that were exposed to carriers for only 24 h; and 95% for carriers that had been stored at 4 °C for 2 months). The mortality of H. abietis adults was affected by the absence/presence of carriers in the cages with spruce plants. The number of dead adults after 4 weeks of treatment in forest clearing were higher in plots with carriers than in the control plot. Mortality was highest for adults trapped in the plot with carriers on trap barks (58%), followed by the plot with carriers and an attractant (48%), and followed by the plots with carriers without an attractant (35%). The results suggest that carriers colonized by the entomopathogenic fungus B. bassiana could be suitable for reducing H. abietis abundance in forests. The results indicated that H. abietis adults may be infected by B. bassiana after only a short exposure to carriers. The B. bassiana-colonized carrier is ready-to-use, i.e., it could be immediately produced and applied.

This is the first report on the occurrence of andromeda lace bug, Stephanitis (Stephanitis) takeyai Drake and Maa, 1955, and rhododendron lace bug, Stephanitis (Stephanitis) rhododendri Horvath, 1905, in Slovakia. Syntopic colonies of both species were found on rhododendron shrubs (Rhododendron sp.) in south-western Slovakia in 2015. The feeding of the lace bugs resulted in damage to infested rhododendrons. Leaves turned yellow and brown, prematurely dropped what led to continuous drying up of twigs and the whole plants. Details on morphology of adult stages of the two species, description of damage symptoms and economic importance of these pests are presented and discussed.

Gypsy moth, Lymantria dispar L., is one of the most important pests of deciduous trees in Europe. In regular cycles, it causes large-scale defoliation mostly of oak, Quercus spp., forests. Government authorities in the most infested countries in Europe conduct large-scale applications of pesticides against gypsy moth. In 1999, a new natural enemy, the entomopathogenic fungus Entomophaga maimaiga, was successfully introduced into a gypsy moth population in Bulgaria. Recent investigations suggest that now E. maimaiga is quickly spreading in Europe. Herein, past studies are reviewed regarding this fungus with special emphasis on its potential for becoming an important factor regulating gypsy moth populations in Europe, focusing on the host's population dynamics in relation to the fungus, the influence of environmental conditions on fungal activity, the influence of E. maimaiga on the native entomofauna, including other gypsy moth natural enemies, and spread of the fungus. Based on this analysis, the potential of E. maimaiga for providing control in European gypsy moth populations is estimated.

The biology of the stable fly is fairly well known, but factors influencing the distribution of adult stable flies within stables are still inadequately investigated. The four experimental stables were located in south western Slovakia. Within each stable, five sticky traps were localized along the stable, and the flies were weekly counted during the flight season of years 2015&ndash, 2017. Seasonal activity and stable fly abundance in relation to temperature, rainfall, light conditions, relative air humidity, and cows per stable were evaluated. The seasonal activity of the stable fly shows one large peak at the end of summer and a second smaller peak just before the end of the flight season. The spatial distribution of stable flies was unique for each stable. All of the environmental variables had significant and mostly positive effect on stable fly abundance. The strongest and most positive effect on stable fly counts was temperature and rainfall five weeks prior to collecting session. Within the stable, cow number, air humidity, and light conditions are the strongest candidates to influence their distribution.

We evaluated the presence and impact of Entomophaga maimaiga on both target and non-target phytophagous larvae. All six study plots, with low gypsy moth population density, were situated in Central and Southeastern European oak forests and E. maimaiga had previously been reported from these plots. Totally, 45 of 4,045 (1.13%) collected non-target larvae died due to fungal infections. No non-target insect specimen was infected by E.maimaiga, although the presence of the pathogen could not be fully excluded in three cadavers. Out of 1,780L.dispar larvae collected, 15individuals (0.84%) were infected by E.maimaiga.

Live plants are often traded in their dormant stage, among other reasons to reduce the introduction of new pests. However, the pests associated with this pathway are largely unknown. Risk mitigation relies on pest risk assessments which can only be performed for known pests. It is therefore important to identify potential pests before the introduction occurs. In addition, post arrival host shifts are very difficult to predict. Several studies attempted to estimate the likelihood of host shifts based on existing literature data on known pest-host associations, but the number of pests in these studies was often limited and the data were unlikely collected in the same place and at the same time. To overcome these problems, we performed a global study which aimed at detecting potential insect pests and fungal pathogens on selected congeneric native and exotic tree species in 33 countries. Samples were taken in botanical gardens and arboreta on the Northern and Southern hemisphere. At each location, twenty 50 cm long twigs were collected from tree species of up to six genera and kept in containers with water at room temperature for insect emergence. Emerged insects were collected and identified based on morphology and DNA barcoding of the mitochondrial DNA COI region. DNA was also extracted from pooled buds, twig parts and needles for the identification of fungi based on the ribosomal DNA ITS region using a high-throughput sequencing approach. Here, we present the first results of this study and discuss them in the context of pest risk assessment.

Leptoglossus occidentalis, a species native to North America, has been expanding through European conifer forests. The fast spread across the European continent demonstrates its great invasive potential and poses a threat to conifer seed orchards. To better understand its potential distribution and population growth in infested regions, the temperature-dependent development in laboratory as well as life history, phenology, and winter survival were investigated in southern Slovakia during 2009–2011. The lower temperature thresholds to complete embryonic and postembryonic development estimated with the linear model were 13.38 and 14.37 °C, respectively. The number of degree-days required for completion of development from egg to adult estimated by the linear model was 533 °C. Overwintered adults or late nymphal instars became active in mid-March, oviposition was not observed before mid-April, and nymphs passing through five stages reached adulthood after mid-July. Individual females laid on average 31 eggs (max 78). The second generation started in August but did not complete its development till winter, although part of the 5th instar nymphs did survive until spring. Using temperature data of 41 localities across Europe and cumulative degree-days for complete life cycle, a prediction of the potential number of generations per year was made. In conditions of southern Slovakia L. occidentalis was univoltine, but up to four generations per year were estimated depending on the locality in Europe.

