First Record of Dendrolimus pini Outbreak on Aleppo Pine in Croatia and Severe Case of Population Collapse Caused by Entomopathogen Beauveria bassiana

Background and Purpose: The pine-tree lappet moth, Dendrolimus pini, is a widely distributed pest in Europe that can cause serious needle defoliation on pines, with outbreaks occurring over large geographical areas. In Croatia the presence of D. pini was recorded only in the continental part, but not in a high level of abundance, and the outbreak of the population has never been recorded so far. Materials and Methods: In autumn 2014 an unexpected and complete defoliation on Aleppo pine (Pinus halepensis Mill.) occurred in the vicinity of Skradin, near Šibenik, and was followed by defoliation in summer and autumn 2017 in Telašćica Nature Park, Dugi otok. Infested areas were inspected and overwintering larvae in soil surface were counted in order to estimate the population and assess whether the critical number of the population has been exceeded. Specimens were collected and transferred to the Laboratory for Entomological Analysis in the Croatian Forest Research Institute for further laboratory analysis. Results: Since more than 10 larvae per m² of soil surface were found, it was concluded that an outbreak occurred at both localities. In both cases natural antagonists played an important role in lowering the pest population. In Skradin, entomopathogenic fungus Beauveria bassiana that occurred on the overwintering larvae broke the pest population, and in Dugi otok it was recorded, but to a much lesser extent and in combination with Drino inconspicua parasitoid. In both cases pines recovered very well in the following spring, with some bark beetle attacks mostly at the edge of the forest. Conclusions: The outbreaks of D. pini resulting with a total defoliation of Aleppo pine stands, and the fact that they occurred in the Mediterranean region which is not an optimal area for its appearance, makes these events unusual. Also, this is the first record of D. pini population breakdown by some antagonist

First Record of Cacopsylla pulchella (Hemiptera, Psyllidae) in Croatia

The aim of this study is to identify plant lice Cacopsylla pulchella a new alien insect in the fauna of Croatia. We propose a new method of infestation level assessment in order to assess the potential of this insect as a pest. In May 2020 samples were collected from eleven locations in Croatia, where Cercis siliquastrum is grown in parks. Adults and nymphs of C. pulchella were collected together with a plant material and brought to entomological laboratory for further analysis. Field research was performed in order to assess the distribution and infestation intensity across various localities in Croatia. The intensity of infestation was assessed heuristically by visual examination and was categorized in four categories: 0) no infestation, 1) low, 2) moderate and 3) high infestation. C. pulchella was confirmed on C. siliquastrum in eleven localities in Croatia. Most of the infested trees had a low or moderate intensity of infestation. Damages caused by this insect are due to the emission of honey dew, a small spherical drop covered with waxy secretion causing necrotic areas which could lead to the premature fall of the leaves. It is not known what kind of progress in infestation intensity could be expected so infested trees found in this study should be checked closely in the coming years. It is strongly suggested to continue the monitoring of this alien insect on C. siliquastrum in Croatia and to estimate its potential of become an invasive pest which could endanger ornamental and other values of its hosts

In situ immunofluorescence localization: A method for rapid detection of Beauveria spp. in the rhizosphere of Quercus robur saplings

