Evidence for a Second Type of Resistance against Cydia pomonella Granulovirus in Field Populations of Codling Moths.

Cydia pomonella granulovirus (CpGV) is an important biocontrol agent for the codling moth (CM) in organic and integrated apple production worldwide. Previously, Z chromosome-linked dominant resistance in at least 38 CM field populations in Europe was reported, threatening organic apple production. Growers responded by switching to a different resistance-breaking isolate of CpGV that could control these populations. Here, we report a nonuniform response of different CM field populations to CpGV isolates from CpGV genome groups A to E. Even more strikingly, one field population, NRW-WE, was resistant to all known CpGV genome groups except group B. Single-pair crossing experiments with a susceptible strain, followed by resistance testing of the F1 offspring, clearly indicated cross-resistance to CpGV isolates that had been considered to be resistance breaking. This finding provides clear evidence of a second, broader type of CpGV resistance with a novel mode of inheritance that cannot be fully explained by Z-linkage of resistance. IMPORTANCE: CpGV is registered and used in virtually all commercial apple growing areas worldwide and is therefore the most widely used baculovirus biocontrol agent. Recently, resistance to CpGV products was reported in different countries in Europe, threatening organic growers who rely almost exclusively on CpGV products. This resistance appeared to be targeted against a 24-bp repeat in the pe38 gene in isolate CpGV-M of genome group A, which had been used commercially for many years. On the other hand, resistance could be broken by CpGV isolates from CpGV genome groups B to E. Here, we report clear evidence of a second type of field resistance that is also directed against resistance-breaking isolates of CpGV genome groups C, D, and E and which appears not to be targeted against CpGV pe38 Therefore, we propose to differentiate between type I resistance, which is targeted against pe38 of CpGV genome group A, and a novel type II resistance with an unknown molecular target. This finding stresses the need for further adoption of resistance management strategies for CpGV, since growers cannot rely solely on the use of resistance-breaking CpGV isolates.

A Third Type of Resistance to Cydia pomonella Granulovirus in Codling Moths Shows a Mixed Z-Linked and Autosomal Inheritance Pattern.

Different isolates of Cydia pomonella granulovirus (CpGV) are used worldwide to control codling moth larvae (Cydia pomonella) in pome fruit production. Two types of dominantly inherited field resistance of C. pomonella to CpGV have been recently identified: Z-chromosomal type I resistance and autosomal type II resistance. In the present study, a CpGV-resistant C. pomonella field population (termed SA-GO) from northeastern Germany was investigated. SA-GO individuals showed cross-resistance to CpGV isolates of genome group A (CpGV-M) and genome group E (CpGV-S), whereas genome group B (CpGV-E2) was still infective. Crossing experiments between individuals of SA-GO and the susceptible C. pomonella strain CpS indicated the presence of a dominant autosomal inheritance factor. By single-pair inbreeding of SA-GO individuals for two generations, the genetically more homogenous strain CpRGO was generated. Resistance testing of CpRGO neonates with different CpGV isolates revealed that isolate CpGV-E2 and isolates CpGV-I07 and -I12 were resistance breaking. When progeny of hybrid crosses and backcrosses between individuals of resistant strain CpRGO and susceptible strain CpS were infected with CpGV-M and CpGV-S, resistance to CpGV-S appeared to be autosomal and dominant for larval survivorship but recessive when success of pupation of the hybrids was considered. Inheritance of resistance to CpGV-M, however, is proposed to be both autosomal and Z linked, since Z linkage of resistance was needed for pupation. Hence, we propose a further type III resistance to CpGV in C. pomonella, which differs from type I and type II resistance in its mode of inheritance and response to CpGV isolates from different genome groups.IMPORTANCE The baculovirus Cydia pomonella granulovirus (CpGV) is registered and applied as a biocontrol agent in nearly all pome fruit-growing countries worldwide to control codling moth caterpillars in an environmentally friendly manner. It is therefore the most widely used commercial baculovirus biocontrol agent. Since 2005, field resistance of codling moth to CpGV products has been observed in more than 40 field plantations in Europe, threatening organic and integrated apple production. Knowledge of the inheritance and mechanism(s) of resistance is indispensable for the understanding of host response to baculovirus infection on the population level and the coevolutionary arms race between virus and host, as well as for the development of appropriate resistance management strategies. Here, we report a codling moth field population with a new type of resistance, which appears to follow a highly complex inheritance in regard to different CpGV isolates.

Hytrosaviridae: a proposal for classification and nomenclature of a new insect virus family.

Salivary gland hypertrophy viruses (SGHVs) have been identified from different dipteran species, such as the tsetse fly Glossina pallidipes (GpSGHV), the housefly Musca domestica (MdSGHV) and the narcissus bulbfly Merodon equestris (MeSGHV). These viruses share the following characteristics: (i) they produce non-occluded, enveloped, rod-shaped virions that measure 500-1,000 nm in length and 50-100 nm in diameter; (ii) they possess a large circular double-stranded DNA (dsDNA) genome ranging in size from 120 to 190 kbp and having G + C ratios ranging from 28 to 44%; (iii) they cause overt salivary gland hypertrophy (SGH) symptoms in dipteran adults and partial to complete sterility. The available information on the complete genome sequence of GpSGHV and MdSGHV indicates significant co-linearity between the two viral genomes, whereas no co-linearity was observed with baculoviruses, ascoviruses, entomopoxviruses, iridoviruses and nudiviruses, other large invertebrate DNA viruses. The DNA polymerases encoded by the SGHVs are of the type B and closely related, but they are phylogenetically distant from DNA polymerases encoded by other large dsDNA viruses. The great majority of SGHV ORFs could not be assigned by sequence comparison. Phylogenetic analysis of conserved genes clustered both SGHVs, but distantly from the nudiviruses and baculoviruses. On the basis of the available morphological, (patho)biological, genomic and phylogenetic data, we propose that the two viruses are members of a new virus family named Hytrosaviridae. This proposed family currently comprises two unassigned species, G. pallidipes salivary gland hypertrophy virus and M. domestica salivary gland hypertrophy virus, and a tentative unassigned species, M. equestris salivary gland hypertrophy virus. Here, we present the characteristics and the justification for establishing this new virus family.

Overcoming the resistance of codling moth against conventional Cydia pomonella granulovirus (CpGV-M) by a new isolate CpGV-I12.

Recently, codling moth (CM, Cydia pomonella L.) populations with a significantly reduced susceptibility to C. pomonella granulovirus (CpGV) products have been observed in Germany. A novel CpGV isolate, designated CpGV-I12, is able to overcome the CpGV resistance. CpGV-I12 originated from Iran and showed superior efficacy in laboratory bioassays against a resistant CM strain (CpR), which has a 100-fold reduced susceptibility to commercially used isolate CpGV-M. Determination of the median lethal concentration (LC(50)) indicated that CpGV-I12 is nearly as efficient in resistant CpR as CpGV-M in a susceptible CM strain (CpS). Beyond, CpGV-I12 caused superior mortality in CpS. Infection experiments showed that the resistance breaking effect can be observed in all instars of CpR. CpGV-I12 is a promising alternative control agent of CM in orchards where conventional CpGV products fail. In addition, we demonstrate in bioassays with recombinant expressed Cry1Ab that cross-resistance to CpGV and Bacillus thuringiensis products is not likely.

Effects of transgenic corn and Cry1Ab protein on the nematode, Caenorhabditis elegans.

The effects of the insecticidal Cry1Ab protein from Bacillus thuringiensis (Bt) on the nematode, Caenorhabditis elegans, were studied with soil from experimental fields cultivated with transgenic Bt corn (MON810) and with trypsinized Cry1Ab protein expressed in Escherichia coli. The content of Cry1Ab protein was above the detection limit of an ELISA test in only half of the soil samples obtained from transgenic plots, ranging from 0.19 to 1.31 ng g(-1) dry weight. In a laboratory bioassay, C. elegans was exposed to rhizosphere and bulk soil from fields with isogenic or transgenic corn or to solutions of Cry1Ab protein (0, 24, 41, 63, 118, and 200 mg l(-1)) over a period of 96 h, with growth and reproduction serving as the test parameters. Nematode reproduction and growth were significantly reduced in rhizosphere and bulk soil of Bt corn compared with soil from isogenic corn and were significantly correlated with concentrations of the Cry1Ab protein in the soil samples. Moreover, the toxicity of pure Cry1Ab protein to the reproduction and growth of C. elegans was concentration-dependent. As significant inhibition occurred at relatively high concentrations of the Cry1Ab protein (41 mg l(-1)), the effects of the soil samples from Bt corn could not be assigned directly to the toxicity of the Cry1Ab protein. The results demonstrate that bioassays with the nematode, C. elegans, provide a promising tool for monitoring the potential effects of Bt toxins in aqueous medium and soils.

Identification and sequence analysis of the integration site of transposon TCp3.2 in the genome of Cydia pomonella granulovirus.

Recently, we described the isolation and characterisation of the novel lepidopteran transposon TCp3.2, which was found to be inserted into the genome of a spontaneous mutant of the baculovirus Cydia pomonella granulovirus (CpGV). Transposon TCp3.2, which is a member of the Tcl/mariner superfamily, is an apparently defective copy which became stably integrated into the viral genome (Jehle et al., 1997. J. Mol. Evol., in press). In this study, we located, cloned and sequenced a genomic region of 2.5 kb of CpGV which encompasses the insertion site of TCp3.2. The TCp3.2 was inserted at a TA dinucleotide as it is typical for many Tcl/mariner-like transposons. The TA insertion site was localised within a non-translated region downstream of the homologous gene of baculovirus late expression factor 2 (lef-2). Additionally, three other complete open reading frames (ORF35Ra, ORF35Rb, and ORF36L) with unknown functions were identified. Transposon insertion into intergenic regions of viral genomes may contribute to the genotypic variability of baculoviruses without any phenotypic effect.

Characterization of the ecdysteroid UDP-glucosyltransferase gene of a single nucleocapsid nucleopolyhedrovirus of Buzura suppressaria.

A putative ecdysteroid UDP-glucosyltransferase (egt) gene was identified in the single nucleocapsid nucleopolyhedrovirus of Buzura suppressaria (BusuNPV). This is the first egt gene to be characterized in a SNPV, suggesting that egt genes are prevalent in nucleopolyhedroviruses and possibly in all baculoviruses. The open reading frame (ORF) of the gene is 1539 nucleotides (nt) long, encoding a putative protein (EGT) of 513 amino acids (aa) with a M of 58922. The 5' noncoding region contains three possible TATA boxes. A polyadenylation signal, AATAAA, was found downstream of the translation stop codon. A putative signal peptide of 16 residues was present at the N-terminus of the EGT. The BusuNPV egt gene has a high degree of nucleotide and amino acid sequence homology to multiple nucleocapsid (M) NPV egt genes, the highest being to the Spodoptera exigua MNPV egt. A phylogenetic tree of eleven known EGTs was constructed using maximum parsimony analysis.

Uterus didelphys with unilateral imperforate vagina: spontaneous rupture of the vaginal septum.

A 13-year-old girl with abnormal vaginal bleeding was found to have uterus didelphys with unilateral imperforate vagina. A brief discussion of this rare anomaly and its treatment accompanies the case report.

Assessing the risk posed to free-living soil nematodes by a genetically modified maize expressing the insecticidal Cry3Bb1 protein.

Before pest-resistant genetically modified maize can be grown commercially, the risks for soil-beneficial, non-target organisms must be determined. Here, a tiered approach was used to assess the risk to free-living soil nematodes posed by maize genetically modified to express the insecticidal Cry3Bb1 protein (event Mon88017), which confers resistance towards western corn rootworm (Diabrotica virgifera; Coleoptera). The toxicity of purified Cry3Bb1 for the nematode Caenorhabditis elegans was determined using a bioassay and gene expression analysis. In addition, a soil toxicity test was used to assess the effects on C. elegans of rhizosphere soil obtained from plots of an experimental field grown with Mon88017, the near-isogenic cultivar, or either of two conventional cultivars. Finally, the indigenous nematode communities from the experimental field site with Mon88017 and from the control cultivars were analyzed. The results showed a dose-dependent inhibitory effect of Cry3Bb1 on the growth and reproduction of C. elegans, with EC50 values of 22.3 mg l⁻¹ and 7.9 mg l⁻¹, respectively. Moreover, Cry-protein-specific defense genes were found to be up-regulated in the presence of either Cry1Ab or Cry3Bb1. However, C. elegans was not affected by rhizosphere soils from Mon88017 compared to the control plots, due to the very low Cry3Bb1 concentrations, as indicated by quantitative analyses (< 1 ng g⁻¹ soil). Nematode abundance and diversity were essentially the same between the various maize cultivars. At the last sampling date, nematode genus composition in Bt-maize plots differed significantly from that in two of the three non-Bt cultivars, including the near-isogenic maize, but the shift in genus composition did not influence the composition of functional guilds within the nematode communities. In conclusion, the risk to free-living soil nematodes posed by Mon88017 cultivation can be regarded as low, as long as Cry3Bb1 concentrations in soil remain four orders of magnitude below the toxicity threshold.

Genomic analysis of Oryctes rhinoceros virus reveals genetic relatedness to Heliothis zea virus 1.

Oryctes rhinoceros virus (OrV) is an unassigned invertebrate dsDNA virus with enveloped and rod-shaped virions. Two cloned PstI fragments, C and D, of OrV DNA have been sequenced, consisting of 19,805 and 17,146 bp, respectively, and comprising about 30% of the OrV genome. For each of the two fragments, 20 open reading frames (ORFs) of 150 nucleotides or greater with no or minimal overlap were predicted. Ten of the predicted 40 ORFs revealed significant similarities to Heliothis zea virus 1 (HzV-1) ORFs, of which five, lef-4, lef-5, pif-2, dnapol and ac81, are homologues of conserved core genes in the family Baculoviridae, and one is homologous to baculovirus rr1. A baculovirus odv-e66 homologue is also present in OrV. Five ORFs encode proteins homologous to cellular thymidylate synthase (TS), patatin-like phospholipase, mitochondrial carrier protein, Ser/Thr protein phosphatase, and serine protease, respectively. TS is phylogenetically related to those of eukarya and nucleo-cytoplasmic large dsDNA viruses. However, the remaining 25 ORFs have poor or no sequence matches with the current databases. Both the gene content of the sequenced fragments and the phylogenetic analyses of the viral DNA polymerase suggest that OrV is most closely related to HzV-1. These findings and the re-evaluation of the relationship of HzV-1 to baculoviruses suggest that a new virus genus, Nudivirus, should be established, containing OrV and HzV-1, which are genetically related to members of the family Baculoviridae.

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.

On the classification and nomenclature of baculoviruses: a proposal for revision.

Recent evidence from genome sequence analyses demands a substantial revision of the taxonomy and classification of the family Baculoviridae. Comparisons of 29 baculovirus genomes indicated that baculovirus phylogeny followed the classification of the hosts more closely than morphological traits that have previously been used for classification of this virus family. On this basis, dipteran- and hymenopteran-specific nucleopolyhedroviruses (NPV) should be separated from lepidopteran-specific NPVs and accommodated into different genera. We propose a new classification and nomenclature for the genera within the baculovirus family. According to this proposal the updated classification should include four genera: Alphabaculovirus (lepidopteran-specific NPV), Betabaculovirus (lepidopteran-specific Granuloviruses), Gammabaculovirus (hymenopteran-specific NPV) and Deltabaculovirus (dipteran-specific NPV).

The granulin gene region of Cryptophlebia leucotreta granulosis virus: sequence analysis and phylogenetic considerations.

The nucleotide sequence of a 2.2 kb genome region of the baculovirus Cryptophlebia leucotreta granulosis virus (CIGV) encompassing the granulin gene and a second open reading frame, designated ORF909, was determined. The putative granulin ORF comprises 744 nucleotides encoding a polypeptide with a predicted M(r) of 29.3 K. The 5' leader of the granulin gene contains a canonical late baculovirus promoter (ATAAG) and differs only in two nucleotide positions from the sequence of Cydia pomonella granulosis virus (CpGV). Sequence comparison with the granulin genes of different granulosis viruses indicated a very close relationship between CIGV and CpGV of about 96% amino acid identity. ORF909 is 909 nucleotides in length and encodes a potential protein of M(r) 36.2K. It contains two zinc finger-like motifs, similar to ME53, which was previously described for Autographa californica multiple nucleocapsid nuclear polyhedrosis virus. ORF909 protein is significantly shorter than ME53 protein, lacking more than 105 amino acids of the amino terminus of ME53. Both genes contain an early and a late promoter motif suggesting a similar temporal regulation. Homologous sequences to ORF909 are also present upstream of the granulin genes of other granulosis viruses (GVs) indicating that this ORF is common in GVs.

Genome organization of the DNA-binding protein gene region of Cryptophlebia leucotreta granulosis virus is closely related to that of nuclear polyhedrosis viruses.

The nucleotide sequence and genomic organization of a conserved genome region within the EcoRI G fragment of cryptophlebia leucotreta granulosis virus (ClGV) is presented. Five open reading frames (ORFs) were identified which were homologous to those of Autographa californica nuclear polyhedrosis virus (AcMNPV), located upstream of the helicase gene (p143) at 63.6 to 65.6 map units. The ORFs of ClGV and AcMNPV share nucleotide sequence homologies of about 47 to 53% and are very similarly arranged. One of the ClGV ORFs potentially encodes a basic DNA-binding protein with an M(r) of about 7.3 K. Its predicted amino acid sequence mainly consists of multiple arginine and serine residues and shows a 52 to 55% identity to the DNA-binding proteins of AcMNPV and other nuclear polyhedrosis viruses. Its amino acid composition conforms to that of the DNA-binding proteins of Plodia interpunctella granulosis virus and Spodoptera litura granulosis virus.

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.

Intra-specific and inter-specific recombination of tortricid-specific granuloviruses during co-infection in insect larvae.

Intra-specific recombination between two genotypes of the Cryptophlebia leucotreta granulovirus (CrleGV), namely CV3 and CV4, was studied by mixed infection experiments of larvae of C. leucotreta, followed by in vivo cloning and DNA restriction enzyme analyses of isolated progeny viruses. As a prerequisite for these studies a comparative restriction map for of CV3 and CV4 was constructed for eight restriction enzymes. The mixed infection experiments resulted in the isolation of the recombinant CrleGV CVR, which contained restriction sites typical for both parental viruses. Inter-specific recombination between two different granulovirus species, namely CrleGV CV3 and Cydia pomonella granulovirus (CpGV), was analogously investigated by mixed infections of larvae of C. leucotreta. A survey of more than 300 isolated CrleGV and CpGV clones did not reveal any recombinant, which indicated an extremely low recombination frequency in these experiments. By using a specific PCR approach, however, chimerical fragments from the highly conserved granulin gene sequence could be observed in DNA preparations of virus progeny. Cloning and sequencing indicated recombination between CrleGV and CpGV DNA. Our results suggest that recombination between granulovirus genotypes and granulovirus species result in eventually viable viruses and may contribute to the genetic diversity in this virus group.

Analysis of the ecdysteroid UDP-glucosyltransferase gene of Heliothis armigera single-nucleocapsid baculovirus.

An ecdysteroid UDP-glucosyltransferase (egt) gene was identified from the single (S) nucleocapsid nucleopolyhedrovirus of Heliothis armigera (HearNPV). In baculovirus-infected insects the viral enzyme (EGT) plays a pivotal role in abrogating the insect molting process. The open reading frame of the egt gene is 1545 nucleotides long, encoding a putative protein of 515 amino acids with a Mr of 59.1. The 5'-noncoding region contains a putative early (CAGT) and late (TAAG) motif for transcription initiation, a transcription enhancer sequence (CGTCGC) and two TATA boxes. A putative polyA signal, AATAAA, was found downstream of the translation stop codon. A putative signal peptide of 21 residues was present at the N-terminus of the EGT. The HearNPV egt gene has a high degree of nucleotide and amino acid sequence homology to the egt genes of Buzura suppressaria SNPV and Spodoptera exigua MNPV. The HearNPV EGT shares ten conserved motifs with other EGTs. A phylogenetic tree of twelve baculovirus EGTs was constructed by using maximum parsimony analysis, suggesting that SNPVs do not form a separate clade within the baculovirus family.

TCl4.7: a novel lepidopteran transposon found in Cydia pomonella granulosis virus.

After the co-infection of larvae of the lepidopteran Cryptophlebia leucotreta with the two baculoviruses C. leucotreta granulosis virus and Cydia pomonella granulosis virus (CIGV and CpGV, respectively), three CpGV mutants and one CIGV mutant carrying insertions of 0.9 to 4.7 kb have been isolated. By cloning, sequencing, and hybridization analysis, one of these insertions was identified as a transposon-like element derived from the C. leucotreta genome. This element, called TCl4.7, was found in the genome of CpGV which naturally replicates in C. pomonella. Sequence analysis suggested that TCl4.7 is 4726 bp in size, flanked by imperfect inverted terminal repeats of 29 bp, and integrated into the target dinucleotide TA. TCl4.7 encompasses an open reading frame sharing homologies to transposase genes of the Tc1-related transposable elements found in Caenorhabditis and in Drosophila species. The open reading frame might represent a pseudogene since it is missing an ATG start codon. The integration site of TCl4.7 is located in a non-protein-coding region of the CpGV genome at m.u. 9.5. In bioassays the TCl4.7-carrying virus and all the other mutants except for one showed LC50 values similar to those of CpGV and CIGV. This is the first report of the horizontal escape of a transposable element during the in vivo infection of lepidopteran larvae by granulosis viruses.

Horizontal escape of the novel Tc1-like lepidopteran transposon TCp3.2 into Cydia pomonella granulovirus.

We characterized an insertion mutant of the baculovirus Cydia pomonella granulovirus (CpGV), which contained a transposable element of 3.2 kb. This transposon, termed TCp3.2, has unusually long inverted terminal repeats (ITRs) of 756 bp and encodes a defective gene for a putative transposase. Amino acid sequence comparison of the defective transposase gene revealed a distant relationship to a putative transposon in Caenorhabditis elegans which also shares some similarity of the ITRs. Maximum parsimony analysis of the predicted amino acid sequences of Tc1- and mariner-like transposases available from the GenBank data base grouped TCp3.2 within the superfamily of Tc1-like transposons. DNA hybridization indicated that TCp3.2 originated from the genome of Cydia pomonella, which is the natural host of CpGV, and is present in less than 10 copies in the C. pomonella genome. The transposon TCp3.2 most likely was inserted into the viral genome during infection of host larvae. TCp3.2 and the recently characterized Tc1-like transposon TC14.7 (Jehle et al. 1995), which was also found in a CpGV mutant, represent a new family of transposons found in baculovirus genomes. The occasional horizontal escape of different types of host transposons into baculovirus genomes evokes the question about the possible role of baculoviruses as an interspecies vector in the horizontal transmission of insect transposons.