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IOBC-WPRS Bulletin Vol. 113, 2016


IOBC-WPRS Bulletin Vol. 113, 2016

Working Group "Microbial and Nematode Control of Invertebrate Pests".
Proceedings of the Meeting "New Challenges for Biological Control" at Riga (Latvia), June 7 - 11, 2015.
Edited by Johannes A. Jehle, Liga Jankevica, Neil Crickmore, Vladimír Půža, Enrique Quesada-Moraga, Oihane Simón De Goñi, Michael Traugott, Jörg Wennmann.
ISBN 978-92-9067-296-8 [XVIII + 171 pp.]


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Influence of multi-year Bacillus thuringiensis subsp. israelensis treatments on the abundance of B. cereus group populations in Swedish riparian wetland soils
Salome Schneider, Tania Tajrin, Niels B. Hendriksen, Jan O. Lundström, Petter Melin, Ingvar Sundh

Abstract: Bacillus thuringiensis subsp. israelensis (Bti) is a soil-borne bacterium affiliated to the B. cereus group (Bcg, a group including the pathogens B. cereus, B. thuringiensis, and B. anthracis) and is used in biocontrol products against nematoceran larvae. However, knowledge is limited on how long-term Bti application affects the structure of indigenous Bcg communities as well as the overall abundance of Bti. Based on new primers, group-specific quantitative PCR assays for Bcg and Bti in environmental samples were developed. On six occasions during the vegetation season, soil samples were collected in forest swamps and wet meadows which had been treated with Bti during the preceding 11 years as well as in untreated forest swamps, wet meadows and well-drained forests. Abundances of Bcg and Bti varied among the different sampling occasions. The highest abundance of Bcg was found in forest swamps and differed significantly from wet meadows while no such variation was found in Bti abundance. The Bti treatments had no effect on the overall Bcg abundance whereas for Bti, the abundances were significantly higher in the treated than in the untreated sites. However, abundances of Bti and Bcg didn’t correlate with the number of Bti applications, indicating that Bti use influenced abundances of Bti in the short term while in the long term the number of treatments had only a limited effect. The findings illustrate the value of such investigations for understanding the ecology of Bti applications, which can facilitate environmental risk assessment as well as approval of biological control agents.


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Bacillus associated with Colorado Potato Beetle – Leptinotarsa decemlineata Say – and the Mottled Umber – Erannis defoliaria Clerck – in Georgia
Medea Burjanadze, Dali Gaganidze, Mariam Arjevanidze, Elena Nakaidze, Giuli Tsereteli, Shorena Kharadze

Abstract: For the identification of endemic species of Bacillus in agricultural and forest ecosystems of Georgia, infected and healthy individuals of Colorado Potato Beetle – Leptinotarsa decemlineata Say – and caterpillars of the Mottled Umber – Erannis defoliaria Clerck – were collected at different larval stages. Ten of the 20 isolates [BZ1, BZ2, BZ3, KM1, KM2, KM3, KM4, KM5, KM5 (1), M5 (2)] were gram-positive. For the evaluation of spore formation isolates were cultivated in nutrient broth and on Selective media and were analyzed under the microscope. For the purpose of establishing the formation of crystal proteins the proteinaceous range of gram-positive isolates was studied by SDS-PAGE. Isolates BZ1, BZ2, KM1, KM2, KM3, KM4, KM5 were found to have a high protein content which ranged in size between 130 kDa and 66 kDa. Microscopic analysis revealed the existence of spores in KM2, KM3 and KM5 isolates. For the purpose of identifying crystal inclusions these isolates were transferred onto selective media. A 24-hour microscopic analysis of culture didn't show the existence of any crystal inclusions.


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Host group adaptation of “Candidatus Rickettsiella isopodorum”, a lineage of intracellular gamma-proteobacterial pathogens carried by woodlice
Andreas Leclerque, Regina G. Kleespies

Abstract: The taxonomic genus Rickettsiella (Gammaproteobacteria; Legionellales) comprises intracellular bacteria associated with a wide range of arthropods including insects, arachnids and crustaceans that are currently under intensive evaluation as possible sources of new biocontrol agents. A multilocus sequence analysis approach has been employed to independently evaluate the previously established 16S rRNA based phylogeny for two Rickettsiella strains associated with isopods from California and Germany. Phylogenetic reconstruction from three protein-encoding marker genes, namely ftsY, gidA, and sucB, confirms the very tight phylogenetic relationship of Rickettsiella bacteria from closely related hosts, but distant geographic origins. These findings are consistent with stable host group – pathogen relationships within the Rickettsiella.


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Effectiveness of antimicrobial compounds produced by entomopathogenic nematode symbiotic bacteria to control pests and bacterial plant diseases
Dávid Vozik, Katalin Bélafi-Bakó, Mária Hevesi, Erzsébet Böszörményi, Zsolt Polgár, András Fodor

Abstract: Entomopathogenic nematode-bacterium symbiotic complexes (EPN-EPB) provide several options for environmentally friendly biological pest control. In the last decades EPN formulations become commercially available for agricultural applications. Relatively less attention was dedicated to the antimicrobial potential of the symbiotic bacteria. The role of the EPB partner is essential for the successful colonization of the insect host cadaver via their production of antibiotics and hydrolytic exoenzymes, and processes the insect’s cadaver into utilisable nutrient for the nematodes. The broad-spectrum antibiotics of peptide nature produced by EPB non-ribosomally keep monoxenic conditions in insect cadavers in soil. These compounds show significant antagonistic activity against numerous plant pathogenic bacteria and fungi in vitro. Potato wilt caused by Ralstonia solanacearum is one of the most challenging plant diseases in several countries, including Hungary. In this project we established some parameters needed prior to experimental field application of antibacterial substances from EPB strains Xenorhabdus budapestensis (EMA) and X. szentirmaii (EMC) in Ralstonia control. We quantitatively determined the (i) optimum inoculum size needed for successful Ralstonia infection; (ii) the minimum phytotoxic concentration and (iii) the minimal inhibitory concentration of EMA cell free conditioned medium (CFCM). The bactericide effect of EMA CFCM against Ralstonia solanacearum test organism was determined under in vitro circumstances. At the light of the results we consider the antibacterial component(s) of EMA CFCM as potential tool(s) of Ralstonia control.


