Iwona Skrzecz

Detection and identification of baculovirus pesticides by multitemperature single-strand conformational polymorphism

The method of single-strand conformational polymorphism (SSCP) was modified in our laboratories for the characterization of baculoviruses, insect viruses with great potential for use as bioinsecticides in biological protection programs. A series of primers were synthesized after the comparison of the polyhedrin gene sequences of over 20 baculoviruses. Polyhedrin is a highly conserved protein which is responsible for the persistence of the virus in the environment. Universal primers were designed which could be used in polymerase chain reactions (PCR) containing genomic DNA from an array of nucleopolyhedrosis viruses (NPVs) including these which are used as biopesticides against important pests of forests and crops, such as Anticarsia gemmatalis, Spodoptera frugiperda, Lymantria dispar, Lymantria monacha and many others. PCR products were denatured and subjected to single-strand DNA electrophoresis at variable temperatures (MSSCP) where, after silver staining, they gave ssDNA band patterns characteristic for each baculovirus species. This technique can be potentially applied to detect baculoviruses in insects collected in the field, as well as to plant tissues and the excrements or bodies of predators without need for sequencing the PCR products. Sometimes MSSCP can be used not only for species determination but also as an indication of genomic variability which can be related to infectivity.

An alphabaculovirus isolated from dead Lymantria dispar larvae shows high genetic similarity to baculovirus previously isolated from Lymantria monacha – An example of adaptation to a new host

A new isolate of baculovirus, Lymantria dispar multiple nucleopolyhedrovirus-BNP (LdMNPV-BNP), was found in dead gypsy moth (L. dispar) caterpillars collected in the Biebrzanski National Park in Poland. Here, we examined its biological activity, structure, genetic content and phylogeny. Multiple nucleocapsids of LdMNPV-BNP are enveloped together in 2-26 virions embedded in occluded bodies (OBs) very similar to the OBs previously described in viruses infecting Lymantriinae. This isolate kills pest larvae in a relatively short time (LT50 of approximately 9days for a dose of 2×10(7)OBs/ml), highlighting the possibility for its use as a biopesticide. Next-generation sequencing of LdMNPV-BNP revealed gene content (e.g. DNA photolyase) that is not present in any LdMNPV isolate sequenced to date. The genome is 157,270 base pairs long and has a notably lower G+C content in comparison to other LdMNPVs (50.3% G+C content compared to an average of 57.4% among other LdMNPVs). According to our phylogenetic analysis based on 37 core genes, LdMNPV-BNP is a member of group II alphabaculoviruses, which are closely related to LdMNPV and LyxyMNPV (Lymantria xylina multiple nucleopolyhedrovirus). Molecular evolution inference based on the partial sequence of lef-8, lef-9 and polh genes shows that LdMNPV-BNP and isolates of Lymantria monacha nucleopolyhedrovirus (LymoNPV) may share a very recent common ancestor or be isolates of the same virus species. LdMNPV-BNP, like other baculoviruses, could be beneficial as an active component of biopesticides that can be used during forest integrated pest management.

Effects of Norway Spruce (Picea abies) Stump Debarking on Insect Colonization in the Polish Sudety Mountains

This study aimed to determine whether debarking of Norway spruce stumps influences the intensity of their colonization by insects. The observed stumps of Picea abies (L.) H Karst were located in the eastern Sudety Mountains at elevations between 600 and 1000 meters above sea level (masl) on clear-cut areas where large-diameter timber and small logging slash had been harvested. In total, 720 fresh (up to 12 months old) P. abies stumps were investigated, of which half were debarked immediately after tree felling. Insects were collected from 0.5 m2 bark samples taken from coarse roots around the stump base and then identified with regard to family, genus, and species. P. abies stumps were colonized by insects from 18 families from 2 orders: Coleoptera and Diptera. Coleopterans were most frequently represented by the families Cerambycidae (52% of all collected insects) and Curculionidae (41%), including the subfamily Scolytinae (15%). Approximately 1.5% of all insects collected were classified as Diptera. Overall, approximately 40% greater insect colonization was observed in the bark samples collected from debarked P. abies stumps. The debarking treatment increased the intensity of stump colonization by both Cerambycidae and Scolytinae but had no effect on colonization by Curculionidae (excluding Scolytinae). These results suggest that debarking Norway spruce stumps does not reduce insect colonization and could be avoided in forest management.

