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IOBC-WPRS Bulletin Vol. 129, 2017


IOBC-WPRS Bulletin Vol. 129, 2017

Working Group "Microbial and Nematode Control of Invertebrate Pests"
Proceedings of the 16th meeting "Is IPM ready for Microbial Control Agents?" at Tbilisi (Georgia), 11 - 15 June, 2017
Edited by Eustachio Tarasco, Johannes A. Jehle, Medea Burjanadze, Luca Ruiu, Vladimír Půža, Enrique Quesada-Moraga, Miguel Lopez-Ferber, Dietrich Stephan
ISBN 978-92-9067-314-9 [XIV + 193 pp]


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An introduction to IOBC
Giselher Grabenweger

Abstract only


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Neem oil inhibits larval and pupal development of Drosophila suzukii in feeding trials under laboratory conditions
Sarah Biganski, Johannes A. Jehle, Regina G. Kleespies

Abstract: Neem oil is widely used for biological control of nearly all kind of leaf mining and sucking pest insects. Its effects, repressing reproduction and inhibiting moulting, could be advantageous for control of Drosophila suzukii if applicable. In larval bioassay with a commercially available Neem product mortality of up to 100% could be achieved when applying a ten times higher concentration than recommended for target insects.


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IPM in practical wireworm control: a challenge!
Hilfred Huiting and Klaas van Rozen

Abstract: Wireworm control in mainstream farming to date consists of applications of chemical compounds. Despite many years of research and development targeting at non-chemical alternatives, mainly including entomopathogenic fungi (EPF) and plant extracts, and some market introductions, there is much room for increase of their market share. Although research has made a huge effort in working towards improvement, market penetration of non-chemical wireworm control is hindered by product properties as difficulty to handle/apply, less than optimal efficacy, and the farming community’s unfamiliarity with the product(s), all of these being interconnected. Following the EU Directive 2009/128/EC, all EU Member States have to comply with stricter guidelines regarding IPM before 2023. In many countries retail parties play an important role in this force field, following from public awareness on crop protection issues. All of these factors imply increased (re-)registration pressure on the current chemical compounds and open new opportunities for introduction of non-chemical alternatives. Use of these alternatives may need a change in approach, and close collaboration between research, extension services and farmers. The result can be a system innovation with a recognised position of both adequate and reliable monitoring and non-chemical wireworm control. Examples are provided of possible ways to achieve the goals described.


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Genetic characterization of cry gene diversity in Bacillus thuringiensis isolates from Kyrgyzstan
Saykal Bobushova, Christina Schuster, Andreas Leclerque, Tinatin Doolotkeldiev

Abstract: A set of Bt strains isolated from insects and soil samples, selected from different ecosystems in Kyrgyzstan was molecular taxonomically characterized using the pycA gene as marker for phylogenetic reconstruction. Within the Bacillus cereus sensu lato species complex, all Kyrgyz isolates were shown to belong to the B. cereus subspecies thuringiensis. Most isolates were assigned to the lineage Bt tolworthi, with two isolates each belonging to the lineages Bt kurstaki and Bt sotto. Targeted amplification of different cry gene variants revealed differences in cry gene frequencies. Whereas cry1 and cry4 genes were regularly detected, cry3 genes were identified in only a small number of Bt strains. Interestingly, the combination of cry1 and cry4 genes in the same strain was frequent, whereas the combination of cry1 and cry3 occurred only in a single Bt strain.


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Effect of UV-stabilized Btk on Helicoverpa armigera larvae after exposure to sunlight
A. Rami Horowitz, Carolina Guzman, Rafael Ischakov, Avishay Pelah, Arie Markus

Abstract: Helicoverpa armigera (Lepidoptera: Heliothinae) is an insect pest, which severely harms vegetables, cotton and flowers. A good solution for controlling H. armigera and other lepidopteran larvae, especially in organic farms, is a treatment with Bacillus thuringiensis (Bt). The problem of spraying with Bt is its sensitivity to UV light emanating from the sun, making the spray preparation short lived. The laboratories of BotanoCap (Israel) have developed a formulation, which is more stable to UV radiation and might be active for a longer period of time, compared with the regular preparation. In our experiments we compared the efficacy of BotanoCap’s encapsulated B.t. var kurstaki formulation, which is considered to be UV-resistant, to a non-encapsulated standard preparation. Both formulations were tested with and without exposure to sunlight.
Exposure to sunlight of the encapsulated formulation did not cause a reduction in mortality of H. armigera larvae (3rd instars) in comparison with the standard (non-encapsulated) preparation. The non-encapsulated formulation lost most of its efficacy after exposure for about four hours to sunlight. It is advisable to repeat the experiments in the field, with direct spraying on the crop plants.


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Recent findings on Brevibacillus laterosporus toxins and virulence factors
Luca Ruiu

Abstract: Brevibacillus laterosporus is a spore-forming bacterium showing significant biopesticidal potential against insect species in different orders including coleoptera, lepidoptera, diptera and against nematodes and molluscs. A main morphological feature of this microbial species is the typical canoe-shaped parasporal body attached to one side of the spore. Recent genome sequencing and analysis of entompathogenic strains led to the identification of several toxins and virulence factors. Among these, some mosquitocidal toxins, vegetative insecticidal proteins, chitinases, and several polyketides and nonribosomal peptides. Besides, highly conserved proteins from the spore coat canoe-shaped parasporal body complex (SC-CSPB) of entompathogenic strains were reported to be involved in pathogenesis. Whilst different strains show varying degrees of virulence against diverse insect pests, some represent a beneficial component of the intestinal bacterial community of certain species like the honey bee Apis mellifera. Unexpectedly, a strain isolated from honeybees was pathogenic to the house fly Musca domestica. These findings, supporting the development of either mutualistic or pathogenic interactions of this bacterium with diverse insect species, as the result of a coevolutionary process, are here presented and discussed.