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Analysis of the bacterial community present in the insect pest Lymantria dispar during the life cycle of the insect
Zane Metla, Monika Maurhofer, Liga Jankevica

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The role of entomopathogenic fungi in the control of citrus pests in South Africa: cause for optimism
Candice Coombes, Veronique Chartier-Fizgerald, Danielle Wiblin, Jo Dames, Martin Hill and Sean Moore

Abstract: Citrus is a highly productive crop in South Africa, but it is damaged by a number of pests that result in yield loss and have the potential to limit market access. Maximum residue limits (MRLs) imposed by importing countries have driven the need for alternative control technologies, including the use of entomopathogenic fungi (EPF). Bioprospecting in citrus orchards and seven years of bioassay trials identified Beauveria bassiana G Ar 17 B3 and Metarhizium anisopliae G 11 3 L6 and FCM Ar 23 B3 as the most virulent fungal isolates. Preliminary trials with these three fungi against the arboreal pests, California red scale, Aonidiella aurantii (Maskell), citrus mealybug, Planococcus citri (Risso) and citrus thrips, Scirtothrips aurantii Faure, have been promising. False codling moth (FCM), Thaumatotibia leucotreta (Meyrick) is the most important citrus pest in South Africa and while considerable research has been conducted on controlling the insect on the tree, the soil borne stages of this insect have largely been ignored. After laboratory bioassays, the three fungal isolates were taken to field trials. All three isolates persisted for at least six months after application to the soil. A large scale field trial, showed that although all three isolates reduced FCM infestation, isolate B. bassiana G Ar 17 B3 performed best, recording a consistent 80% reduction in FCM infestation throughout the trial period. The results of nearly 10 years of research on the potential of EPFs in the control of citrus pests in South Africa are certainly cause for optimism.


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Endophytic Metarhizium strains against herbivorous insect pests: development of novel fermentation and formulation strategies
Desiree Jakobs-Schönwandt, Vivien Krell, Stefan Vidal and Anant V. Patel

Abstract: Biocontrol of insect pests with entomopathogenic fungi is challenging because of the lower efficacy, difficult handling and limited shelf life of these organisms compared to synthetic pesticides. However, recent studies have provided evidence that some of these fungi can grow endophytically in plant tissues, paving the way for novel plant protection measures. One strategy could be the use of the endophytic entomopathogenic fungus Metarhizium brunneum isolate F52. For application as a commercial biocontrol agent, the fungus has to be mass-produced. M. brunneum was cultivated in shake flasks in media based on agricultural residues to produce submerged spores to a maximum of 1.15 ± 0.06x107 spores/ml. By the use of UV protectants, the viability of submerged spores was significantly increased by 71.6 ± 12.1% after a UV-B radiation of 120 min. Submerged spores were formulated in a novel spray that delivered the fungus on tomato leaves and allowed penetration of the leaves verified by a polyphasic approach using light microscopy and PCR.


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Biological control of pollen beetles with the entomopathogenic fungus Beauveria bassiana formulated in oil
Deborah Kaiser, S. Bacher, Giselher Grabenweger

Abstract: Pollen beetles (Meligethes spp.) are a major pest in oilseed rape throughout Europe, able to cause substantial yield loss. A laboratory screening of isolates of entomopathogenic fungi revealed high potential of Beauveria bassiana isolates for biological pollen beetle control. To improve the efficacy of entomopathogenic fungi in field applications, we explored synergistic interactions between submerse spores of Beauveria bassiana and other natural compounds that have been shown to reduce pollen beetle abundance in oilseed rape, such as stone dusts or vegetable oils. The combined application of Beauveria bassiana submerse spores and an oil formulation in a spray chamber showed an increase in pollen beetle mortality of up to 40%, relative to single treatments. This was 11% more than expected by mere addition of the effects of single-treatments, suggesting a synergistic interaction between the fungal submerse spores and the oil formulation. The latter seems to foster the fungus’ reproductive phase since twice as many mycosed pollen beetles were recorded in the combined treatment.


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Occurrence and characterization of entomopathogenic fungi from the complex of sugarcane borer Diatraea spp. in Colombia
Paula Sotelo, Paola Cuartas, Gloria Barrera, Adriana Santos, Emiliano Barreto, Laura Villamizar

Abstract: Panela is a solid piece of unrefined sucrose obtained from evaporation of sugarcane juice, a very important industry and source of employment in Colombia. Panela yield depends of sucrose content in sugarcane, characteristic seriously affected by the presence of the stem borers’ complex, difficultly controlled by chemical insecticides. Biological control by using microorganisms is an interesting tool for controlling this pest, which has been poor developed. The aim of this work was to isolate and characterize entomopathogenic fungi naturally infecting Diatraea spp. larvae in sugarcane crops for panela production in Colombia. Larvae were collected from three different production areas and maintained in quarantine until dead. A total of 445 larvae were collected and 23 presented fungal growth after dead. One isolate of Beauveria sp. (Bc3j-22) was obtained, which caused 83.3% and 73.3% of mortality of Diatraea indiginella and Diatraea saccharalis larvae, respectively. Sequence analysis of three genomic regions (rDNA 18S, ITS and EF1-α) allowed to classify Bc3j-22 isolate as Beauveria bassiana. This isolate showed high susceptibility to ultraviolet radiation type B (900 μW/cm2), with 33%, 49% and 81% of inactivation after 30, 40 y 50 minutes of irradiation, respectively. Optimal conditions for fungal growth were 25 °C and pH 7.0. Isolate Bc3j-22 showed high potential for controlling different species of the sugarcane borer and constitute the base for the development of a new biopesticide.