Zur biologischen Bekämpfung von Pflanzenschädlingen, z.B. Maikäfer-Engerlingen, in der Rhizosphäre von Eichen, Apfelbäumen oder Kiefern werden zunehmend entomopathogene Beauveria-Spezies eingesetzt. Für eine erfolgreiche Anwendung ist es wichtig, die Ausbreitung und Persistenz der ausgebrachten Pilze qualitativ und quantitativ zu erfassen. Die Bestimmung beider Größen durch Ausplattieren auf selektiven Nährmedien oder durch mole­kulare Methoden wie PCR ist mühsam und oft ungenügend. Ziel der vorliegenden Studie war daher, eine spezifische In-situ-Methode durch Immunfluoreszenzmarkierung von Beauveria spp. zu entwickeln, hier an jungen Feinwurzeln dreijähriger Stieleichen. Durch Anfärben mit dem unspezifischen Farbstoff Blankophor wurde sichtbar, dass alle untersuchten Feinwurzeln ein dichtes Netz von Bodenpilzen trugen. Polyklonale Beauveria-Antikörper markierten an nicht beimpften Wurzeln keinen dieser natürlich wachsenden Pilze. Mit Beauveria brongniartii beimpfte Wurzeln zeigten bis zu zehn Monate nach der Inokulation eine spezifische Markierung. Während die natürlich vorkommenden Rhizosphären-Pilze in den Interzellularräumen der Wurzelrinde wuchsen, waren Hyphen von inokulierter B. brongniartii nie im Wurzelgewebe zu finden, sondern nur oberflächlich auf der Rhizodermis. Diese Beobachtungen zeigen, dass B. brongniartii bei Eichenwurzeln nicht endophytisch wächst, und dass die verwendete Methode die Unterscheidung von B. brongniartii von der in der Eichen-Rhizosphäre lebenden Pilzflora ermöglicht. Immunfluoreszenzmarkierung, wie in der aktuellen Studie eingesetzt, kann eine nützliche Methode sein, um B. brongniartii in der Rhizosphäre nachzuweisen und quantitativ zu erfassen und somit eine Langzeitkontrolle von Schädlingen mit Entomopathogenen zu ermöglichen.For biological control of plant pests, e.g. cockchafer grubs, in the rhizosphere of oak, apple or pine trees, ento­mopathogenic Beauveria spp. are increasingly applied. For successful use, it is important to monitor the spread and persistence of the inoculated fungi, both qualitati­vely and quantitatively. The determination of both para­meters by plating on selective nutrient media or by mole­cular methods such as PCR of soil samples are quite laborious and often do not yield satisfactory results. Therefore, the aim of the present study was to develop a spe­cific in situ method using immunofluorescence labelling of Beauveria spp. growing on young fine roots of three-year old oak saplings. All fine roots investigated were covered with a dense net of soil rhizosphere fungi, as visua­lized by staining with the nonspecific dye blankophor. On non-inoculated roots, polyclonal Beauveria anti­bodies did not label any of these naturally growing fungi. Only samples of roots inoculated with Beauveria brongniartii displayed specific labelling up to ten months after inocu­lation. Whereas the natural rhizosphere fungi were detected growing in the intercellular space of the root cortex in an ectomycorrhiza-like manner up to the endodermis, hyphae of the inoculated B. brongniartii were never seen within the root tissue but only growing on the surface of the rhizodermis. These observations indicate that B. brongniartii does not grow endophytically, and that the method used allows to discriminate B. brongniartii from the resident fungal flora in the oak tree rhizosphere. Detection by immunofluorescence labelling employed in the current study may be a useful tool to follow B. brongniartii in experiments aimed at establishing the entomopathogen in the rhizosphere and to monitor its fate in long-term control of entomopathogens

Dieback of Aleppo Pine and Outbreak of Bark Beetle Orthotomicus erosus in Forest Park Marjan