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Genetic diversity of Bacillus thuringiensis isolates from Cuba
María Elena Márquez, Christina Schuster, Andreas Leclerque, Yohana Gato, Yamilé Baró

Abstract: The molecular taxonomy and cry gene diversity of a set of Bacillus thuringiensis isolates stemming from different sources and provinces of Cuba has been investigated. Within the Bacillus cereus sensu lato complex, most isolates were assigned to the B. thuringiensis lineages “Kurstaki” or “Sotto” when using the pycA gene encoding pyruvate carboxylase for phylogenetic reconstruction. Targeted amplification of different cry gene variants revealed that most Bt strains in a mutually exclusive way contained either cry1 or cry4 gene copies, whereas cry3 genes were not identified. The distribution of cry genes was consistent with taxonomic assignment. The results indicate an elevated degree of local biodiversity among B. thuringiensis isolates from Cuba.


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Commercial production of conidia emitting granules for soil application of Metarhizium brunneum
Arne Peters, Stefanie Fiege

Abstract: Metarhizium brunneum Cb15 was produced in liquid culture and formulated into an extruded granule for controlling wireworms (Agriotes sp.) in potatoe-fields by applying the granules at planting. The production was optimized by changing the production medium for the fungus. Best conidia emission of resulting granules was achieved in media where the highest concentration of submerged conidia was produced. More submerged conidia were produced if the liquid culture was inoculated with aerial conidia than if started from a liquid preculture with submerged conidia. In the full grown fermentation broth a concentration of 5e7 submerged conidia/ml was achieved. The granules were produced directly from the fermentation broth by adding soy-flour, glucose, potassium-mono-phosphate and sodium-alginate. The resulting granules were dried at 45 °C inlet – and max. 38 °C outlet temperature to a residual moisture of approx. 7%. Conidia production of the resulting granules was tested on water agar. While 100% of the granules produced aerial conidia after 10 days incubation at16 °C, the concentration of conidia was 4.2e9 ± 7e8 conidia per g granules. The method for producing these conidia emitting granules is highly efficient and can eassily be adapted to other entomopathogenic or disease suppressing fungi.


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Preliminary study of selected entomopathogenic fungi for corn leaf aphid, Rhopalosiphum maidis, control
Nana Bitsadze, Ketevan Pavliashvili, Rusudan Khazaradze, Mzia Beruashvili, and Mariam Betsiashvili

Abstract: Rhopalosiphum maidis Fitch. (Hemiptera, Aphididae), corn leaf aphid, is a pest of maize (corn) and other related crops with worldwide distribution. This insect can infest all aboveground parts of the corn plant and cause serious damage to the yield. The aim of this study was to determine the pathogenicity of selected entomopathogenic fungi (Beauveria bassiana (Bals.-Criv.) Vuill., Isaria fumosorosea Wize and three different strains of Metarhizium anisopliae (Metschn.) Sorokin against R. maidis in the second nymphal stage under controlled conditions (23 °C temperature, 60% humidity). Maize local variety ‘Adjametis tetri’ was used for the assay. Maize seeds were grown in separate pots containing growth substrate mix. Two weeks old corn seedlings` stems and leaves were sprayed with suspension of entomopathogenic fungi at concentration 1.0 × 106 conidia/ml. Controls were sprayed with distilled water with 0.01% Tween 80 as a wetting agent. Ten minutes later, ten R. maidis aphids were confined on each corn seedling previously infested by fungal entomopathogens and pots were placed in micro-perforated polypropylene bags to avoid migration of the aphids from plant to plant. Aphid mortality and the progeny production were studied 7 days later. Four replicates have been done per treatment. Significantly high aphids’ mortality rate has been shown by B. bassiana and I. fumosorosea and relatively low influence was caused by M. anisopliae strains. Almost all studied fungi caused suppression in aphid progeny production, but in different extent. B. bassiana and strain MA2 caused highest suppression, while effect of strain MA3 on progeny production was not significantly different from the control sample. Number of aphids’ new generation after I. fumosorosea and MA1 treatment was almost the same.


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First report of entomopathogenic fungi on the insect vector Hyalesthes obsoletus Signoret (Hemiptera: Cixiidae) in Georgia
Nona Chkhaidze, Medea Burjanadze, Mzagho Lobghanidze, Ketevan Koridze

Abstract: Hyalesthes obsoletus Signoret,1879 (Hemiptera: Cixiidae) is the vector of phytoplasma diseases in various crops of economic importance. Among them is grapevine bois noir (BN) disease, caused by stolbur phytoplasma (16SrXII-A group). Two phytoplasma strains, are associated with grapevine yellows ‘Candidatus Phytoplasma solani’ and ‘Са. P.a convolvuli” in Georgia. H. obsoletus were found on the grapevine (Vitis vinifera), sunflower (Helianthus annuus), liquorice (Glycyrrhiza glabra) and bindweed (Convolvulus arvensis) in Sighnaghi district of Eastern Georgia, 2014-2016. Its imagines were met in June, July and August. Adults were collected on vineyard 2014 and on sunflower crop in Sighnaghi district, village Bodbe, while I and II decades of 2016. Laboratory researches shown, that some examples had visual symptoms of fungal infection. The pure culture consisted entomopathogenic fungi Beauveria bassiana and Beauveria pseudobassiana .
The is first report of Beauveria bassiana and Beauveria pseudobassiana on the phytoplasma diseases vector Hyalesthes obsoletus in Georgia.


