Characterization of nuclear polyhedrosis viruses obtained from Adoxophyes orana and from Barathra brassicae
Jurkovicova, M. - \ 1979
Landbouwhogeschool Wageningen. Promotor(en): J.P.H. van der Want, co-promotor(en): D. Peters. - Wageningen : Jurkovicova - 133
baculovirus - biologische bestrijding - organismen ingezet bij biologische bestrijding - insecten - lepidoptera - noctuidae - kernpolyedervirussen - plantenplagen - tortricidae - virusmorfologie - virologie - virussen - adoxophyes orana - mamestra brassicae - microlepidoptera - baculovirus - biological control - biological control agents - insects - lepidoptera - noctuidae - nuclear polyhedrosis viruses - plant pests - tortricidae - viral morphology - virology - viruses - adoxophyes orana - mamestra brassicae - microlepidoptera
ln infectivity experiments some A. orana larvae died after being inoculated with an inoculum containing NW isolated from B. brassicae. The polyhedra formed upon infection occluded single virus particles, whereas the inoculum contained polyhedra with bundles of virus particles. This change could be explained either by activation of a virus in A. orana, which is singly embedded, or the inoculum from B. brassicae had infected A. orana and consequently the inclusion of virus particles in outer membranes is controlled by the hosts. This thesis describes studies performed to discriminate between both possibilities. Therefore, the first task was to characterize the virus particles from B. brassicae and A. orana NPV and their polyhedra by different techniques (Chapter 1).The properties of the NW of A. orana and of B. brassicae as observed with the electron microscope and polyacrylamide gel electrophoresis are similar to those found for many other NPVs. The polyhedra of both NPVs differ in size and shape. Most of the A. orana polyhedra are globular and range in diameter from 1-2 μm. Most of the B. brassicae polyhedra are hexagonal or pentagonal in outline and range in diameter from 1.5-4 μm. Analysis of polyhedral protein by polyacrylamide gel electrophoresis shows the presence of two polypeptides of molecular weight 28,000 and 54,000 Daltons.Treatment of the polyhedra of both viruses with sodium carbonate ruptures the polyhedral membrane and the virus particles and polyhedral proteins are released. The virus particles of A. orana polyhedra are singly embedded in the polyhedral matrix and have a size of 250 x 60 nm. The multiply embedded virus particles of B. brassicae have a size of 347 x 113 nm. Analysis of the viral proteins by SDS-polyacrylamide gel electrophoresis showed that NW of A. orana has 5 polypeptides of 68,000, 48,000, 39,000, 32-34,000, and 28,000 Daltons, respectively. Those of the NPV of B. brassicae were 69,000, 57,000, 46,000, 34-39,000, and 28,000 Daltons, respectively.In the polyhedral membrane fractions of both polyhedra one polypeptide of molecular weight of 28,000 Daltons as estimated by polyacrylamide gel electrophoresis, was found.Due to proteolytic activity associated with the polyhedra, which is evident after dissociation of the polyhedra, it was difficult to establish the number of polyhedral proteins and their molecular weight (Chapter 2). The electrophoretic pattern of polyhedral proteins of A.orana and B. brassicae polyhedra dissociated in alkali differed from those proteins obtained by other means. Six to seven polypeptides with molecular weights between 28,000 and 8,000 Daltons were found after incubation at pH 10.5. After inactivation of the enzyme only two polypeptides with molecular weights of 28,000 and 26,000 Daltons were observed. When the polyhedral proteins were analysed without incubation at pH 10.5 also two proteins were found, but their molecular weight was 54,000 and 28,000 Daltons.On the basis of the results described in Chapters 1 and 2 it can be concluded that the virus particles of B. brassicae and A.orana NPV differ with respect to size, the way of occlusion, and the form and size of the polyhedra involved. Protein analysis by polyacrylamide gel electrophoresis reveal some difference in molecular weight of viral protein but no significant difference in the protein composition of their polyhedra. Further analyses of amino acid composition and sequence of these proteins is necessary to elucidate possible differences.To differentiate further between both viruses their genomes were analysed (Chapter 3). Both genomes are circular double-stranded DNA molecules. The molecular weights of A.orana and of B. brassicae NPV-DNAs are 6.7 x 10 7and 8.9 x 10 7Daltons, respectively as determined by electron microscopy and by renaturation kinetic analysis. The renaturation also indicated that both genomes contain only unique sequences. The buoyant density in CsCl of the NPV-DNA of A. orana and of B. brassicae is 1.694 and 1.696 g/cm 3, respectively. These values are in good agreement with (G+C) contents of 34.5 and 37%, respectively as determined by thermal denaturation. The digestion of the A. orana and of the B. brassicae NPV-DNA with endonuclease Eco RI resulted in completely different electrophoretic patterns. Also in experiments on