Intenzivno sušenja borova na Marjanu primijećeno je 2017. godine, a vjerojatno je započelo 2016. godine pojedinačnim sušenjem stabala alepskog bora. Uzimajući u obzir vrlo ozbiljne razmjere i širenje sušenja stabala u Park šumi Marjan, cilj ovog istraživanja utvrditi je moguće uzroke sušenja alepskog bora s fokusom na determinaciju uzročnika, dobnu i debljinsku strukturu sušaca te utjecaja klime na sušenje. U 2017. godini oboreno je nekoliko stabala zaraženih borova, s kojih su sakupljani uzorci sa simptomima bolesti iglica i grana. Uzorci su se obilježili i transportirali u laboratorij Hrvatskog šumarskog instituta, gdje su inkubirani i pripremani za daljnju analizu. Sakupljao se zaraženi materijal svih dijelova stabla te se evidentirala zaraza. Zaražen materijal inkubiran je u kaveze na kontroliranim uvjetima temperature (20 °C ± 2 °C) te odnosa dana i noći (L : D = 18 : 6). U kavezima su se sakupljale izašle jedinke kukaca, koje su pohranjene u alkoholu ili odmah determinirane. Iz svih dijelova stabala u klima komori izašli su potkornjaci Orthotomicus erosus i Pityogenes calcaratus. Obje vrste spadaju u štetne organizme, ali u radu se, ipak, više pažnje daje mediteranskom potkornjaku, koji je u gradacijskoj fazi te je vjerojatno počeo napadati i zdrava stabla. Na cijelom području Park šume Marjan, pod šumom, položene su primjerne pruge, dijagonalno kroz svaki odjel/odsjek u 2017., 2018. i 2019. godini. Svakom stablu na koje se naišlo izmjeren je promjer i visina, te je vizualnim pregledom i korištenjem dalekozora određena kategorija zdravstvenog stanja, pri čemu su sušci borova raspoređeni u jednu od 5 kategorija stupnja sušenja. Najveći broj potkornjacima zaraženih stabala srednjeg su promjera 27 cm, dok je za cijelu šumu srednji promjer signifikantno veći i iznosi 31 cm. U 2017. godini na Marjanu se sušilo oko 7.500 stabala borova, što predstavlja 13 % svih stabala, a sušenje se nastavlja i u sljedećim godinama, dosežući kumulativno 17 % u 2018. te 23 % u 2019. godini. Sezonska odstupanja temperature i oborina u percentilima ukazuju na višegodišnji pomak normale prema toplijem i sušnijem. Više temperature i ekstremne suše stvaraju kumulativni stres na stabla. S druge strane, kukci su poikilotermne životinje, brzo reagiraju na promjene u temperaturi, što kod vrsta s visokim biološkim potencijalom, poput nekih vrsta potkornjaka, može dovesti do brze populacijske i prostorne ekspanzije, odnosno gradacije. Uočeni fenomen upućuje na zaključak da je uzrok sušenju borova na Marjanu ulančavanje nekoliko nepovoljnih čimbenika (suša, produljenje vegetacije uslijed promjena u klimi), što je rezultiralo gradacijom potkornjaka O. erosus. Nova saznanja proizašla iz ovog istraživanja pokazuju da se mediteranski potkornjak najprije pojavljuje u krošnji, a zatim u deblu i donjim dijelovima stabla. Predlaže se da se donji nezaraženi dijelovi stabla koriste kao lovna. Gradacija potkornjaka O. erosus kakva je zabilježena u ovom istraživanju nikada prije pojave na Marjanu nije evidentirana u Hrvatskoj, te je moguće da će ovaj štetnik, ustraju li aridifi kacijske promjene, postati permanentan problem u mediteranskim šumama.The intensive dieback of pine trees on Marjan was observed in 2017, and it probably began in 2016 with dieback of individual Aleppo pine trees. Given the very serious extent and spread of drying of trees in the Forest Park Marjan, the aim of this study is to determine the possible causes of Aleppo pine dieback with the focus on identification of the cause, the age and the thickness of the dead trees, as well as the effect of the climate on this event. In 2017, several infested pine trees were cut down from which samples of symptomatic needles and branches were collected. Samples were marked and transported to the Croatian Forest Research Institute laboratory, where they were incubated and stored for further analysis. The infested material from all parts of the tree has been collected and infestation was recorded. The infested material was incubated in cages at controlled temperature conditions (20 ° C ± 2 ° C) and day and night regime (L : D = 18 : 6). Thus, the emerging insects were collected from the cages, where upon stored in alcohol or immediately determined. Bark beetles Orthotomicus erosus and Pityogenes calcaratus emerged from all parts of the trees in the climate chamber. Both species are harmful pests, but more attention here is given to the Mediterranean bark beetle, which is in the gradation phase and probably started to attack healthy trees. In the whole forest area of Marjan sample stripes have been laid out, diagonally throughout each compartment/subcompartment in 2017, 2018 and 2019. To each encountered tree the diameter and height was measured, and a certain category of health condition was determined by visual inspection and using of the binoculars for each pine tree, dividing them into one of the 5 categories of drying stage. The largest number of infected and dried trees are about 25 cm in diameter. In 2017, about 7,500 pine trees were showing symptoms of dieback on Marjan, representing 13% of all trees, and drying continued in the following years, reaching cumulative 17% in 2018 and 23% in 2019. Seasonal deviations of temperature and precipitation in percentile indicate the perennial shift of normal to hotter and drier. Higher temperatures and extreme droughts create cumulative stress on the trees. On the other hand, insects are poikilotherm animals, they react quickly to temperature changes which in species with high biological potential such as some types of bark beetles can lead to rapid population and spatial expansion, i.e. gradation. The observed phenomenon suggests that the cause of the pine trees dieback on Marjan lies in chaining of the several unfavorable factors (drought, the prolongation of vegetation due to climate changes), resulting in the gradation of the bark beetle O. erosus. Climate changes enabled a higher number of generations and an increase in population that led to the attack of healthy trees and their drying in groups in 2017, i.e. the gradation of bark beetles. New findings emerged from this research show that the Mediterranean bark beetle first appears in the tree crown and then in the trunk and lower parts of the tree. It is suggested that the lower uninfected parts of the tree are used as trap trees. Gradation of bark beetle O. erosus that was observed in this study has never been recorded so far and it is possible that this pest, in case that aridifi cation changes continue, will become a permanent problem in Mediterranean forests