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Group-I intron based strain-specific diagnosis of entomopathogenic Metarhizium spp. fungi from Cuba
Yohana Gato, Christina Schuster, Andreas Leclerque, María Elena Márquez, Yamilé Baró

Abstract: Mitosporic entomopathogenic ascomycetes from the genus Metarhizium (Ascomycota: Hypocreales) are of particular interest as biological control agents of numerous pest insects. Bioprospection for these fungi in Cuba has given rise to a set of strains from a wide range of hosts. Internal transcribed spacer (ITS) based molecular taxonomic analysis locates the strains within the Metarhizium anisopliae species complex. In order to facilitate the development of strain LBM-41 into a myco-insecticide, a PCR-based diagnostic tool allowing the reliable and fast differentiation of this strain from the other Cuban isolates was needed. Screening of this set of fungal strains for the presence or absence of self-splicing group-I introns disrupting the 18S and 28S rRNA encoding genes at previously identified intron insertion hot-spots revealed a unique intron constellation comprising two group-I introns in insertion positions 1 and 4, for the 28S rRNA gene of strain LBM-41. These findings permit to develop an identification assay for this strain. Primer pairs hybridizing against the 28S rRNA intron sequences were designed and used to amplify partial rRNA gene sequences in a strain specific manner. The approach was shown to unambiguously identify strain LBM-41 among all strains investigated. In conclusion, the feasibility of strain-specific identification based on group-I intron sequences has been demonstrated for a potential Metarhizium biocontrol strain from Cuba.


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Effect of ultraviolet radiation and screening tolerance on native isolates of Beauveria spp.
Mikheil Gogebashvili, Nazi Ivanishvili, Medea Burjanadze, Mariam Arjevanidze, Ketevan Koridze

Abstract: The objective of this study was to explore diverse habitats as potential sources of local strains of the Beauveria, their relative distribution in various geographical areas and their potential application as biological control agents against the target insect. The original 10 isolates were obtained from 3 different geographical sites at different altitudes and climate zones. In experimental trials, selected B. bassiana isolates (Bb002, Bb007, Bb010, Bb017, Bb026, Bb027, Bb029, Bb070, Bb114, Bb Geometridae), were weighted by UV-B irradiance at dosages 850 mW/cm2, while 1-6 h, with 1 h interval. As a control not exposed to UV radiation were used.
As a result, after 6 h irradiation, achieved in 50% growth inhibition of selected isolates compared to control. Isolates from the different ecosystems and sea level are characterized with different tolerance, in particular, high-sensitive isolates were more adapted to the UV-B radiation. In order to establish, two different isolates – B.b 029 (214 m asl) and B.b 070 (1200 m asl) were evaluated. By providing to analysis the relative increase of the intensity, these two isolates are differing only by individual tolerance, but restorative processes in both cases were going equally.


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The effects of natural substrates and artificial media on the production of conidiospores and blastospores of the entomopathogenic fungus Isaria fumosorosea, strain CCM 8367
Jana Konopická, Andrea Bohatá, Rostislav Zemek, Vladislav Čurn

Abstract: Entomopathogenic fungus Isaria fumosorosea WIZE (Hypocreales: Cordycipitaceae) is among the most common species used in biological control. The aim of this study was to find the most suitable media for both surface and submerged productions of spores of the strain CCM 8367 of I. fumosorosea isolated in the Czech Republic from the horse chestnut leaf miner, Cameraria ohridella (Lepidoptera: Gracillariidae). For the surface cultivation we used the following commercial agar media: Sabourad dextrose agar (SDA), V8 juice agar (V8J), Corn meal agar (CMA), Czapek dox agar (CDA), Agar malt extract (MEA) and Potato dextrose agar (PDA) and also natural substrates (hulled barley, rice and oatmeal) were tested. For submerged cultivation three standard liquid media were used: Sabourad dextrose broth (SDB), Czapek dox broth (CDB) and Potato dextrose broth (PDB) and two modified media (A and B). The radial growth of the strain and the production of conidiospores was evaluated after 7, 14, and 21 days. Production of blastospores was evaluated daily for 7 days. Best growth of the strain CCM 8367 was observed when two agar nutrient media CDA and SDA were used, while the highest production of spores was observed on medium V8J when fungus produced after 21 days of cultivation 6.28 × 105 conidiospores per mm2 of culture. Production of conidiospores on natural substrates was highest on rice. After 21 days, the production of coniodiospores was 2.58 × 109 per 1 gram of rice. Blastospores production of the strain CCM 8367 was the highest in the modified medium of our own recipe (medium A). After 4 days of cultivation, amount of blastospores per 1 ml in this medium was 3.34 × 108. The best medium producing the highest amount of blastospores among the commercially available artificial media was SDB. The effect of C:N ratio (glucose:peptone) on agarized and liquid media is currently evaluated.


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Exoenzymes improve penetration and colonization of potato plants by endophytic entomopathogenic Metarhizium brunneum
Vivien Krell, Desirée Jakobs-Schoenwandt, Laurenz Hettlage, Stefan Vidal, Anant V. Patel

Abstract: Biocontrol with endophytic entomopathogenic fungi is a promising option to protect plants systemically from insect herbivores. Yet, current applications are limited especially by low plant colonization. Inspired by penetration mechanisms of plant pathogenic fungi, we aimed at enhancing Metarhizium brunneum strain Cb15 potato plant penetration and colonization by supplementation of plant cell-wall degrading enzymes pectinase, hemicellulase or cellulase. As part of a pre-conditioning strategy of “endophytically competent” biomass, we further looked into the possibility of inducing these enzymes during submerged cultivation of Metarhizium brunneum mycelial biomass. We found that after hemicellulase or cellulase supplementation to mycelial biomass on potato tubers, the number of plants with endophytic fungal colonization in the roots was enhanced by 20% and even by 60%, respectively. This correlated with a rise in fungal endophytic DNA levels by 4% after hemicellulase and by 25% after cellulase treatments. Pectinase had no beneficial effect on plant colonization. Supplementation of cellulose derivatives during submerged cultivation of Metarhizium brunneum led to induction of cellulase activity. Maximum enzymatic activity depended significantly upon the water solubility of the cellulosic materials. Our study provides first evidence that refined formulations of endophytic entomopathogenic fungi could contribute to a more effective use of these fungi in strategies priming plants against pests and diseases.