competition hybridization no homology between these genomes was found. The conclusion of these studies is that these two NPVs can be clearly differen tiated by their DNA properties.In order to study the occurrence of viral DNA in uninfected larvae the DNA of A. orana and B. brassicae was isolated and the complexity studied (Chapter 4). The genomes of A.orana and of B. brassicae differ in their kinetic complexity as estimated from the reassociation data on hyperchromicity, but they are both relatively small and show remarkable similarity in the extent of intragenome homology. A haploid cell of A. orana has a DNA equivalent of 4.2 x 10 10and that of B. brassicae of 8.4 x 10 10Daltons. The intragenome homology was estimated to be 10 and 9% for A.orana and B. brassicae genome, respectively. The (G+C) content, estimated by thermal denaturation, was found to be 36.2% for the A. orana genome and 35.8% for the B. brassicae genome.The results obtained during rearing of insects from surface-sterilized eggs and from untreated eggs showed that the NPV of A.orana and of B. brassicae can be transmitted to the progeny of these insects on the outside of the eggs (transovum) as well as inside the eggs (transovarially) (Chapter 5). Evidence for transovarial transmission was also obtained from reassociation of viral DNA with the host DNA of homologous insects reared from surface-sterilized eggs. These experiments revealed the presence of viral sequences in host DNA: 0.03 and about 2.5 viral copies for the diploid quantity of the A. orana and of the B. brassicae host DNA, respectively.Results obtained in infectivity experiments with insects in various developmental stages showed that transstadial transmission is a prerequisite for generation-to-generation transmission.The presence of a latent virus infection in both insects could also be demonstrated in cross-inoculation experiments (Chapter 6). When the larvae of A. orana and of B. brassicae were inoculated with polyhedra of the reciprocal species, the number of larvae containing polyhedra increased compared with that of the control. Comparison of the restriction endonuclease Eco RI pattern of DNA isolated from polyhedra used as inocula with that from polyhedra obtained after cross-inoculation indicated that both viruses are not cross-infective but that they activate a latent virus infection in both insects. Because the cross- inoculation experiments were done under laboratory conditions (as aseptic as possible), it could be concluded that the B. brassicae NPV is not suitable for biological control of A. orana in the field, because this virus is not cross-infective.
Studies on dispersal of Adoxophyes orana F.v.R. in relation to the population sterilization technique
Barel, C.J.A. - \ 1973
Landbouwhogeschool Wageningen. Promotor(en): J. de Wilde. - Wageningen : Veenman - 107
insecten - plantenplagen - vruchtbomen - boomgaarden - tortricidae - sterilisatie - steriele insecten techniek - genetische gewasbescherming - dieren - territorium - habitats - milieu - plantenziekten - epidemiologie - distributie - adoxophyes orana - microlepidoptera - insects - plant pests - fruit trees - orchards - tortricidae - sterilization - sterile insect release - genetic control - animals - territory - habitats - environment - plant diseases - epidemiology - distribution - adoxophyes orana - microlepidoptera
In apple growing areas, in the Netherlands, Panonychus ulmi Koch and Adoxophyes orana FvR. are the most important pests. Preliminary experiments had shown that P. ulmi could be controlled by predators, but these predators are killed by insecticide sprayings against other pests.As A. orana had already been cultured on an artificial medium, the application of the population sterilization technique was within the realms of possibility. However, the high population density of the species was a disadventage. But if this was counterbalanced by a poor capacity for dispersal, the application of this technique, on a small scale, would be possible. In a simulation model, it was demonstrated that only 100 immigrants per generation per ha, was already too high to make the technique feasible.Three modes of dispersal could be expected: flight, aerial transport of larvae with wind and transport of larvae together with plants and packaging materials. The last possibility was not investigated, it was assumed that it could be reduced to a great extent by appropriate measures.Dispersal by flight was studied in release-recapture experiments. The released moths were cultured on a meridic diet. For marking, either 32P or the dye Calco oil red D was added to the medium. In order to recapture the moths, light traps and sex traps were used. The experiments were carried out in several places, an open field, orchards, and hedgerows. The results obtained indicated that the movement from the release points was small. The greatest distance at which a male was recaptured in these experiments was 250 m. Only in the experiment in which 75500 males were released homogeneously throughout an orchard of 1.5 ha, were the distances, at which males were still recovered, somewhat greater, but the maximum distance was 435 m. For this particular experiment it was calculated that about 1 to 2 percent of the males had left the orchard. It is assumed that, for this kind of biotope, this a representative percentage.In light traps, in general, the numbers of males captured were higher than the numbers of females captured. This could be the result of, either a lower flight activity of the females compared to the males, or an eventual difference in the diurnal flight period of males an females. In laboratory experiments, evidence was obtained for both possibilities.Concerning larval dispersal by means of wind, only qualitative data were obtained. It was demonstrated that it occurs frequently and that characteristic behaviour elements are involved. The larva spins a silken thread when it drops off from a leaf. This thread can be broken by the force of the wind, exerted on the thread. It subsequently breaks near the end at which it is attached to the leaf. The buoyancy of the larva in the air depends on the length of the thread. It was demonstrated that wind velocities of more than 3 m per sec have an immobilizing effect on the locomotion of the newly-hatched larvae. It is likely that this behaviour protects the larva against being blown away from the leaf, with a very short thread. Experiments on phototaxis and geotaxis yielded indications that the behaviour of young larvae makes them vulnerable to this mode of dispersal. The behaviour of older larvae was different in this respect. It is likely that, because of the greater weight, the chances for aerial transport of older larvae are reduced. In a qualitative way, aerial transport can be important. At 20 °C and 70 % R.H. the newly-hatched larva can survive 6 hours starvation. With an air speed of 2 m per sec, transport over more than 40 km is possible. The quantative aspects will be determined by the vegetation that surrounds the egg-mass. In an orchard, a large proportion of the larvae will be sieved out from the air, by the surrounding trees.A. orana has a wide hostplant range. In preliminary experiments the possibility of a variability of response towards different hostplants was investigated. No indications were found.When there is no variability in response towards apple, between A. orana in orchards and A. orana in other biotopes, it is important to know the population densities in these biotopes, especially in the fruit growing areas, where eventually the population sterilization technique is going to be applied. The population densities were estimated in 23 different places, hedgerows, lanes and woods, by releasing a known number of marked moths into the wild population. From the numbers recaptured by means of sex traps, the number of moths of the wild population was calculated, with the aid of the Lincoln-index. The density in woods was found to be lower than the average population density in orchards, but for some hedgerows higher densities were calculated. The calculated densities were highest when a great part of the hedgerows consisted of alder ( Alnus spp.) or hawthorn ( Crateagus sp.).The conclusion of these studies was, that with regard to the dispersal of the adults, there is a fair chance that the population sterilization technique can be applied on a small scale, when the area is surrounded by a zone of 500 m, free from other populations of A.orana. If this is not possible, sterilized adults also have to be released in these populations. In this conclusion, the dispersal of larvae by wind, is not taken into consideration. It is assumed that quantitatively, this mode of dispersal is of minor importance.
|The production of polyhedral viruses from Barathra brassicae to control Adoxophyes reticulana
Ponsen, M.B. - \ 1966
Wageningen : [s.n.] (Mededeling / Laboratorium voor virologie, Landbouwhogeschool no. 40) - 5
biologische bestrijding - organismen ingezet bij biologische bestrijding - insecten - plantenplagen - tortricidae - virussen - adoxophyes orana - microlepidoptera - biological control - biological control agents - insects - plant pests - tortricidae - viruses - adoxophyes orana - microlepidoptera