Dieback of Aleppo Pine and Outbreak of Bark Beetle Orthotomicus erosus in Forest Park Marjan

Intenzivno sušenja borova na Marjanu primijećeno je 2017. godine, a vjerojatno je započelo 2016. godine pojedinačnim sušenjem stabala alepskog bora. Uzimajući u obzir vrlo ozbiljne razmjere i širenje sušenja stabala u Park šumi Marjan, cilj ovog istraživanja utvrditi je moguće uzroke sušenja alepskog bora s fokusom na determinaciju uzročnika, dobnu i debljinsku strukturu sušaca te utjecaja klime na sušenje. U 2017. godini oboreno je nekoliko stabala zaraženih borova, s kojih su sakupljani uzorci sa simptomima bolesti iglica i grana. Uzorci su se obilježili i transportirali u laboratorij Hrvatskog šumarskog instituta, gdje su inkubirani i pripremani za daljnju analizu. Sakupljao se zaraženi materijal svih dijelova stabla te se evidentirala zaraza. Zaražen materijal inkubiran je u kaveze na kontroliranim uvjetima temperature (20 °C ± 2 °C) te odnosa dana i noći (L : D = 18 : 6). U kavezima su se sakupljale izašle jedinke kukaca, koje su pohranjene u alkoholu ili odmah determinirane. Iz svih dijelova stabala u klima komori izašli su potkornjaci Orthotomicus erosus i Pityogenes calcaratus. Obje vrste spadaju u štetne organizme, ali u radu se, ipak, više pažnje daje mediteranskom potkornjaku, koji je u gradacijskoj fazi te je vjerojatno počeo napadati i zdrava stabla. Na cijelom području Park šume Marjan, pod šumom, položene su primjerne pruge, dijagonalno kroz svaki odjel/odsjek u 2017., 2018. i 2019. godini. Svakom stablu na koje se naišlo izmjeren je promjer i visina, te je vizualnim pregledom i korištenjem dalekozora određena kategorija zdravstvenog stanja, pri čemu su sušci borova raspoređeni u jednu od 5 kategorija stupnja sušenja. Najveći broj potkornjacima zaraženih stabala srednjeg su promjera 27 cm, dok je za cijelu šumu srednji promjer signifikantno veći i iznosi 31 cm. U 2017. godini na Marjanu se sušilo oko 7.500 stabala borova, što predstavlja 13 % svih stabala, a sušenje se nastavlja i u sljedećim godinama, dosežući kumulativno 17 % u 2018. te 23 % u 2019. godini. Sezonska odstupanja temperature i oborina u percentilima ukazuju na višegodišnji pomak normale prema toplijem i sušnijem. Više temperature i ekstremne suše stvaraju kumulativni stres na stabla. S druge strane, kukci su poikilotermne životinje, brzo reagiraju na promjene u temperaturi, što kod vrsta s visokim biološkim potencijalom, poput nekih vrsta potkornjaka, može dovesti do brze populacijske i prostorne ekspanzije, odnosno gradacije. Uočeni fenomen upućuje na zaključak da je uzrok sušenju borova na Marjanu ulančavanje nekoliko nepovoljnih čimbenika (suša, produljenje vegetacije uslijed promjena u klimi), što je rezultiralo gradacijom potkornjaka O. erosus. Nova saznanja proizašla iz ovog istraživanja pokazuju da se mediteranski potkornjak najprije pojavljuje u krošnji, a zatim u deblu i donjim dijelovima stabla. Predlaže se da se donji nezaraženi dijelovi stabla koriste kao lovna. Gradacija potkornjaka O. erosus kakva je zabilježena u ovom istraživanju nikada prije pojave na Marjanu nije evidentirana u Hrvatskoj, te je moguće da će ovaj štetnik, ustraju li aridifi kacijske promjene, postati permanentan problem u mediteranskim šumama.The intensive dieback of pine trees on Marjan was observed in 2017, and it probably began in 2016 with dieback of individual Aleppo pine trees. Given the very serious extent and spread of drying of trees in the Forest Park Marjan, the aim of this study is to determine the possible causes of Aleppo pine dieback with the focus on identification of the cause, the age and the thickness of the dead trees, as well as the effect of the climate on this event. In 2017, several infested pine trees were cut down from which samples of symptomatic needles and branches were collected. Samples were marked and transported to the Croatian Forest Research Institute laboratory, where they were incubated