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Isolation and identification of fungal community of alfalfa pest weevils (Coleoptera: Curculionidae) in the Republic of Moldova
Anna Moldovan, Ion Toderas, Andreas Leclerque, Natalia Munteanu Molotievskiy

Abstract: The main objective was to investigate the fungal community of alfalfa pest weevils Hypera postica, Sitona lineatus and Protapion apricans in the Republic of Moldova and identify strains with potential for biological control development. Based on the nucleotide sequence of the ITS region, 19, 14, and 5 fungal strains were identified from H. postica, S. lineatus and P. apricans, respectively. Of these, 28 strains were taxonomically assigned at the species and 10 further strains at the genus level. In particular, four isolates were identified as entomopathogenic fungi Beauveria bassiana (3) and Paecilomyces fumosoroseus (1). This is the first exploration of fungal communities of pest weevils in the Republic of Moldova. Revealed data offer useful information for subsequent studies in the development of microbial control agents.


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Potential of entomopathogenic fungi and nematodes against the two cryptic species Parahypopta caestrum and Cossus cossus in laboratory assays
Eustachio Tarasco, Monica Oreste

Abstract: Preliminary assays were performed in laboratory conditions in order to evaluate the infectivity of several entomopathogenic fungi (EPFs) and entomopathogenic nematodes (EPNs) autochthonous strains against P. caestrum and C. cossus larvae. Results revealed the efficacy of these microbial control agents in killing the larvae, although a wide inter- and intra-specific variability in virulence was detected among different microbial strains. Considering the lack of effective chemical control means, the microbial control of the Asparagus moth and the goat moth by EPNs and EPFs reveals promising perspectives and needs further investigations.


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Potential of entomopathogenic fungi and nematodes against Drosophila suzukii in laboratory assays
Monica Oreste, Nuray Baser, Khalid Ibouh, Vincenzo Verrastro, Eustachio Tarasco

Abstract: The effect of several entomopathogenic fungi (EPFs) and entomopathogenic nematodes (EPNs) autochthonous strains against D. suzukii individuals (larvae, pupae and adults) was evaluated in laboratory assays. Results showed a moderate effect against D. suzukii larvae and adultsand an higher virulence against pupae. Furtheremore, the deterrent effect on D. suzukii oviposition activity of several EPFs strains was evaluated in laboratory conditions (no-choice and double-choice test), in comparison with other mineral compounds (potassium silicate and kaolin). Results reveals promising perspectives for the biological and integrated management of this pest.


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Using insect-pathogenic fungi to manage insect pests, where are we going? (Where SHOULD we be going?)
Stefan T. Jaronski

No abstract


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Biological control of adult Diabrotica – Spray experiments with Metarhizium brunneum strain BIPESCO 5 under real farm conditions
Hermann Strasser, Hannes Rauch1, Roland Zelger

Abstract: The western corn rootworm Diabrotica virgifera virgifera (WCR) is causing economic damages in maize production in parts of the European Union (EU), so in Austria. The control of adults is becoming increasingly cumbersome as the WCR has a history of adapting to several management practices. The European Union has directed Member States to consider Integrated Pest Management as a strategy for controlling WCR. Entomopathogenic fungi Metarhizium brunneum (BIOPESCO 5) have been found to have a real potential also for adult control. First studies conducted in Styria (Austria) showed the potential of adult spray application using safe, water dispersible Metarhizium brunneum formulations (Hypocreales: Clavicipitaceae), under real farm conditions as an innovative direct biological control method for WCR in maize production.


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Entomopathogenic fungus Entomophaga aulicae as agents in classic biological control of browntail moth in some broadleaf forest in Serbia
Mara Tabaković-Tošić, Marija Milosavljević

Abstract: In some forest areas of Central Serbia, the entomopathogenic fungus Entomophaga aulicae was first discovered in 2015. In the literature, this species is referred as entomopathogen of browntail moth larvae, and there is no data about its pathogenicity for other development stages, primarily for pupae.
The studies were conducted during the growing season in the period 2015-2016, in some broadleaved forests in Novi Pazar region. A detailed quantitative and qualitative analysis of sampled browntail moth Euproctis chrysorrhoea caterpillars litters were conducted at the laboratory of the Institute of Forestry. In the litters, there were an average of 9.7 pupae, of which 15% were alive, 23% were parasites by species from order Diptera, and 62% dead (causing ocular invisible). By the detailed microscope survey of the causes of the mortality of the browntail moth pupae, the presence of hyphal bodies, primary conidia and resting spores of the entomopathogenic fungus E. aulicae were confirmed in them. As entomopathogenic fungus on two development stages of the host, larvae and pupae, presented results indicate that E. aulicae is a promising microbial control agent against the browntail moth in some broadleaf forests and orchards in central part of Serbia.


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Potential of anamorphic entomopathogenic fungi and biological products on their base for the control of greenhouse whitefly Trialeurodes vaporariorum West. (Homoptera: Aleyrodidae) in Belarus
Dmitry Voitka, Alena Yankouskaya

Abstract: Among the phytophages of the family Aleyrodidae greenhouse whitefly Trialeurodes vaporariorum West. is one of the most dangerous pests of greenhouse agrobiocenoses. The results of long-term laboratory, vegetative and field research of entomocidal activity of the mycopathogens against T. vaporariorum conducted on the base of the collection of anamorphic entomopathogenic fungi indicate their availability for application in biological control of Aleyrodidae family phytophages. Depending on the pest population number the biological efficiency of entomopathogenic fungi has made 22.6-100%. Using these entomopathogens at the initial stage of greenhouse whitefly population development allows a long-term limit of the population number. The application of the studied mycopathogens during the population number increase is also highly effective.
On the basis of high-active and technological strains of entomopathogens the biological preparations Pecilomycin-B (based on Isaria fumosorosea Wize), Boverin-BL (based on Beauveria bassiana (Bals.) Vuill), Entolek (based on Lecanicillium lecanii (Zimm.) Zare & W. Gams) have been developed. These products have passed through the state registration procedure and have been approved for use in agriculture.