Complete Genome Sequence of Lymantria dispar multiple nucleopolyhedrovirus Isolated in Southwestern Poland

ABSTRACT We report here the complete genomic sequence of Polish alphabaculovirus isolated from dead gypsy moth caterpillars. Its genome structure and G+C content indicate that it is a Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) strain which possesses the shortest sequence among eight other (similar to reference sequence AF081810) LdMNPV sequences available in GenBank; it is 159,729 bp long.

The role of fungus Beauveria bassiana in reducing the number of Pissodes castaneus (Col., Curculionidae) in young forests

Abstract Small banded pine weevil Pissodes castaneus is one of the most dangerous pests of Pinus sylvestris plantations and thickets. The lack of effective and environmentally safe methods of limiting the number of the pest justified to undertake the studies aimed at the laboratory and field evaluation of biological activity of entomopathogenic fungus Beauveria bassiana used to reduce the numbers of small banded pine weevil. In laboratory, the beetles were reared on the sections of fresh Scots pine twigs that were treated with five suspensions containing from 1 × 104 to 1 × 108 conidia of B. bassiana in 1 ml. During the 3-week rearing, insect mortality was determined and median lethal concentration LC50 was calculated. The field treatments consisted of spraying 4-year-old P. sylvestris trees with two formulations of fungus containing 1 × 108 conidia ml−1 of suspension. Treatments consisted of spraying 4-year-old P. sylvestris trees with two fungus formulations containing 1 × 108 conidia ml−1. High insecticidal activity of B. bassiana was found because the pathogen caused the death of 14-94% of P. castaneus beetles, LC50 = 6.51 × 105 conidia ml−1. Field treatments did not result in the reduction of plant damage caused by small banded pine weevil; therefore, the spraying of trees with B. bassiana cannot be recommended to protect the young stands of P. sylvestris against pest.

Insecticidal activity of alpha-cypermethrin against small banded pine weevil Pissodes castaneus (Coleoptera: Curculionidae) in forest plantations and thickets

Abstract Scots pine (Pinus sylvestris) plantations and thickets damaged by biotic and abiotic factors are particularly attractive to small-banded pine weevil Pissodes castaneus, whose larvae excavate feeding tunnels in the stems of young trees, causing their death. There are no chemical methods that can be applied to protect forest plantations and thickets against this pest. Therefore, the studies were undertaken aimed at the assessment of the efficacy of alpha-cypermethrin used to reduce the numbers of this pest within restock areas. The scope of work included laboratory and field estimation of insecticidal activity of alpha-cypermethrin. In laboratory, the beetles of P. castaneus were reared on P. sylvestris twigs treated with five concentrations of alpha-cypermethrin (0.0001–1%) formulated as Fastac Forest 15 SC. Insect mortality was calculated during 7-day rearing and median lethal concentration LC50 was calculated. The field treatments consisted of spraying of four-year old Scots pines with the insecticide in concentrations of 2% and 4%. In laboratory conditions, the insecticide used at five different concentrations caused a 7–95% mortality of the beetles (LC50 = 0.266%), while field sprays resulted in a 1.5–3.5-fold reduction in the colonization of trees and in higher pest mortality rates. The results indicate the possibility of using of alpha-cypermethrin in protecting the forest against P. castaneus and can be the basis for the development of chemical method used in the forestry practice.

