Eva Fritsch

Rapid emergence of baculovirus resistance in codling moth due to dominant, sex-linked inheritance.

Insect-specific baculoviruses are increasingly used as biological control agents of lepidopteran pests in agriculture and forestry, and they have been previously regarded as robust to resistance development by the insects. However, in more than a dozen cases of field resistance of the codling moth Cydia pomonella to commercially applied C. pomonella granulovirus (CpGV) in German orchards, resistance ratios exceed 1000. The rapid emergence of resistance is facilitated by sex-linkage and concentration-dependent dominance of the major resistance gene and genetic uniformity of the virus. When the gene is fixed, resistance levels approach 100,000-fold. Our findings highlight the need for development of resistance management strategies for baculoviruses.

Physical map of the Cryptophlebia leucotreta granulosis virus genome and its relationship to the genome of Cydia Pomonella granulosis virus

A physical map of the genome of Cryptophlebia leucotreta granulosis virus (CIGV) was constructed for the restriction enzymes BamHI, EcoRI, KpnI, NdeI, NruI, SacI and XhoI using hybridization techniques. The size of the viral genome was determined to be 112.4 kbp. A restriction fragment library covering almost the entire genome of CIGV was constructed, and the position of the granulin gene was identified by cross-hybridization with granulin coding fragments of Cydia pomonella granulosis virus (CpGV). Two further regions of intergenomic similarity between CIGV and CpGV were mapped. These regions were aligned and show a collinear arrangement.

The occurrence and distribution of resistance of codling moth to Cydia pomonella granulovirus in Europe

Thirty‐five codling moth (CM, Cydia pomonella L., Lepidoptera, Tortricidae) populations collected in different commercial orchards in six European countries were tested for their susceptibility to Cydia pomonella granulovirus (CpGV‐M). Including previously published data on CpGV‐M resistance, a total of 38 CM colonies showed considerably elevated LC50 values, independent of the country origin. When only few test individuals are available, determination of mortality of neonate larvae at a discriminating concentration range of 104 to 106 OB/ml (>log4) as a direct measure of percentage susceptible individuals in a CM population is more advisable than calculation of LC50 values. The >log4 mortality alone or in combination with the LC50 value can be used for identification of resistance in a population. Results indicated a locally separated but widely spread occurrence of CM populations with low susceptibility to CpGV‐M. The most plausible hypothesis for the emergence of CpGV‐M resistance is its selection by repeated use of CpGV products.

High stability and no fitness costs of the resistance of codling moth to Cydia pomonella granulovirus (CpGV-M)

Resistance against the biocontrol agent Cydia pomonella granulovirus (CpGV-M) was previously observed in field populations of codling moth (CM, C. pomonella) in South-West Germany. Incidental observations in a laboratory reared field colony (CpR) indicated that this resistance is rather stable, even in genetically heterogeneous CM colonies consisting of both susceptible and resistant individuals. To test this hypothesis, the resistance level of CpR that was 1000times less susceptible to CpGV-M was followed for more than 60 generations of rearing. Even without virus selection pressure, the high level of resistance, expressed as median lethal concentration, remained stable for more than 30 generations and declined only by a factor of 10 after 60 generations. When cohorts of the F32 and F56 generations of the same colony were selected to CpGV-M for five and two generations, respectively, the resistance level increased to factor of >1,000,000 compared to a susceptible control colony. Laboratory reared colonies of CpR, did not exhibit any measurable fitness costs under laboratory conditions in terms of fecundity and fertility. Resistance testing of seven selected codling moth field populations collected between 2003 and 2008 in commercial orchards in Germany that were repeatedly sprayed with CpGV products gave evidence of different levels of resistance and a more than 20-fold increase of the resistance in 1-3 years when selection by CpGV-M was continued. A maximum 1,000,000-fold level of resistance to CpGV-M that could be induced in the laboratory under virus pressure had been also observed in one field population. The high stability of resistance observed in the genetically heterogenous colony CpR indicates that resistance to CpGV-M is not very costly.

Novel resistance to Cydia pomonella granulovirus (CpGV) in codling moth shows autosomal and dominant inheritance and confers cross-resistance to different CpGV genome groups

Commercial Cydia pomonella granulovirus (CpGV) products have been successfully applied to control codling moth (CM) in organic and integrated fruit production for more than 30 years. Since 2005, resistance against the widely used isolate CpGV-M has been reported from different countries in Europe. The inheritance of this so-called type I resistance is dominant and linked to the Z chromosome. Recently, a second form (type II) of CpGV resistance in CM was reported from a field population (NRW-WE) in Germany. Type II resistance confers reduced susceptibility not only to CpGV-M but to most known CpGV isolates and it does not follow the previously described Z-linked inheritance of type I resistance. To further analyze type II resistance, two CM strains, termed CpR5M and CpR5S, were generated from parental NRW-WE by repeated mass crosses and selection using the two isolates CpGV-M and CpGV-S, respectively. Both CpR5M and CpR5S were considered to be genetically homogeneous for the presence of the resistance allele(s). By crossing and backcrossing experiments with a susceptible CM strain, followed by resistance testing of the offspring, an autosomal dominant inheritance of resistance was elucidated. In addition, cross-resistance to CpGV-M and CpGV-S was detected in both strains, CpR5M and CpR5S. To test the hypothesis that the autosomal inheritance of type II resistance was caused by a large interchromosomal rearrangement involving the Z chromosome, making type I resistance appear to be autosomal in these strains; fluorescence in situ hybridization with bacterial artificial chromosome probes (BAC-FISH) was used to physically map the Z chromosomes of different CM strains. Conserved synteny of the Z-linked genes in CpR5M and other CM strains rejects this hypothesis and argues for a novel genetic and functional mode of resistance in CM populations with type II resistance.

The potential of novel African isolates of Phthorimaea operculella granulovirus for the control of Tuta absoluta

Phthorimaea operculella granulovirus (PhopGV) is infectious for larvae of different Gelechiidae insect species, including Tuta absoluta and Phthorimaea operculella. As these are major economic pests in North and sub‐Saharan Africa as well as in the Mediterranean area, the development of locally suitable biocontrol agents is essential. We have studied five isolates of PhopGV from Tunisia (Tns16, Tu1.11 and Tu2.11), Kenya (Ken13) and Yemen (Ym14) for their biological activity and the sequence polymorphism of their granulin and ecdysteroid UDP‐glucosyltransferase (egt) genes and allocated the isolates to two different egt types. Infection experiments with neonate larvae of T. absoluta and P. operculella demonstrated their pathogenicity to both host species. The isolate PhopGV Tu1.11 was the most virulent one for T. absoluta but had a relatively low infectivity to two P. operculella populations originating from Italy and Tunisia.

Using Next Generation Sequencing to Identify and Quantify the Genetic Composition of Resistance-Breaking Commercial Isolates of Cydia pomonella Granulovirus

The use of Cydia pomonella granulovirus (CpGV) isolates as biological control agents of codling moth (CM) larvae is important in organic and integrated pome fruit production worldwide. The commercially available isolates CpGV-0006, CpGV-R5, and CpGV-V15 have been selected for the control of CpGV resistant CM populations in Europe. In infection experiments, CpGV-0006 and CpGV-R5 were able to break type I resistance and to a lower extent also type III resistance, whereas CpGV-V15 overcame type I and the rarely occurring type II and type III resistance. The genetic background of the three isolates was investigated with next generation sequencing (NGS) tools by comparing their nucleotide compositions to whole genome alignments of five CpGV isolates representing the known genetic diversity of the CpGV genome groups A to E. Based on the distribution of single nucleotide polymorphisms (SNPs) in Illumina sequencing reads, we found that the two isolates CpGV-0006 and CpGV-R5 have highly similar genome group compositions, consisting of about two thirds of the CpGV genome group E and one third of genome group A. In contrast, CpGV-V15 is composed of equal parts of CpGV genome group B and E. According to the identified genetic composition of these isolates, their efficacy towards different resistance types can be explained and predictions on the success of resistance management strategies in resistant CM populations can be made.

Neue Erkenntnisse zur Verbreitung der Resistenz des Apfelwicklers gegen das Cydia pomonella Granulovirus (CpGV)

Die Daten der Vergleichsbetriebe stellen den Status quo im Pflanzenschutz in Deutschland dar. Der Behandlungsindex in Mohre und Frischkohl betrug im Durchschnitt der Jahre (2007-2014) bei 6,2 bzw. 9,2. Mohren wurden vorrangig mit Herbiziden und Frischkohl mit Insektiziden behandelt. Unter dem Aspekt des Pflanzenschutzes und zur Reduktion der Behandlungsintensitat mit Insektiziden kann Abbildung 1: Behandlungsindex der Herbizide (Herbizid-BI) in Abhangigkeit der Vorfrucht in den Vergleichsbetrieben fur Mohre, 2007-2014, in Deutschland, ungleiche Buchstaben symbolisieren signifikante Unterschiede (p<0,05), nBlattfrucht=58, nGemuse=28, nHalmfruchte=115 208 72. ALVA-Tagung, Seminarhotel Wesenufer, 2017 der Fruhkohlanbau empfohlen werden. Im Kohl- und Mohrenanbau konnen Halmvorfruchte Schadlingsbefall vorbeugen. Die Mittelaufwandmengen wurden vorrangig bei Herbizidanwendungen reduziert In beiden Kulturen wurden Abweichungen vom notwendigen Mas verzeichnet. Die Pflanzenschutzberatung sowie die regelmasige Durchfuhrung von Bonituren konnen Pflanzenschutzmittelanwendungen dem notwendigen Mas naher bringen und die Umsetzung des IPS in der Praxis verbessern. Fur die Ausschopfung nichtchemischer Masnahmen sind Praxisdemonstrationen und der intensive Austausch zwischen Wissenschaft und Praxis notig, genauso wie Forschungsforderung zur Entwicklung neuer und Optimierung bestehender integrierter Verfahren und neuer Wirkstoffe. Vorbildhaft wird der IPS von den Demonstrationsbetrieben u. a. in den Kulturen Mohre und Weiskohl, seit 2014 umgesetzt. Die Betriebe werden individuell und intensiv betraten, um Pflanzenschutzmittelanwendungen auf das notwendige Mas zu beschranken. Hierfur werden Bonituren unter Nutzung von Schadschwellen, Warndiensthinweisen und Monitoringverfahren durchgefuhrt.

Identifizierung neuer Isolate des Phthorimaea operculella Granulovirus (PhopGV) zur kombinierten Bekämpfung von Phthorimaea, Tuta und Tecia

Die Daten der Vergleichsbetriebe stellen den Status quo im Pflanzenschutz in Deutschland dar. Der Behandlungsindex in Mohre und Frischkohl betrug im Durchschnitt der Jahre (2007-2014) bei 6,2 bzw. 9,2. Mohren wurden vorrangig mit Herbiziden und Frischkohl mit Insektiziden behandelt. Unter dem Aspekt des Pflanzenschutzes und zur Reduktion der Behandlungsintensitat mit Insektiziden kann Abbildung 1: Behandlungsindex der Herbizide (Herbizid-BI) in Abhangigkeit der Vorfrucht in den Vergleichsbetrieben fur Mohre, 2007-2014, in Deutschland, ungleiche Buchstaben symbolisieren signifikante Unterschiede (p<0,05), nBlattfrucht=58, nGemuse=28, nHalmfruchte=115 208 72. ALVA-Tagung, Seminarhotel Wesenufer, 2017 der Fruhkohlanbau empfohlen werden. Im Kohl- und Mohrenanbau konnen Halmvorfruchte Schadlingsbefall vorbeugen. Die Mittelaufwandmengen wurden vorrangig bei Herbizidanwendungen reduziert In beiden Kulturen wurden Abweichungen vom notwendigen Mas verzeichnet. Die Pflanzenschutzberatung sowie die regelmasige Durchfuhrung von Bonituren konnen Pflanzenschutzmittelanwendungen dem notwendigen Mas naher bringen und die Umsetzung des IPS in der Praxis verbessern. Fur die Ausschopfung nichtchemischer Masnahmen sind Praxisdemonstrationen und der intensive Austausch zwischen Wissenschaft und Praxis notig, genauso wie Forschungsforderung zur Entwicklung neuer und Optimierung bestehender integrierter Verfahren und neuer Wirkstoffe. Vorbildhaft wird der IPS von den Demonstrationsbetrieben u. a. in den Kulturen Mohre und Weiskohl, seit 2014 umgesetzt. Die Betriebe werden individuell und intensiv betraten, um Pflanzenschutzmittelanwendungen auf das notwendige Mas zu beschranken. Hierfur werden Bonituren unter Nutzung von Schadschwellen, Warndiensthinweisen und Monitoringverfahren durchgefuhrt.

Characterization of the Tunisian PhopGV isolate TU1.11; molecular identification and biological activity on Phthorimaea operculella larvae

The project is dealing with the improvement of a universal pneumatic seeder which is suitable for sowing a wide range of different seeds in order to decrease particulate emissions of seed dressings. Aim of the project is to identify leakages in the pneumatic system in order to develop opportunities for the subsequent improvement of existing equipment to fulfill the high demands of user and environmental protection. Pneumatic seeders are characterized by a central, funnelshaped hopper. The batch feeder being an airlock and dosing feeder is located in the outlet of the seeder. The metered seed is fed into the air stream and transported to the sowing distributor by a conveying air stream.Due to climatic change, phenology traits are becoming increasingly important in grapevine breeding, since a premature flowering and ripening time could be observed for grapevine in the last decades. However, knowledge about these traits is still limited as they are genetically very complex and highly influenced by environmental factors. The analysis of the genetic basis of flowering time therefore will enable the development of tightly linked molecular markers useful for markerassisted selection of especially late flowering breeding lines.One recently occurred invasive insect pest has caught the attention for investigating biological control mechanisms and systems: The spotted wing drosophila (SWD, Drosophila suzukii Matsumura) is endemic in East China and Japan but has been introduced to the western hemisphere about 10 years ago and has been found in Europe since 2009. Nowadays, it has emerged to one of the most harmful pests to commercially grown fruit plants like stone fruits and nearly all kind of berries while it prefers ripe and overripe fruits. Our intention is to investigate the possible usage of natural antagonists for biological control. Therefore, we examine the natural load of parasites and pathogens (i.e. fungi, bacteria, viruses, microsporidia and protista) in fruit flies, isolate them and re-infect lab populations of D. suzukii for investigating the antagonistic potential. Furthermore, we will integrate the fruit pest codling moth (Cydia pomonella), which is an ongoing problem in apple orchards also because the pest develops resistance against commercially available insecticides. The long-term aim is to establish a stable system for pathogen detection that can be used for rapid identification of microbial antagonists in natural populations.Two main problems have to be considered in the development of control agents against the Fire Blight pathogen Erwinia amylovora: First, the ability of exponential growth leads to high cell densities in a short amount of time. Second, the most critical phase of Fire Blight infection occurs during blossoming, when the pathogen is transported to open flowers by various insects. To prevent infection, it is important to avoid invasion inside the plants tissue by interfering with growth of E. amylovora cells.The entomopathogenic fungus Isaria fumosorosea, formerly known as Paecilomyces fumosoroseus, has got a relatively wide host range. Within the scope of the EU project BIOCOMES investigations were done to validate the use of I. fumosorosea as a BCA against several pest insects. Under laboratory conditions it could be shown that I. fumosorosea seems to be a suitable BCA against Bemisia tabaci (silverleaf whitefly) and Spodoptera exigua (beet armyworm).Since the last two decades a number of fingerprinting methods have been developed to analyze microbial communities and their dynamics, including Terminal Restriction Fragment Length Polymorphism (T-RFLP), Length Heterogeneity-Polymerase Chain Reaction (LHPCR) and Automated Ribosomal Intergenic Spacer Analysis (ARISA). Because the latter provides a quick and cheap way together with high accuracy, we have chosen this method to investigate the fungal communities on grapevine, wood, leaves and berries.Saoussen Ben Tiba, Andreas Larem, Eva Fritsch, Karin Undorf-Spahn, Asma Laarif, Sami Fattouch, Johannes A. Jehle 1 Julius Kuhn-Institut, Institute for Biological Control, Darmstadt, Germany 2 Regional Center for Agriculture and Biological Agriculture, Chott Meriem, Tunisia 3 National Institute for applied technological Science Tunis (INSAT), Carthage University, Tunisia Email of corresponding author: saoussen.ben.tiba@jki.bund.deOne promising approach to a more environmentally friendly viticulture is growing fungus resistant cultivars with the novel cultivation method of the minimal pruning of trellis trained grapevines (MPTS). This practice reduces the tremendous amounts of fungicides needed to protect traditional cultivars and is expected to increase biodiversity compared to vineyards with traditional trellis trained grapevines (TS).Meiosis as the specialized cell division of sexual reproduction plays a crucial role in the exchange and reorganization of genetic material between two individuals by dividing the chromosome set in half and forming gametes. Even though in the last years major findings in the field of meiosis have been achieved, especially in plants, some key questions remain concealed. For a proper meiosis the initiation of double strand breaks (DSBs) during early prophase I is essential. Without DSBs no physical connection can occur between homologous chromosomes and recombination, pairing, and crossing over are excluded. So far in all analyzed eukaryotes SPO11, a meiosis specific transesterase, is the key enzyme inducing DSBs. But other than in animals and fungi where a single SPO11 is sufficient, plants need at least two different SPO11, referred to as SPO11-1 and SPO11-2, for proper meiosis. In Arabidopsis thaliana both have crucial functions and are essential in a functional form for the induction of meiotic DSBs as single knock out mutants are leading to near sterility by random chromosome distribution. Despite the same function of the homologs SPO111 and -2, the identity between both proteins is quite low. Homology of the orthologous SPO11 from different organisms is much higher. By exchanging SPO11-1 and -2 in Arabidopsis by their orthologs from various organisms we could demonstrate a species specific function of each SPO11, as a functional complementation of sterility could only be achieved with SPO11 from closely related species from the Brassicaceae. By exchanging non conserved regions between SPO11-1 and -2 of Arabidopsis we additionally could show a sequence specific function for each SPO11, as a functional rescue could not be achieved with all chosen regions. Interestingly, we could reveal a specific pattern of aberrant spliced isoforms for each SPO11 which are also sequence as well as species specific. By producing antibodies against AthSPO11-1 and -2 we were able to analyze for the first time the binding of SPO11-2 onto the DNA and perform co-immunolocalization studies with SPO11-1 and -2.European Stone Fruit Yellows (ESFY) is one of the most serious diseases in European fruit production. Infected Prunus cultivars yield poorly and lead to high economic losses. ESFY is caused by a specialized bacterium located in the phloem tissue of Prunus ssp., the Phytoplasma ‘Candidatus Phytoplasma prunorum’. It is spread by the phloemfeeding plum psyllid (Cacopsylla pruni) which acquires the bacterium by feeding on infected plants and is able to transmit it to healthy plants.Leaf rust caused by Puccinia triticina can cause yield losses up to 60 % and is the most common rust disease of wheat in the world. Vertical leaf rust resistance genes (Lr-genes) have been introduced in cultivars. Many of these resistances are broken down by virulent pathotypes. Horizontal resistances which are independent from races of a pathogen are known but show a quantitative characteristic which is carried by a few cultivars.