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Encapsulation of Metarhizium brunneum as basis for an attract and kill strategy within the EU project INBIOSOIL
Miriam Hanitzsch, Michael Przyklenk, Anant Patel

Abstract: The EU funded project INBIOSOIL aims at developing innovative beads containing entomopathogenic fungi (EPFs) as well as novel synergistic co-formulations of EPFs with efficacy enhancing agents to control soil-borne insect pests. We developed an EPF formulation with 80% spore survival after drying on lab scale and technical scale. Furthermore, we developed a co-formulation that is based on biopolymer beads or granules combining CO2 releasing baker’s yeast, an effective attractant compound for various insect pests, with M. brunneum aerospores (BIPESCO5 or ART 2825) as a kill component. Both components were encapsulated separately as well as co-encapsulated to implement an “attract and kill”-strategy to control wireworms and other soil-borne insect pests.


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Attract and kill kept simple: Fungus colonized barley kernels in cover crops for microbial wireworm control
Sina Rogge, Giselher Grabenweger

Abstract: The multiannual belowground development of wireworms is characterized by a sequence of active and inactive phases. During the former, wireworms are found feeding and foraging in upper soil levels. It follows that a control measures against wireworms may hit its target only when applied during active phases of the pest insect.
Wireworms typically show two peaks of activity per year, one in springtime and a second one in late summer. Considering this seasonal activity, we developed a simple “attract and kill” approach, which was tested in a semi-field pot experiment. Metarhizium-colonized, sterile barley kernels were applied into the upper substrate layers of the pots in late summer, immediately before sowing of summer oat as a cover crop. The CO2-emitting roots of the oat seedlings should attract artificially released late instar larvae of Agriotes obscurus into fungus infested upper substrate layers during their late summer activity peak. After hibernation in deeper substrate layers, wireworms were assumed to migrate upwards again during springtime, facing the risk of getting infected a second time, right before planting of potatoes as the main crop. Establishment of the fungus was estimated by counting colony forming units in substrate samples. The efficacy of the control measure was evaluated by assessing the mortality of recaptured wireworms from the pots. We were able to confirm establishment of the fungus in the substrate after a few weeks and to show up to 70% of wireworm mortality, depending on fungus inoculum dose applied. The simple yet promising biocontrol strategy against wireworms is currently tested in a follow-up experiment under field conditions.


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Group-I intron based strain-specific diagnosis of entomopathogenic Lecanicillium fungi for aphid biocontrol
Christina Schuster, Katharina Saar, Romina Manfrino, Juan Aguilera Sammaritano, Julieta Tornesello Galván, Juan J. García, Claudia C. López Lastra, Andreas Leclerque

Abstract: Mitosporic fungal entomopathogens of the genus Lecanicillium (Ascomycota; Hypocreales) are of particular interest as biological control agents for phloem-sucking plant pests including aphids. Bioprospection for these fungi in Argentina has given rise to a set of isolates from a wide range of hosts. Molecular taxonomic analysis using a Multilocus Sequence Typing approach has confirmed previous morphology based assignments to the genus Lecanicillium.
For further research in Lecanicillium fungi and development of a bio-aphicide, a PCR-based diagnostic tool allowing the reliable and fast differentiation of strain CEP419 from the further Argentine isolates was highly solicited. Screening of this full set of fungi and additional reference strains from other geographic origins 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 for the strain of particular interest. In contrast to all other Lecanicillium isolates investigated, the rRNA genes of CEP419 were found to comprise at least two group-I introns each. These findings were exploited in the development a double identification assay for CEP419. Primer pairs hybridizing against the 18S and 28S rRNA intron sequences, respectively, were designed and used to amplify partial rRNA gene sequences in a strain specific manner. Each of both diagnostic PCR reactions alone unambiguously identified strain CEP419 across the full set of Lecanicillium isolates investigated. In conclusion, the feasibility of strain-specific identification based on group-I intron sequences has been demonstrated for a potential biocontrol strain of Lecanicillium.


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Entomophaga maimaiga caused the crash of the gypsy moth outbreak in the forests of Central Serbia in the 2014
Mara Tabaković-Tošić

Abstract: Entomophaga maimaiga Hamber, Shimazu & Soper is not a native entomopathogenic fungus in Europe. In 1999, it was introduced for the first time in Bulgaria. Recent data suggest that E. maimaiga is spreading across Europe. Since 2011 the fungus has been found in several other European countries. First time this fungus was reported in the European part of Turkey in 2011 and in the same year it was also found in Serbia. During the culmination phases (2014) of the latest outbreak of the gypsy moth in the central Serbia, the greatest area (339,989 hectares) was subject to the very high infection rates. The weather plays an important role in the anticipation of the effectiveness of E. maimaiga. April and May 2014 were the favourable months to the germination of the azygospores and to the infection of the gypsy moth larvae which caused the massive epizootics and mortality of gypsy moth larvae (L2-L4 stage). By the microscopic analysis of the dead caterpillars, the presence of the conidiospores and azygospores of the E. maimaiga was confirmed.


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Metarhizium stationary surface cultivation and encapsulation
Michael Przyklenk, Lenka Capova, Miloslav Nesrsta, Miriam Hanitzsch, Hynek Stodulka, Arne Peters, Anant Patel

Extended abstract


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Testing effects of natural resin and synthetic terpenes on conidial germination and growth of entomopathogenic fungi
Ariadne Svoboda, Cezary Tkaczuk, Rudolf Wegensteiner

Abstract: The effects of natural resin and synthetic terpenes on conidial germination and growth of selected entomopathogenic fungi was tested under laboratory conditions.
Liquid resin, solidified “scrape” resin and colophony inhibited conidia germination and fungal growth of B. bassiana. All three substances inhibited conidia germination of M. anisopliae and liquid resin inhibited fungal growth of M. anisopliae, but only colophony inhibited conidia germination of I. fumosorosea, no inhibition of fungal growth were found with the latter species.
All synthetic substances (-)-α Pinene, (-)-β Pinene, Myrcene and R-Limonene inhibited germination of conidia up to 48 hours at least within the area of 10 mm next to the test substance. However, no inhibition of fungal growth was found with (-)-α Pinene and (-)-β Pinene, Myrcene caused a very small inhibition zone (1-2 mm) in B. bassiana and M. anisopliae, but only for a period of one week, R-Limonene caused a very small inhibition zone (1-2 mm) only in B. bassiana for one week; after that time no inhibition was found in fungal growth with any of the terpenes.


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Occurrence of entomopathogenic fungi in agricultural and forest soils and vermicompost samples
Lelde Grantina-Ievina

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A survey of microbial antagonists of Otiorhynchus species from Germany
Regina G. Kleespies, Peter Sprick

Abstract: Several species of the genus Otiorhynchus Germar (Coleoptera: Curculionidae) are serious polyphagous pests in horticulture. Not much is known about their natural microbial antagonists. During 2011 and 2013 a survey of naturally occurring Otiorhynchus species in Germany and their antagonists was conducted. Altogether, 3,821 beetles of 9 selected species were collected. 178 dead specimens were diagnosed by light microscopy for insect pathogens: 31 beetles contained unspecified bacterial infections, and 86 specimens displayed fungal growth with 33 infected by the entomopathogenic taxa Beauveria bassiana (15), Isaria sp. (1) and Lecanicillium sp. (17). On one beetle, a nematode infecting fungus, Arthrobotrys sp., was found, and another one carried an infection by an Entomophthoralean species. Microsporidia, a protist of the genus Mattesia, and nematodes were each observed in two dead individuals, 57 individuals showed no pathogen infections. The role of these entomopathogens as control factors in the field is still unknown.


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Testing entomopathogenic fungi against Hylobius abietis (Coleoptera, Curculionidae)
I. Kölmel, Cezary Tkaczuk & Rudolf Wegensteiner

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Development of validated analytical methods for the assessment of Metarhizium brunneum metabolites in different matrices
Judith Taibon, Sonja Sturm, Christoph Seger, Hermann Stuppner, Hermann Strasser

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Five years of experience in biocontrol of the western corn rootworm, Diabrotica virgifera virgifera, by entomoparasitic nematodes
Andreas Kahrer, Christina Pilz, Udo Heimbach, Katharina Wechselberger, Giselher Grabenweger

Abstract: Between 2009 and 2013 several field experiments were carried out in order to show efficiency and reliability of various application rates of the commercial product Dianem™ based on insect parasitic nematodes (Heterorhabditis bacteriophora). Artificial infestation of maize plants with 300 to 500 WCR eggs per plant resulted in about 40 beetles per plant emerging from soil (and was a basis for reliable results). Application of Dianem reduced populations of WCR up to 65% in some trials, however, it was less effective in root protection. In order to achieve high efficacy application rates of 2-3 x 109 nematodes/ha are necessary. Proper application of nematodes was crucial for good efficacy.


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Efficacy assessment of Heterorhabditis bacteriophora (Nematoda: Heterorhabditidae), Metarhizium brunneum (Hypocreales: Clavicipitaceae), and chemical insecticides for Diabrotica virgifera virgifera larval management under real farm conditions
Hannes Rauch, Roland Zelger, Hermann Strasser

Abstract: A two-year field study using a blend of entomopathogens in conjunction with chemical insecticides was carried out to determine to which extent they affect indigenous western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte) survival, maize root damages, and grain yield. The products applied consisted of formerly in Austria registered clothianidin dressed maize seeds (Poncho®), the currently approved products Belem® (active ingredient cypermethrin) and dianem® (nematodes of the species Heterorhabditis bacteriophora), and of two innovative products based on the insect pathogenic fungus Metarhizium brunneum. The experiments were conducted in two (2013) and four (2014) naturally heavily WCR infested maize fields in the southeast of Styria, Austria. The lowest number of WCR adults was found, when the two biological control agents were combined with clothianidin dressed maize seeds. Apart from this promising result, further IPM management strategies and operational necessities aiming to bring WCR population below its stipulated economic threshold are discussed.


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Development of complex protection of vegetable crops from T. absoluta in greenhouse conditions
M. Chubinishvili, R. Skhirtladze, M. Kakhadze, Ts. Chkhubianishvili, I. Malania, I. Rizhamadze

Abstract: A new invasive insect pest, the South American tomato moth, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), is causing the great damage (80-90%) of vegetable crops in Georgian greenhouse farming. The complex protection of tomato plants from the pest insect was elaborated by using of biological and biotechnical means. The experiments and observations at the stationary plot (200-250 m² greenhouse, village Misaktsieli, Mtskheta region) planted by Dutch tomato sort "Rose pinkinikum" and cucumber sort "Georgian Mukhranuli", were carried out. The biotechnical means – insect yellow traps for pests monitoring – were placed in greenhouse. Towards T. absoluta the new generation of highly efficient biological formulations – Proclaim (application rate of 0.4 kg/ha), Vertamectin Forte WG (application rate of 0.16 kg/ha) and entomopathogenic nematodes "Geo-nema", Steinerma feltiae Georgian strains (application rate 25 million IJs/ha) – were tested. The treatment of vegetable plants with mentioned solutions was carried out two times. The biological effectiveness (B.E.) of formulations – Proclaim and Vertamectin Forte WG – 71.8-91.2% and 72.5-82.7%, respectively, was established. The B.E. of nematode suspension was 53.2-54.1%, while in case of chemical product Aqtara (etalon) 91.1-97.8% has been achieved. The preliminary data in laboratory and natural conditions on the based of tested environmentally safe, biological and biotechnical means, has been developed for T. absoluta complex control in greenhouse farms. The elaborated approach for complex protection will take the determined and important place in the integrated pest management system (IPM) of vegetable crops from pest organisms.


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Characterization of heat shock protein 90 gene in native entomopathogenic nematodes from Southern Italy
Eustachio Tarasco, Elena Fanelli, Monica Oreste, Alberto Troccoli, Francesca De Luca

Abstract: Heat shock protein 90 (hsp90) family is an evolutionarily conserved, abundant cytosolic protein that has been shown to be involved in chaperone function and cell cycle control. It also plays an essential role in response to stress conditions such as heat or cold shock and presence of heavy metals. The partial hsp90 genes of Steinernema apuliae, S. ichnusae and Heterorhabditis bacteriophora were amplified by using degenerate primers and sequenced. Expression analysis of hsp90 gene will be determined at different temperatures by qRT-PCR.


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Nematode fauna of Rhynchophorus ferrugineus (Oliver) in Southern Italy
Eustachio Tarasco, Monica Oreste, Alberto Troccoli, Elena Fanelli, Francesca De Luca

Abstract: This study reports a survey conducted to evaluate the nematode fauna associated to the red palm weevil Rhynchophorus ferrugineus (RPW) in Southern Italy, in order to select potential biological control agents. Several infested Phoenyx canariensis exemplars were sampled in different locations, particularly in the Apulia region, collecting each stage of RPW. Insects were dissected and nematodes were isolated using the water bait method. Individual nematodes were identified by morphological and molecular traits. Results reveal that the RPW shows a specific nematode fauna, including some new species.


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Microbial control of winter moth in Georgia
Miranda Tserodze, Nikoloz Meskhi

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Can entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) function as an indirect plant defense?
Žiga Laznik, Stanislav Trdan

Abstract: In a laboratory investigation, we have tested the chemotactic responses of 4 EPN species (Steinernema and Heterorhabditis) to 6 compounds released by insect (wireworms and grubs) damaged and undamaged carrot roots. Our results indicate that all of the tested EPN species exhibited attraction (or repulsion) to volatiles, irrespective of their foraging strategy. Our current results suggest that responses to distinct volatile cues are a species-specific characteristic. Terpinolene (VOC released from undamaged roots) was a repellent for Steinernema and Heterorhabditis species in our investigation. Our results suggest that healthy plant roots release specific VOCs into the soil, which signal to natural insect enemies (EPNs) to keep away.


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Pathogenicity, penetration and reproduction of two species of entomopathogenic nematodes, Heterorhabditis bacteriophora and Steinernema carpocapsae, on pistachio root beetle larvae, Capnodis cariosa hauseri (Coleoptera: Buprestidae)
Elham Salari, Javad Karimi

Abstract: The pistachio root beetle, Capnodis cariosa hauseri causes general weakness in the pistachio trees and may even kill the infected trees. This insect was distributed in many pistachio growing regions of Iran and causes heavy damage to pistachio trees, particularly when the infection is accompanied by gummosis. Cryptic behavior of the larvae reduce efficacy of common control methods. Due to hidden habitat of pest and penetration ability of entomopathogenic nematodes (EPNs) to these habitat types of some insects, we investigated efficacy of two EPN species, Heterorhabditis bacteriophora and Steinernema carpocapsae against last instars larvae of the pest in laboratory. Pathogenicity test in petri dish were performed with five concentrations: 5, 10, 25, 50 and 100 infective juveniles (IJs) per larva. The LC50 values indicated that S. carpocapsae (12.6 IJs/larva) was comparatively more virulent than H. bacteriophora (17.1 IJs/larva) against C. cariosa larvae after 72 h. Larval mortality was significantly influenced by the EPN species and concentration. The highest mortality were recorded at the concentration of 50 IJs/larva for S. carpocapsae (97.1%). Both EPNs successfully penetrated and reproduced in the C. cariosa larvae at the concentrations of 5 and 25 IJs/larva. The highest reproduction rate was recorded for H. bacteriophora at 5 IJs/larva in C. cariosa (562,881.5 ± 7,773.1 IJs) and the highest penetration into C. cariosa was observed for S. carpocapsae at 25 IJs/larva (19 IJs). The current study demonstrated susceptibility of C. cariosa to both nematode species as well as their reproduction capability and penetration on the pest larva.