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Metarhizium brunneum BIPESCO 5: a sustainable and preventive biological control agent to control Diabrotica virgifera virgifera larvae
Maria Zottele, Roland Zelger, Hermann Strasser

Abstract: The fungal biocontrol agent GranMet-GRTM containing Metarhizium brunneum BIPESCO 5 is used against larvae of the maize pest Diabrotica virgifera virgifera. The product was used for a preventive application in maize fields in Styria (Dietzen, Laafeld) and Tyrol (Oberndorf, St. Johann in Tirol, Austria). Until the end of the planting season 2016 the Metarhizium spp. abundance was significantly raised in three of four treatment sites (field size: 2 to 12 ha). With a SSR-PCR based molecular technique the applied strain BIPESCO 5 could be identified out of 14 different genotypes isolated from all soil samples (n = 192). In Styria fewer emerging Diabrotica adult beetles were caught compared to the previous years. No damage of maize plants was detected in Laafeld, less than 10% lodging was assessed in the treated field in Dietzen. In the untreated control field more than 80% lodging occurred with a yield reduction of more than 30% (- 4.2 t/ha). In Tyrol no emerging beetles got trapped but a high number of D. v. virgifera was assessed on PAL sticky traps for the first time x = 259.83 beetles per trap; n = 6).


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Entomopathogenic nematodes to control Sciarid flies in mushroom-cropping systems of Georgia
Mariam Chubinishvili, Manana Kakhadze, Tsisia Chkhubianishvili, Iatamze Malania, Rusudan Skhirtladze, Irine Rizhamadze, Mikola Butirin

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Exon-intron structure and sequence variation of the hsp-90 gene in the entomopathogenic nematode Heterorhabditis bacteriophora
Elena Fanelli, Monica Oreste, Francesco Capriglia, Alberto Troccoli, Eustachio Tarasco, Francesca De Luca

Abstract: HSP90 is a highly conserved molecule across all species, from bacteria to humans. It plays critical roles in the development, survival, chaperone function, cell cycle control, oncogenic transformation and also in response to abiotic stresses as heat or cold, or the presence of heavy metals. Little is known about biochemical and molecular responses to cold and heat stresses in entomopathogenic nematodes (EPN). In the present study, the isolation and the molecular characterization of the partial cDNA of the hsp-90 and its corresponding gene from the entomopathogenic nematode Heterorhabditis bacteriophora are reported. Four cDNA variants of the Hb-hsp-90 gene were isolated and sequenced. The unexpected finding of intron presence-absence in the Hb-hsp-90 genes of H. bacteriophora opens some questions about their function and their evolutionary significance.


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Challenges to the implementation of microbial pesticides with emphasis on entomopathogenic nematodes as biocontrol agents in Iran
Javad Karimi, Shokoofeh Kamali

Abstract: Iranian agriculture is pesticide-dependent and pest control usually involves chemical products. This extensive pesticide use is increasingly raising health, safety, and environmental concerns while at the same market demand for organic products is growing. Strong negative feedbacks to synthetic pesticide use have led to a tendency among Iranian farmers for using IPM approach by emphasis on biological control application in recent years. Biological control against agrarian pest in the country is a practice that started in 1930s. Work on microbial biopesticides in Iran has recently increased rapidly, and about 20 biopesticide products are being produced by companies. However, no registered products based on entomopathogenic nematodes are currently available. Here we will discuss about the current status of the work in term of research issue. Major challenges facing the progress of microbial pesticides use implement in the country will be another part of the presentation. Finally, some suggestions are made for the improvement of nematodes and other microbial insecticides for biological control of insect pest within IPM plans of the country.


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Are entomopathogenic nematodes (Steinernema and Heterorhabditis) compatible with acaricides?
Žiga Laznik, Stanislav Trdan

Abstract: Entomopathogenic nematodes (EPNs) are organisms that can be used in biological control programmes. To expand our knowledge about the compatibility of EPNs to pesticides, we studied the compatibility of EPNs infective juveniles (IJs) to five acaricides under laboratory conditions. The direct exposure of acaricides to EPNs was studied in Petri dishes at 15, 20, and 25 °C. EPNs were exposed to acaricides for 1, 4, and 24 hours. Four EPNs species were included in our investigation: Steinernema feltiae, S. carpocapsae, S. kraussei, and Heterorhabditis bacteriophora. The results of our laboratory investigation showed that H. bacteriophora was the most tolerant EPN species. On the other hand, the most sensitive EPN species was S. feltiae. Our observations showed that S. feltiae can be mixed with only two acaricides. The active ingredient fenpyroximate proved to be the most suitable for mixing with EPNs. Our results showed that fenpyroximate was only lethal to S. feltiae (44% mortality) at 25 °C. The mortality of EPNs was highest in the active ingredients abamectin and pyrethrin. Our results confirmed that the compatibility of EPNs to acaricides is a species specific characteristic. The mortality of EPNs was also influenced by the exposure time, active ingredient, and temperature. The combined use of EPNs and acaricides could represent an advantage in integrated plant protection programmes. Combinations of EPNs and acaricides could save time and money in the simultaneous control of various pests (insects, mites) in glasshouses.


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Susceptibility of the cutworm, Agrotis segetum (Lepidoptera: Noctuidae) to entomopathogenic nematodes
Manana Lortkipanidze, Oleg Gorgadze, Vugar Babayev, Nick Nwolisa, Nino Gabroshvili

Abstract: The cutworm, Agrotis segetum Schiff. (Lepidoptera: Noctuidae) is a species widely spread in Europe that damages the cultivated plants belonging to more than 15 families and including host plants such as okra, cabbage, cauliflower, rutabaga, bell pepper, tomato, potato, maize and cotton. The effect of entomopathogenic nematodes (EPNs), Heterorhabditis bacteriophora and a new species from Georgia, Steinernema thesami was investigated in the last instar of cutworm larvae and pupae in the laboratory conditions. The nematodes were used in the following doses: 50 and 100 infective juveniles (IJs) per insect. The mortality of tested insects was estimated on the third day after the EPN application. The last instar of larvae turned out to be more susceptible in the course of laboratory experiments. No significant differences were observed between H. bacteriophora and S. thesami at a low concentration of 50 IJs per insect during all exposure times, whereas S. thesami was more pathogenic against larvae at a high concentration of 100 IJs per insect as compared with H. bacteriophora and the mortality reached 97.2-100%. The emerging IJs were harvested and counted throughout the interval of 11-15 days. The experiments were carried out in the laboratory conditions at a temperature of 22 °C and 80% RH.


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Compatibility of bio-nematicide and plant stimulant of microbial origin with Heterorhabditis bacteriophora
Ivana Majić, Ankica Sarajlić, Tamás Lakatos, Tímea Tóth, Emilija Raspudić, Gabriella Kanižai Šarić, Žiga Laznik

Abstract: Entomopathogenic nematodes (EPN) are often combined with plant stimulants, inorganic and organic plant fertilizers in the integrated pest management. Previous studies mostly evaluated a compatibility of chemical pesticides and fertilizers with EPN. We evaluated the compatibility of bio-nematicide and plant stimulant of microbial origin on viability of Heterorhabditis bacteriophora infective juveniles (IJs) under controlled conditions. The viability of the IJs of H. bacteriophora was determined 1, 3, 24 and 72 hours after direct exposure to bio-nematicide and plant stimulant used in their commercial formulations. To assess infectivity, IJs were tested for their virulence against Achroia grisella larvae for 96 h. We found that bio-nematicide was highly toxic to H. bacteriophora. Three hours after direct exposure to bio-nematicide IJs were paralyzed, and level of mortality was 100% after 24 h, respectively. The plant stimulant did not affect nematode viability or infectivity. Our results demonstrate that H. bacteriophora is highly susceptible, intolerant to bio-nematicide, and compatible with the plant stimulant with no loss in viability and infectivity up to 72 h of exposure.


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The assessment of the effect of entomopathogenic nematodes on bulb mites, pests of garlic and onion
Jiří Nermuť, Vladimír Půža, Rostislav Zemek, Zdeněk Mráček

Abstract: In the present study the screening of entomopathogenic nematodes in the fields of onion and garlic specialized farms in the Czech Republic and Israel was performed and more than thirty strains of Steinernema and Heterorhabditis nematodes were isolated. Selected strains were tested for infectivity and pathogenicity against garlic and onion pest mites (Rhizoglyphus) and the potential of the use of EPNs against bulb mites of the genus Rhizoglyphus was assessed.


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Diversity of mollusc-parasitic nematodes of the genus Phasmarhabditis. How to identify them?
Jiří Nermuť, Vladimír Půža, Zdeněk Mráček

Abstract: As the mollusc pests cause increasing damage in agriculture and horticulture parasites of molluscs are getting much attention. Phasmarhabditis nematodes as one of the most common slug and snail parasites do not fall behind. P. hermaphrodita is the best known mollusc-parasitic nematode of the family Rhabditidae (Nematoda). However during the last few years several new species from different parts of the world have been discovered and described. Only during the last year four descriptions were published with two species originating from the Czech Republic, one from Italy and one from the USA. Up to these days (January 2017) we know nine species and other new species will probably accrue in the close future.


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Management of leaf curl plum aphid, Brachycaudus helichrysi (Hemiptera: Aphididae) by entomopathogenic nematodes
Nona Mikaia

Abstract: The aim of this study was to determine the biological control effect of entomopathogenic nematode species, Steinernema carpocapsae, Steinernema feltiae and Heterorhabditis bacteriophora against the adult stage of LCPA (Leaf curl plum aphid). Experiments were evaluated under laboratory conditions. The tests were conducted in 10 cm Petri dishes lined with a moistened filter paper. One infested plum leaf containing 100-120 LCPA adults was placed in each Petri dish and the nematodes were applied as 50, 100 or 150 infective juveniles (IJs)/ml per cm2. Plates were incubated at 15, 20 and 25 °C and the insect mortality was checked 24, 48, 72 and 96 hours after the treatment. Ten Petri dishes were used for each nematode concentration and temperature. The results showed that entomopathogenic nematodes were quite effective against LCPA and the mortality was related to nematode concentration and temperature. At 15 °C, S. carpocapsae, S. feltiae and H. bacteriophora exhibited 59, 24 and 23% mortality, respectively. S. carpocapsae showed the highest mortality (79%) following S. feltiae (58%) and H. bacteriophora (32%). For all nematode species, the highest virulence was observed 85%, 69% and 58% on the temperature 25 °C and 150 IJs/ml cm2 concentration for S. carpocapsae, S. feltiae and H. bacteriophora, respectively. It was determined that LCPA can be controlled by S. carpocapsae, but further studies should be conducted under greenhouse and field conditions.