and stored for further analysis. The infested material from all parts of the tree has been collected and infestation was recorded. The infested material was incubated in cages at controlled temperature conditions (20 ° C ± 2 ° C) and day and night regime (L : D = 18 : 6). Thus, the emerging insects were collected from the cages, where upon stored in alcohol or immediately determined. Bark beetles Orthotomicus erosus and Pityogenes calcaratus emerged from all parts of the trees in the climate chamber. Both species are harmful pests, but more attention here is given to the Mediterranean bark beetle, which is in the gradation phase and probably started to attack healthy trees. In the whole forest area of Marjan sample stripes have been laid out, diagonally throughout each compartment/subcompartment in 2017, 2018 and 2019. To each encountered tree the diameter and height was measured, and a certain category of health condition was determined by visual inspection and using of the binoculars for each pine tree, dividing them into one of the 5 categories of drying stage. The largest number of infected and dried trees are about 25 cm in diameter. In 2017, about 7,500 pine trees were showing symptoms of dieback on Marjan, representing 13% of all trees, and drying continued in the following years, reaching cumulative 17% in 2018 and 23% in 2019. Seasonal deviations of temperature and precipitation in percentile indicate the perennial shift of normal to hotter and drier. Higher temperatures and extreme droughts create cumulative stress on the trees. On the other hand, insects are poikilotherm animals, they react quickly to temperature changes which in species with high biological potential such as some types of bark beetles can lead to rapid population and spatial expansion, i.e. gradation. The observed phenomenon suggests that the cause of the pine trees dieback on Marjan lies in chaining of the several unfavorable factors (drought, the prolongation of vegetation due to climate changes), resulting in the gradation of the bark beetle O. erosus. Climate changes enabled a higher number of generations and an increase in population that led to the attack of healthy trees and their drying in groups in 2017, i.e. the gradation of bark beetles. New findings emerged from this research show that the Mediterranean bark beetle first appears in the tree crown and then in the trunk and lower parts of the tree. It is suggested that the lower uninfected parts of the tree are used as trap trees. Gradation of bark beetle O. erosus that was observed in this study has never been recorded so far and it is possible that this pest, in case that aridifi cation changes continue, will become a permanent problem in Mediterranean forests

Transformer-based biomarker prediction from colorectal cancer histology:A large-scale multicentric study

Deep learning (DL) can accelerate the prediction of prognostic biomarkers from routine pathology slides in colorectal cancer (CRC). However, current approaches rely on convolutional neural networks (CNNs) and have mostly been validated on small patient cohorts. Here, we develop a new transformer-based pipeline for end-to-end biomarker prediction from pathology slides by combining a pre-trained transformer encoder with a transformer network for patch aggregation. Our transformer-based approach substantially improves the performance, generalizability, data efficiency, and interpretability as compared with current state-of-the-art algorithms. After training and evaluating on a large multicenter cohort of over 13,000 patients from 16 colorectal cancer cohorts, we achieve a sensitivity of 0.99 with a negative predictive value of over 0.99 for prediction of microsatellite instability (MSI) on surgical resection specimens. We demonstrate that resection specimen-only training reaches clinical-grade performance on endoscopic biopsy tissue, solving a long-standing diagnostic problem