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Evaluation of entomopathogenic nematode Steinernema carpocapsae (Weiser), against fern scale Pinnaspis aspidistrae Sign
Nona Mikaia

Abstract: The aim of this study was to determine the biological control effect of entomopathogenic nematode Steinernema carpocapsae on fern scale Pinnaspis aspidistrae under laboratory conditions. The experiments were conducted in 10 cm Petri dishes lined with a moistened filter paper. One infested Laurel (Laurus nobilis) leaf containing approximately 100-120 P. aspidistrae adult and 500, 1000 and 1500 infective juveniles/ml nematode placed in each Petri dish. Insect mortality was checked 3, 5 and 7 days after the treatment. The results showed that S. carpocapsae (Weiser), was highly virulent against P. aspidistrae adult and the mortality was related with time and nematode concentrations. At 7 days after treatment, 500, 1000 and 1500 IJs/ml applications exhibited 22, 62 and 82% mortality, respectively. It was concluded that P. aspidistrae adult can be controlled by S. carpocapsae (Weiser). Further studies should be conducted in greenhouse and filed conditions to expand on this knowledge.


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Efficacy evaluation of Heterorhabditis bacteriophora against click beetle (Coleoptera: Elateridae)
Manana Lortkipanidze, Oleg Gorgadze, Nino Gabroshvili, Medea Burjanadze, Madona Kuchava

Abstract: The efficacy of the entomopathogenic nematode Heterorhabditis bacteriophora was evaluated in the control of click beetles (Coleoptera: Elateridae). The species of harmful insects, responsible for the damage, are Agriotes lineatus, A. obscurus, A. sputator and A. gurgistanus in many European regions. The efficiency of H. bacteriophora was tested against different stages of click beetle of the species A. gurgistanus-wireworms, pupae and adults in the laboratory. H. bacteriophora with 100% mortality has been shown to be pathogenic towards wireworms. We also evaluated the effect of nematode infection on wireworm’s organism and studied histological changes of the fatty tissues.


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Screening of bacteria isolated from Galleria mellonella (Lepidoptera: Pyralidae) larvae infected with novel entomopathogenic nematodes
Luca Ruiu, Barbara Virdis, Maria Elena Mura, Maria Giovanna Marche, Ignazio Floris, Alberto Satta, Eustachio Tarasco

Abstract: Major bacterial species developing in Galleria mellonella larvae infected with different entomopathogenic nematodes (EPN) were isolated by plating serial dilutions of insect haemolymph. On average 1, and at most 2 different bacterial isolates, were collected from each insect-EPN association. Nematodes involved in the research were collected from soil in different areas of Italy and Algeria, and were previously identified as Steinernema and Heterorhabditis species.
Bacterial cultures were screened against insects of different orders, including Lepidoptera (Malacosoma neustria and Lymantria dispar) and Diptera (Musca domestica and Ceratitis capitata). In total 80 bacterial isolates were assayed by incorporating either the cell fractions or the culture supernatants in comparative experiments involving reference strains of Bacillus thuringiensis, Photorhabdus luminescens and of Xenorhabdus nematophila. The majority of bacterial isolates were toxic to lepidopteran larvae, while around 20 of them showed significant toxicity against fly larvae and/or adults. Bacteria showing the highest mortality levels (> 50%) on flies were submitted to identification through 16S rRNA gene sequencing, which highlighted the effectiveness of species belonging to different genera including Serratia, Pseudomonas, Alcaligenes, and Stenotrophomonas.


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Effect of Spodoptera exigua iflavirus co-inoculation on the insecticidal properties of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV)
Arkaitz Carballo, Salvador Herrero, Trevor Williams, Rosa Murillo, Primitivo Caballero

Abstract: The Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) is a species-specific pathogen that can efficiently control this pest in sweet pepper crop systems of southern Spain. Studies on SeMNPV persistence in covertly infected adults revealed the presence of RNA viruses in insect colonies. These were identified as two iflaviruses (SeIV1 and SeIV2). Subsequent experiments indicated that SeMNPV and SeIV could be detected in both field and laboratory insect populations using PCR-based techniques. In this study, we evaluated the effect of SeIV1 co-infection on the insecticidal characteristics of SeMNPV. For this, bioassays were performed on second instar larvae by inoculating at the same time one of five SeMNPV inoculum concentrations and one of the following iflavirus treatments: i) SeIV1; ii) SeIV2; iii) SeIV1+SeIV2; and mock-infected control. Overall, iflavirus co-inoculation consistently reduced median lethal concentrations (LC50) for SeMNPV compared to larvae infected with SeMNPV alone. However, the speed of kill of SeMNPV was similar in the presence or absence of the iflaviruses. Adults’ survivors of a sublethal SeMNPV treatment were examined for covert infection and SeMNPV DNA was found to be present at a high prevalence in all treatments. In conclusion, simultaneous SeMNPV-SeIV co-infection resulted in improved pathogenicity of SeMNPV and did not alter the capacity of SeMNPV for vertical transmission.


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Mixtures of vertically and horizontally transmitted variants of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) as the basis for biological insecticides
Cristina Virto, David Navarro, Mª del Mar Tellez, Trevor Williams, Rosa Murillo, Primitivo Caballero

Abstract: The Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) is a highly pathogenic host-specific virus that can be used to control its natural host, the beet armyworm S. exigua (Lepidoptera: Noctuidae). Recent studies have demonstrated that distinct genotypes of SeMNPV differ in their insecticidal properties and could be associated with different routes of transmission. In the present study we evaluated the value of using mixed genotypes of SeMNPV that had favorable insecticidal properties (Se-G25), or the capability for vertical transmission (Se-Al1). Interestingly, mixed genotypes containing 25 and 75% of Se-G25 improved the pathogenicity of mixed genotype occlusion bodies (OBs) compared to OBs of Se-Al1 genotype alone, although no differences were observed in speed of kill or OB production (OBs/larva). The capacity to produce covert infections was evaluated in adult survivors of a sublethal dose. The Se-Al1 genotype was the most efficient in producing sublethal infections (90% infection) and the mixture of 50% Al1 + 50% G25 (87%), which were significantly more efficient than the Se-G25 (51%) genotype alone. The mixture of 25%Al1 + 75% G25 and 75% Al1 + 25% G25 resulted in 72% and 80% of adults positive for covert infection by qPCR, respectively. A greenhouse experiment revealed that sublethal infections in adult survivors increased with the proportion of Se-Al1 genotype present in inoculum OBs. We conclude that the presence of vertically-transmitted variants is of limited interest for the development of biological insecticides based on SeMNPV.


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A novel co-occluded binary mixture of Helicoverpa armigera single nucleopolyhedrovirus (HearSNPV) genotypes from Spain is a highly effective insecticide
Maite Arrizubieta, Oihane Simón, Trevor Williams, Primitivo Caballero

Abstract: The cotton bollworm, Helicoverpa armigera, is a major polyphagous pest. The Helicoverpa armigera single nucleopolyhedrovirus (HearSNPV) can be an effective agent for control of this insect. In the present study, the genotypic diversity present in two Spanish HearSNPV isolates was evaluated with the aim of identifying mixtures of genotypes with improved insecticidal characteristics. Biological characterization of the different genotypes and
co-occluded mixtures revealed that a co-occluded mixture of two genotypes in equal proportions named HearSP1B:LB6 showed the highest insecticidal activity, and therefore was selected as the basis for a bioinsecticide. In order to optimize the conditions for scaled virus production, the effects of several factors on the production of HearSP1B:LB6 occlusion bodies (OB) were evaluated. Results indicated that inoculation of newly molted L5 larvae with an LC80 concentration of OBs followed by rearing at 30 °C resulted in the highest production of OBs. Finally, the insecticidal efficacy and persistence of HearSP1B:LB6 OBs in greenhouse and open-field tomato crops in the Iberian Peninsula were compared to those of two commercial biorational insecticides. Application of HearSP1B:LB6 resulted in similar levels of crop protection to those provided by commercial insecticides against H. armigera in both types of crops. OBs showed good persistence in both crop settings, although persistence was markedly longer in greenhouse crops. We conclude that HearLB6:SP1B deserves to be registered as a biological insecticide for control of H. armigera on tomato crops in this region. The unique HearSP1B:LB6 binary genotypic mixture has been the subject of a patent application (P201430956).


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Occurrence and characterization of two betabaculoviruses isolated from Tuta absoluta (Lepidoptera: Gelechiidae) in Colombia
Juliana Gómez-Valderrama, Gloria Barrera, Mariano Belaich, Pablo Daniel Ghiringhelli, Miguel López-Ferber, Laura Villamizar

Abstract: Tuta absoluta is a devastating invasive pest of tomato plants, causing losses of up to 100%. The high use of chemical pesticides for its control, the risk of contamination and the generation of resistance make it necessary to find control alternatives. An interesting tool is the use of entomopathogenic viruses of Baculoviridae family, which are insect-specific and do not generate environmental impacts. Despite its high potential, its use on T. absoluta larvae has been poorly studied. In this work, two betabaculoviruses (granuloviruses) were isolated from 1,186 T. absoluta larvae sampled in tomato crops in Colombia (virus occurrence: 0.3%), which were coded as VG012 and VG013 and morphological, biological and molecularly characterized. The granules of these isolates showed ovoid shape with an approximate size of 514 nm x 250 nm, with a single virion enclosing one nucleocapsid. Restriction endonuclease analysis showed the same pattern compared to Phthorimaea operculella granulovirus but different compared to the granulovirus isolated from Tecia solanivora, although they were classified in the same monophyletic group. The estimated genome size was 120 kbp. The mean lethal concentration (LC50) of viral isolates was 1.6 x 104 and 1.9 x 104 OBs/ml for VG012 and VG013, respectively, and LC90 of 7.0 x 106 and 8.5 x 106 OBs/ml, with an average productivity of 3.0 x 1010 OBs per gram of tissue.


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Overwinter transmission of CpGV on infected diapausing larvae
Benoît Graillot, Sandrine Bayle, Myriam Siegwart, Christine Blachere-Lopez, Samantha Besse, Miguel Lopez-Ferber

Abstract: Different granuloviruses are used as biological control against insect pests. Among them, Cydia pomonella granulovirus (CpGV) is widely sprayed in apple orchards to limit codling moth proliferation. Resistance to CpGV-M has developed in some European orchards. A better understanding of the biology of the virus should allow a better rational use leading to increased durability of this control method.
Transmission of CpGV from one year to the next in natural conditions relies in the presence of reservoirs in orchards, allowing young larvae of the first generation in the spring to enter in contact with the virus. A possible way is the survival over winter of infected larvae that could die upon metamorphosis or yield infected adults able to vertically transmit the virus.
We have explored using qPCR the level of infection in overwintering five instar codling moth larvae that survive to the late summer treatments in commercial orchards. About 8% of such larvae were found to be positive for virus.


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Determination of reapplication frequency required for the Cryptophlebia leucotreta granulovirus: a factor of rate of virus breakdown and larval behavior
Patrick Mwanza, Storm Hilliar, Gill Dealtry, Michael Lee, Martin Hill, Sean Moore

Abstract: The Cryptophlebia leucotreta granulovirus (CrleGV) has now been used commercially for the control of the false codling moth, Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), on citrus in southern Africa for more than 10 years. Farmers need improved clarity on frequency of application required. Consequently, the rate of breakdown in the field, determined to be mainly due to ultraviolet radiation, was determined. This was done at regular intervals from zero to 28 days after application, by washing occlusion bodies off Navel orange fruit from both the northern (sunny) and southern aspects of trees and conducting dose-response bioassays against neonate T. leucotreta larvae in the laboratory. At 21 days after application, LD50 of CrleGV recovered from the northern side of trees was 15 times higher than from the southern side of trees. By 28 days after application, virulence of CrleGV on the northern side of trees was indeterminable, whereas on the southern side of trees, there was still a clear dose response. In a separate study, neonate T. leucotreta larvae were placed onto molasses-treated and water only-treated Navel oranges and behaviour filmed, recorded and analysed. Although distance traversed by the larvae on the fruit surface was significantly greater without molasses, larvae on molasses-treated fruit fed more actively, thus increasing the probability of viral ingestion before penetration. Combining the rate of breakdown of virus and larval feeding behaviour with and without molasses, a model is proposed for estimating necessary time intervals between CrleGV sprays.


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Detection and quantitation of alpha- and betabaculovirus in Spodoptera frugiperda mixed infections
Gloria Barrera, Paola Cuartas, Jorge Cerón, Laura Villamizar

Abstract: The fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) is an important pest of the Americas. This insect has a very wide host range and has been able to attack several crops; however, the most frequently hosts are maize and sorghum causing an economic impact. Alphabaculoviruses (lepidopteran nucleopolyhedroviruses, NPV) and betabaculoviruses (granuloviruses, GV) of the family Baculoviridae have been isolated causing co-infection in fall armyworm populations in Colombia. This condition has a considerable potential as biological control strategy, as in some cases GVs are able to enhance the infectivity and virulence of NPVs. In this work a sensitive tool for detection and quantitation of NPVs and GVs in mixed infections in S. frugiperda was developed. We used a multiplex real-time PCR (q-PCR) method based on highly specific oligonucleotides and Taq-man probes that recognize fragments of polyhedrin (polh) and granulin (gran) genes. The specificity of individual probes showed non-cross amplification between NPVs and GVs. Besides, each probe was able to detect NPVs or GVs from different geographical origins. Samples of mixed SfMNPV/SfGV genomes at different ratios were analysed and interferences were not observed. The minimum detection limit of the technique was 6.4 x 10-4 ng /μl of DNA, equivalent to 1.25 x 103 gene copies (gran and polh) and minimal variation inter-assays showed that the technique was reproducible. This technique is essential in the process of biopesticide developing based on alpha- and betabaculoviruses, including the detection of larvae infected in field until the quality control of final formulated product.


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Possible natural betabaculovirus coinfections in Gelechiidae insects from Colombia based on pif gene determination
Cecilia S. Turco, Gloria P. Barrera, Mariano N. Belaich, Laura F. Villamizar, Pablo D. Ghiringhelli

Abstract: Baculoviruses are pathogens distributed worldwide and classified in four genera (Alpha-, Beta-, Gamma- and Deltabaculovirus) which infect insects many of them considered as pests in agriculture. In view of that, these entities are excellent candidates to formulate bioinsecticides. Tecia solanivora, Phthorimaea operculella and Tuta absoluta are invertebrates belonging to the potato tuber moth complex (Lepidoptera: Gelechiidae) and are considered the main pests in potato and tomato crops. Thus, the characterizations of baculoviruses that can multiply in these insects comprise a relevant focus required to assist in the development of biological control strategies.
Considering the above, three betabaculoviruses recovered in Colombia from T. solanivora, P. operculella and T. absoluta respectively were studied to add information with respect to the host range specificity associated with the identity of the per os infectivity complex, constituted by seven proteins encoded in all baculovirus genomes (P74, PIF1, PIF2, PIF3, ODV-e28, ODV-e56 and PIF6) which are essential to support the primary infection into larvae. For this, fragments of the aforementioned seven pif genes were amplified by PCR and sequenced to assist in the virus classification and to study sequence factors related to virus host range. Results showed that three isolates were closely related to the previously described Phthorimaea operculella granulovirus (PhopGV) based on sequence data of six pifs, but surprisingly some amplicons of pif1 also revealed a sequence relationship with Helicoverpa armigera granulovirus (HearGV). This observation could suggest the presence of natural coinfections and/or horizontal gene transfer processes associated to expand the host range.


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Conditions adjustment for in vivo mass production of a Colombian Spodoptera frugiperda nucleopolyhedrovirus
Jenny Carolina Ruiz, Juliana Gómez-Valderrama, Martha Chaparro, Paula Sotelo, Laura Villamizar

Abstract: The Colombian Spodoptera frugiperda nucleopolyhedrovirus isolate SfNPV003, was used for developing a biopesticide with great potential for the control of this pest in the field, becoming an alternative to replace chemical pesticides. However, its commercialization has been limited due to high costs for in vivo virus production, which have a significant impact on the product price. Traditional in vivo production involved feeding the virus to insect larva, temperature should be maintained carefully to keep the larva alive and to produce the highest yield and quality of virus, and larvae must be harvested at the proper time. In this sense, the aim of the present work was to adjust the conditions of SfNPV003 multiplication system, in order to increase yield and reduce costs. Initially, the effect of larval age and viral inoculum concentration on viral productivity was determined. Subsequently, the effect of diet for larvae rearing and incubation temperature was established. Finally, the effect of harvest time on productivity and contaminant content was evaluated. Sixteen days-old larvae were selected for inoculation with a viral suspension adjusted to a concentration of 1x107 occlusion bodies (OBs) per ml and wheat germ-based diet was selected for larvae rearing. Implementation of a temperature control system for incubation did not improve viral production. Seven days post- inoculation was selected as the optimum time for infected larvae harvest.


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