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Entomopathogenic nematodes can replace soil insecticides in western corn rootworm control
Stefan Toepfer , Michael Zellner

Abstract: In an attempt to replace insecticides against the maize-root feeding larvae of the western corn rootworm (Diabrotica virgifera virgifera, Coleoptera: Chrysomelidae), biological control solutions based on entomopathogenic nematodes have been developed. Currently, the fluid application of the living nematodes into the sowing furrow is the most developed and validated application technique. This study investigated whether fluid applications of Heterorhabditis bacteriophora (Nematoda: Rhabditida) can be an alternative to older as well as recently registered synthetic soil insecticides in the management of established D. v. virgifera pest populations in maize. Five field trials were therefore implemented in southern Hungary between 2013 and 2015 using growers` machinery. Results revealed that all agents tested, regardless of biological or chemical, were able to reduce adult emergence of D. v. virgifera; but none is highly effective. However, entomopathogenic nematodes can, if applied at a relatively high dosage, be as effective as the synthetic soil insecticides in controlling the larvae. All agents were in most occasions also able to significantly prevent some of the root damage caused by D. v. virgifera larvae. As for economic root damage (node injury scale data), Tefluthrin soil granules best prevented such heavy root damage followed by the nematodes; whereas Cypermethrin and Chlorpyrifos soil granules were usually not able to prevent heavy root damage. Nematode treatments as well as soil insecticides slightly increased yield. In conclusion, the recommended commercial dose of 2 billion nematodes per hectare appeared likely to be enough for pest management in most cases; this is, keeping D. v. virgifera and damage below thresholds. This is, however, only true when nematodes are correctly applied and when D. v. virgifera infestations are significant. To assure a higher security in treatment efficacy across locations, conditions, and different grower skill-levels, a higher dose might be applied. Findings support a nematode-based solution for the biological control of D. v. virgifera larvae in maize fields in European regions as one among the alternative options to replace synthetic insecticides.


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Microbial and nematode control of the Colorado potato beetle
Rostislav Zemek, Jana Konopická, Vladimír Půža, Andrea Bohatá, Hany M. Hussein, Oxana Skoková Habuštová

Abstract: The Colorado potato beetle, Leptinotarsa decemlineata, is the most widespread and best-known insect pest that causes great economic losses especially on potatoes. As a result of the intensive use of insecticides this species has gradually developed resistance to most pesticides and its regulation is thus currently very difficult. In addition to the use of disputable genetically modified crops as a promising solution to the problem of pest resistance, development of biocontrol methods using natural pathogens might be solution for sustainable potato production. The aim of our study was to assess the efficacy of entomopathogenic fungus Isaria fumosorosea 163-strain CCM 8367 and entomopathogenic nematode Steinernema feltiae strain Ustinov against L. decemlineata under laboratory conditions. Petri dish trials revealed the highest susceptibility in the last-instar larvae followed by pre-pupae and pupae. The median lethal concentration (LC50) of I. fumosorosea was estimated to be 1.03 × 106 blastospores/ml. Simultaneous application of the fungus with the nematodes increased the mortality of L. decemlineata larvae up to 98% and shortened the median lethal time to two days while no obvious changes in development of nematodes in cadavers were found. When, however, nematodes were applied more than 24 hours after fungus treatment, their development was negatively affected and adults were smaller in comparison to control. In soil application experiments, standard soil substrate was inoculated by either I. fumosorosea, S. feltiae or both before last instar larvae, which finished feeding and searched for place to pupate, were placed individually into the pots. The uncorrected mortality of L. decemlineata was 44% and 45% when 1 × 108 blastospores of I. fumosorosea and 1000 IJ of S. feltiae were applied to the pot, respectively. Combined application of the fungus and the nematode at the same doses increased mortality to 84%. We can conclude that both entomopathogens are prospective biocontrol agents against L. decemlineata and that they could be applied together for higher efficacy.


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Measuring the fitness cost of type I resistance breaking by CpGV
Benoit Graillot, Christine Blachère-Lopez, Samantha Besse, Myriam Siegwart, Miguel Lopez-Ferber

Abstract: In a previous work we have measured virus productivity and pathogenicity of two virus isolates:CpGV-M, and CpGV-R5 (a type I resistance breaking isolate). We were not able to find significative differences between them. Another way of testing fitness differences is to analyse genotype replacement in mixed genotype experimental populations over successive generations. We constructed five experimental populations with various proportions of each isolate and followed them through six succesive passages on insects. In absence of selection, that is when replicating in an insect colony susceptible to both virus isolates, the ability to kill resistant larvae is not lost. After 6 passages, the pathogenicities of the 5 experimental virus populations on the resistant insect colony RGV converge to a common level χ2 = 13.35, 8 dof, P > 0.05). In addition, markers specific to both isolates are detected. Our results suggest again that, at least in laboratory conditions, the cost for the virus to break type I resistance -if it exists- is under the level of detection. All these observations, on the virus and on the host, suggest the existence of multiple choices with no cost for the players (virus and insect).


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Baculovirus synergy: mixed Alphabaculovirus and Betabaculovirus infections for the control of Thaumatotibia leucotreta in South Africa
Michael D. Jukes, Lukasz Rabalski, Caroline M. Knox, Sean D. Moore, Martin P. Hill, Boguslaw Szewczyk

Abstract: The recent identification of a nucleopolyhedrovirus (NPV) capable of establishing infection in three major pests, namely Cryptophlebia peltastica (litchi moth), Thaumatotibia leucotreta (false codling moth) and Cydia pomonella (codling moth) provides an opportunity for improved methods such as synergism for control of agricultural pests in South Africa. Genetic characterisation identified the NPV as Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV), a newly identified Alphabaculovirus. In this study, we report the development of a multiplex PCR assay which can distinguish Cryptophlebia leucotreta granulovirus (CrleGV) and CrpeNPV in mixed samples. Furthermore, the biological activity of CrpeNPV and CrleGV-SA against FCM larvae was evaluated both in isolation and in combinations at ratios of 3:1 and 1:3. The LC50 values for all treatments varied only slightly, ranging from 7.95 × 104 to 1.23 × 105 OBs/ml. At the LC90, a combination of CrleGV and CrpeNPV showed a three- to four-fold improvement in virulence compared to pure viruses. Trials are currently underway to evaluate the effect of mixed CrleGV/CrpeNPV applications under field conditions.


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The isolation of a novel alphabaculovirus and its potential for microbial control of key tortricid moth pests
Tamryn Marsberg, Michael Jukes, Craig Chambers, Christiaan Hendriks, Caroline Knox, Martin Hill, Sean Moore

Abstract: A laboratory culture of Cryptophlebia peltastica, a major pest of litchis in southern Africa, was established for the first time. During the rearing of C. peltastica, larvae showing symptoms of baculovirus infection were collected and analysed. Occlusion bodies (OBs) were purified from the symptomatic larvae and observed by transmission electron microscopy to be a nucleopolyhedrovirus. Genetic characterisation through creating restriction profiles and sequencing the whole genome, revealed that this was a novel alphabaculovirus, with no close relative, and was thus named the Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV). This is the first known record of an NPV naturally infecting any species within the Grapholitini tribe of the Tortricidae. Bioassays were used to determine the virulence of CrpeNPV against C. peltastica, Thaumatotibia leucotreta (false codling moth) and Cydia pomonella (codling moth). The LC90 of CrpeNPV for neonate larvae of these three species was respectively, 3.33 × 105, 9.97 × 104 and 1.26 × 104 OBs/ml. The average speed of kill to cause 90% mortality of each species was 5 days. As the bioassay results indicated superior virulence against T. leucotreta and C. pomonella than against the homologous host, C. peltastica, field trials were conducted against T. leucotreta in citrus and C. pomonella in apples at rates of between 5 × 1011 and 5 × 1013 OBs/ha. Very promising results were achieved. Consequently, CrpeNPV is considered to have significant potential for effective biological control of these, and potentially other, key tortricid pests of agriculture.


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Including baculoviruses in insect control strategies – opportunities & challenges
Silvana Niedermann, Silvio Crameri, Eric T. Natwick, Heiri Wandeler

Abstract: Baculovirus-based insecticides have rarely been included in integrated pest management (IPM) in open-field vegetable production. However, due to the unique mode of action and its efficacy, baculovirus-based insecticides have great potential for IPM, especially for resistance management strategies. In close collaboration with researchers and distributors, Andermatt Biocontrol is developing strategies, which allow using baculovirus (BV) products for the control of pests in complexes. Field trials are performed in the USA with the two products Spexit® (SeNPV) and Loopex® (AcNPV), applied alone or in combination. In fresh market tomato, a high dose of Spexit® as well as a low dose of Spexit® in tankmix with Bta showed the best results with around 70% efficacy for beet armyworm control. Furthermore, Spexit® was an excellent rotation partner for spinosad. In a second trial with Loopex® on broccoli, efficient cabbage looper (Trichoplusia ni) control was found. The development of late larval instars, which are mainly responsible for feeding damage, was successfully avoided. Likewise, good efficacy of Loopex® in combination with Btk was shown, also controlling secondary pest. Both products Spexit® and Loopex® were applied in a third trial in cabbage in combination with organic and conventional products. Results show that NPV’s are valuable tools for IPM production systems with similar efficacy to growers’ standard.


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Effect of temperature on long-term storage of Cydia pomonella Granulovirus (CpGV-M)
Annette J. Sauer, Johannes A. Jehle

Abstract: Codling moth (CM) is the major pest in apple and pear production worldwide. The application of Cydia pomonella granulovirus (CpGV) products is an ecologically safe alternative to chemical treatments to control CM. In this study the long-term activity of occlusion bodies (OBs) of the commonly used CpGV-M isolate, stored at room temperature (RT), +4 °C, -20 °C, and -80 °C for five years was assessed. Median lethal concentration (LC50) of the tested OB suspensions was elucidated in full-range bioassays with neonates of the susceptible CM strain CpS after seven and 14 days. The LC50 values at +4 °C, -20 °C and -80 °C storage temperature were between 225 OBs/ml (4 °C) and 7991 OBs/ml (-20 °C) after seven days and comparable to the LC50 determined at the beginning of the storage test (LC50 1871 OB/ml). Only the virus-induced mortality of OBs stored at RT decreased that the LC50 could not be determined. This research indicates that the storage of OBs of CpGV-M at 4 °C or colder will ensure the bioactivity for at least five years.


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Integrated pest and resistance management of Helicoverpa zea (Lepidoptera: Noctuidae) with a Helicoverpa armigera nucleopolyhedrovirus
Silvana Niedermann, Silvio Crameri, Brian Jensen, Marcel Tanner

Abstract: Andermatt Biocontrol is developing strategies to use Helicovex® for the control of Helicoverpa zea, a key pest in sweet corn. Field trials are performed in the USA to compare Helicovex® in different doses, stand-alone and in combination with grower’s standard products. In the first trial Helicovex® showed significant better protection than the pyrethroid-based product which suggests the presence of pyrethroid-resistant larvae. Even though pest pressure was exceptionally high, Helicovex® could almost double the amount of undamaged ears. For resistance management, Helicovex® can also be applied in rotation with pyrethroids, indicated in the second trial. There is no significant difference between stand-alone pyrethroid and the rotation of the pyrethroid and Helicovex®. In a third trial a Bta-based product and Helicovex® were compared. Helicovex® had significantly more marketable ears (76.8%) than the Bta (50%). The findings underline that with prevalent resistances to pyrethroids and other insecticides, Helicovex® is an essential tool for resistance management in organic and IPM production systems and may be applied in rotation with grower standard insecticides.


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