The entomopathogenic fungus Entomophaga maimaiga was found for the first time in Slovakia in 2013. Late instar larvae of gypsy moth, Lymantria dispar, from two sites with different population densities were dissected to evaluate the presence of pathogens. The presence of conidia and resting spores of E. maimaiga in gypsy moth cadavers was confirmed from both sites.

Neozygites osornensis is a recently described aphid pathogen characterized by morphological criteria and compared with two morphologically close taxa, Neozygites cinarae and Neozygites turbinata. Neozygites species are traditionally classified based on morphological characteristics and molecular data that would confirm these taxa and permit investigation of their phylogenetic relationships are scarce. In this study, we successfully evaluated a genetic variation within partial 18S rDNA sequences among the three Neozygites species and supported the recent erection of N. osornensis as a new species. A phylogenic analysis was also included to support our results.

We tested the combined effect of the fungus Beauveria bassiana and the microsporidium Nosema pyrausta on the European corn borer larvae, Ostrinia nubilalis, in the laboratory. The first instar of O. nubilalis larvae was the most sensitive to the B. bassiana infection followed by the fifth, second, third, and fourth instar (LC50s were 4.91, 6.67, 7.13, 9.15, and 6.51 × 105 conidia/ml for the first to fifth instars, respectively). Mortality of each instar increases positively with concentration of conidia. When B. bassiana and N. pyrausta were used in combination, mortality increased significantly in all instars. Relative to the B. bassiana treatment alone, the B. bassiana + N. pyrausta treatment decreased the LC50s by 42.16%, 37.63%, 21.60%, 27.11%, and 33.95% for the first to fifth instars, respectively. The combined effects of the two pathogens were mostly additive. However, at the two highest concentrations the pathogens interacted synergistically in the first and second instar. Individuals that survived the B. bassiana and B. bassiana + N. pyrausta treatments and developed into adults had significantly shorter lifespans and females oviposited fewer eggs than non-exposed insects. The effects on the longevity and the egg production were most pronounced at high concentration of B. bassiana conidia.

The occurrence and distribution of Dothistroma needle blight (DNB) were studied in 2014–2017 around Slovakia. A total of 84 localities, both native and planted, were investigated, and the presence of DNB was confirmed in 73 of them. In all positive locations, symptoms typical of DNB were observed and the Dothistroma species was confirmed using species‐specific primers either from fungal cultures or directly from needles. Both Dothistroma species—D. septosporum and D. pini—were identified. Both species occurred together in 29 locations, only D. septosporum in 42 and only D. pini in two locations. The host range of D. septosporum included 10 pine species and two spruce species. The host range of D. pini comprised the same number of pine hosts but only one spruce species. Five pine hosts, P. aristata, P. coulteri, P. densiflora, P. jeffreyi, P. × schwerinii, and one spruce host P. abies are new hosts species of D. pini. P. densiflora and Picea pungens have earlier been reported to be susceptible for DNB. In this study, D. septosporum was found from both tree species.

Leptoglossus occidentalis, an insect species native to North America, is a pest of conifer seed orchards in its natural distribution area. Recently, it was accidentally introduced into Europe where its populations have been expanding throughout the continent. In this study we evaluated the pathogenicity of Beauveria bassiana, Isaria fumosorosea, and Metarhizium anisopliae to this pest under laboratory and outdoor conditions. Pathogenicity varied depending upon isolates, exposure methods and location of bioassay. In the laboratory, LC50 values were highest for I. fumosorosea and lowest for M. anisopliae, and an indirect exposure was less effective than a direct one. In outdoor experiments the overall mortality was in all isolates significantly lower than mortality in the laboratory, and inter-specific variability in pathogenicity was not as prominent outdoors as in the laboratory. The results of this bioassay showed that I. fumosorosea has the potential as a microbial control agent of L. occidentalis.

Seasonal development of entomophthoralean infection in colonies of Diuraphis noxia and Rhopalosiphum maidis was evaluated on summer barley under conditions of south-western Slovakia in 2001 and 2002. D. noxia was infesting summer barley during both years, while R. maidis was only recorded in 2002. Further three cereal aphids, Rhopalosiphum padi, Metopolophium dirhodum and Sitobion avenae, were also observed on summer barley. Complex of entomophthoralean fungi identified in cereal aphid colonies included four species, Pandora neoaphidis, Entomophthora planchoniana, Conidiobolus obscurus and Neozygites fresenii, with P. neoaphidis as the most prevalent fungus during both years. Two parasitic fungi, P. neoaphidis and E. planchoniana, were identified from D. noxia, but P. neoaphidis was predominant. Infection rate in D. noxia colonies was low, not exceeding 15% and 10% in 2001 and 2002, respectively. Only nine R. maidis individuals were killed with entomophthoralean fungi. The fungi were identified as P. neoaphidis and C. obscurus. No epizootics were observed in the aphid colonies. The fungi could not control development of the aphid populations since they did not appear until high host densities had been established in the crops.