Effects of botanical antifeedants on Melolontha melolontha grub feeding on Scots pine roots

Abstract The aim of the study was to evaluate the possibility of using botanic antifeedants to reduce the damage caused by Melolontha spp. grubs. To achieve the objective, the experiments were established in semi-field conditions to estimate the antifeedant activity of rutin, quercetin (flavonoids from buckwheat Fagopyrum esculentum) and an extract from black alder Alnus glutinosa leaves against Melolontha melolontha grubs. The grubs were placed individually in the pots with a soil in which 2 year old Pinus sylvestris trees were planted. The pots were put in garden pavilions placed in the open area. Then the soil in the pots were watered with the emulsions of rutin, quercetin, an extract from A. glutinosa leaves, and with pure water-comparative variant. After 4 months, the weight and mortality of grubs were compared, as well as the weight of tree roots in all pots. There was no effect of the antifeedants on the development and extent of damage caused by M. melolontha grubs. The results do not indicate the use of botanic antifeedants in the protection of forests against the cockchafer grubs

The effect of initial dose on the recovery and final yields of Heterorhabditis megidis (Rhabditida: Heterorhabditidae) in larvae of the great wax moth, Galleria mellonella

The aim of this study was to determine the effect of different initial doses of the infective juveniles (IJs) (50 IJs, 200 IJs, 1000 IJs) of Heterorhabditis megidis Poinar (Rhabditida: Heterorhabditidae) strain IsM15/09 on recovery, final yields and percent final yields in larvae Galleria mellonella ( L.). Percent recovery was not directly related to initial dose. Final yields also did not change with the initial dose. However, percent yields was highly negatively correlated with initial dose of nematodes and was the highest with the 50 IJs dose. Additional point of the study was to investigate whether the nematodes are able to produce progeny from one hermaphroditic individual. The results showed that the invasive larvae resumed growth and transformed into hermaphroditic individuals that reproduced without cross-fertilisation.

Evaluation of biological activity of biopreparations containing nematodes from the genera Steinernema and Heterorhabditis used for reducing large pine weevil Hylobius abietis L. population in pine Pinus sylvestris L. stumps

The use of entomopathogenic nematodes (EPNs), such as Steinernema and Heterorhabditis spp., against the large pine weevil Hylobius abietis has been mainly studied in the United Kingdom and Ireland. The results of these studies show a great potential of nematodes for H. abietis control. Similar research conducted in Poland aimed at evaluating effectiveness of commercially produced biopreparations that contained entomopathogenic nematodes and were applied against the large pine weevil. The treatments consisted of spraying soil around Pinus sylvestris stumps with an aqueous suspension of Steinernema carpocapsae, S. feltiae and Heterorhabditis bacteriophora, H. downesi, H. megidis at a dose 3.5 million IJs/1000 ml of water per stump. The roots of experimental stumps were analyzed 4 weeks subsequent to the treatments. After roots debarking, H. abietis larvae were isolated and the percentage of insects parasitized by EPNs was assessed. All tested nematodes showed ability to parasitize H. abietis larvae overwintering in P. silvestris stumps. The highest extensivity of parasitizm was observed in H. abietis larvae parasitized by S. carpocapsae and H. downesi, and the lowest – in the case of larvae collected from the roots of the stumps sprayed with H. megidis. There were no nematodes found in H. abietis larvae collected from untreated – control stumps.

First all-Poland Conference on Insect Pathology “State of the art and future directions of research on insect pathology in Poland”

First all-Poland Conference on Insect Pathology was held on 6– 7 March 2012 at the Forest Research Institute (FRI) in Sekocin Stary. The Conference was organized by FRI in cooperation with the University of Humanities and Sciences in Siedlce, Poland. Obtaining research funding both from national and foreign financing mechanisms requires establishing research teams including researchers from various scientific institutions, who can together make the best use of knowledge capacity and research infrastructure of these organizations. Moreover, there is a necessity for information and experiences exchange among scientists which facilitates collaboration in projecting and realizing research and elaborating publications. Also, friendliness, honesty and cooperation in research carried out on daily basis are very important in accomplishing scientific projects’ goals. All the above justified organization of the conference with the main objective to join together Poland’s scientific centres involved in research on insect pathology. The Conference was aimed at all scientific workers active in the field of insect pathology. The meeting gathered 60 persons from 15 universities and research institutes. Conference agenda embraced four thematic sessions: 1. History of insect pathology in Poland. 2. State of the art and directions of research undertaken in national scientific institutions. 3. Cooperation in research and fund raising (Discussion). 4. Poster Session. The Conference started from presentations prepared by distinguished guest speakers – the scientists involved in research on insect pathology for many years who talked about the history of research in this field (1st Session). The following papers were presented: