Diversity of Baculoviruses isolated from cutworms ( Agrotis spp.)

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Baculoviruses (Baculoviridae) are double-stranded DNA viruses which infect the larval stages of insects belonging to the orders Lepidoptera, Hymenoptera and Diptera. Due to their narrow host range and high virulence to target insects, different baculoviruses have been used as biological control agents in pest control. Severe soil pests of many agricultural and horticultural crops, which are difficult to be controlled, are larvae of the genus Agrotis (Lepidoptera: Noctuidae), also called cutworms. Their habitat is the soil or soil surface where they feed on seedlings, stems, and other parts of plants. So far, cutworms are mainly control by chemical pesticides, but biological control agents would be highly desirable for environmental reasons. Two of the most important cutworm pests are the common cutworm, Agrotis segetum (Denis & Schiffermuller), and the black cutworm, A. ipsilon (Hufnagel). At least four distinct baculovirus species, three of the genus Alphabaculovirus and one of the genus Betabaculovirus, were isolated from these two cutworm species. The alphabaculoviruses are the Agrotis segetum nucleopolyhedrovirus A (AgseNPV-A), Agrotis segetum nucleopolyhedrovirus B (AgseNPV-B), and Agrotis ipsilon nucleopolyhedrovirus (AgipNPV). The Agrotis segetum granulovirus (AgseGV) represents the betabaculovirus. Together with their two host species, from which they were first isolated and characterized, they form the so-called Agrotis baculovirus complex. Agrotis baculoviruses have the potential to be used as biological control agents for the control of Agrotis cutworms. However, in-depth knowledge and a full characterization of their biology, molecular setup, and virulence parameters are required for a successful registration and application in the field. In this study the genome sequence of AgseNPV-B was fully sequenced. Its genome is 148,981 bp in length and codes for 150 putative open reading frames. Whole genome comparisons with AgseNPV-A and AgipNPV, whose genome sequences have been published previously, suggested that AgseNPV-B belongs to a new species of the Agrotis baculovirus complex. Phylogenetic analysis indicated a very close relationship to AgipNPV and it could be concluded that both viruses are two distinct species at an early stage of separation. Whole genome alignments revealed a different number of viral enhancing factor (vef) gene copies in AgseNPV-A, AgseNPV-B and AgipNPV as one of the most striking distinguishing features between their genomes. VEFs are known to affect the virulence of baculoviruses. A putative site of genomic recombination was found in the region of the cathepsin and chitinase genes where the high co-linearity of the genomes of all three Agrotis nucleopolyhedroviruses was interrupted by inversions, deletions, or insertions. A putative fifth Agrotis baculovirus, the Agrotis exclamationis nucleopolyhedrovirus (AgexNPV), was suggested to be an isolate of AgseNPV-B due to high nucleotide sequence similarities of partial genomic regions. Based on the genome sequences of AgseNPV-A, AgseNPV-B, AgipNPV and AgseGV a multiplex PCR based method for the identification of these Agrotis baculoviruses was established. Highly specific oligonucleotide primers specific for the polyhedrin (polh) or granulin (gran) genes of the four viruses were developed and resulted in discriminating PCR fragments. Furthermore, this method allowed the quantitation of AgseGV and AgseNPV-B by quantitative PCR (qPCR). Since co-infections of AgseGV and AgseNPV-B have been observed and a combination of both viruses was considered as a combined biological control agent, the potential interaction of both viruses in mixed infections was investigated. Potential interactions between AgseNPV-B and AgseGV were examined in activity studies using single virus as well as combined virus infections of neonate A. segetum larvae. Mortality rates were determined and the virus progeny produced in individual larvae was quantified by using the newly established qPCR method of quantitation of AgseNPV-B and AgseGV. As a result, combinations of AgseNPV-B and AgseGV did not exhibit an advantageous effect in terms of pest control. Neither an increase in mortality rates in mixed virus treatments in comparison with single virus treatments, nor an increase in production of AgseNPV-B or AgseGV progeny in coinfected larvae was observed. On the contrary, a competitive behavior of both viruses in mixed infections could be concluded. The present thesis contribute to the biological control of Agrotis cutworms by providing extensive insight into the molecular setup of these viruses and the characterization of AgseNPV-B as a new Alphabaculovirus species. The use and registration of baculoviruses as biocontrol agents rely on such virus characterizations as well as on virus activity studies that were performed for AgseNPV-B and AgseGV. The new technique in Agrotis baculovirus detection and quantitation will facilitate future single and mixed infection studies of AgseNPV-B and AgseGV, as well as other combinations of Agrotis baculoviruses. Auf Wunsch des Autors / der Autorin ist diese Dissertation nur als Druckausgabe verfugbar.