Ana M. Espinoza

Genetic Variability of Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) Populations from Latin America Is Associated with Variations in Susceptibility to Bacillus thuringiensis Cry Toxins

ABSTRACT Bacillus thuringiensis strains isolated from Latin American soil samples that showed toxicity against three Spodoptera frugiperda populations from different geographical areas (Mexico, Colombia, and Brazil) were characterized on the basis of their insecticidal activity, crystal morphology, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of parasporal crystals, plasmid profiles, and cry gene content. We found that the different S. frugiperda populations display different susceptibilities to the selected B. thuringiensis strains and also to pure preparations of Cry1B, Cry1C, and Cry1D toxins. Binding assays performed with pure toxin demonstrated that the differences in the toxin binding capacities of these insect populations correlated with the observed differences in susceptibility to the three Cry toxins analyzed. Finally, the genetic variability of the three insect populations was analyzed by random amplification of polymorphic DNA-PCR, which showed significant genetic diversity among the three S. frugiperda populations analyzed. The data presented here show that the genetic variability of S. frugiperda populations should be carefully considered in the development of insect pest control strategies, including the deployment of genetically modified maize in different geographical regions.

Cauliflower mosaic virus gene II product forms distinct inclusion bodies in infected plant cells

Turnip leaves infected with the aphid transmissible isolate of cauliflower mosaic virus (CaMV Cabb B-JI) showed two types of virus-containing inclusion bodies (IBs), which differed morphologically and in their protein composition when analyzed by immunogold labeling of ultrathin sections. Vacuolated IBs, typical of CaMV infections, contained P62 (the generally accepted IB protein) but lacked P18 (the aphid transmission factor), while electron-lucent IBs did not contain P62 but were the only detectable sites of P18 accumulation within the infected leaf cells. Both types of inclusions were detected in cells of the epidermis, vascular bundles, mesophyll, and spongy parenchyma. Electron-lucent IBs were not found in the aphid nontransmissible isolates of CaMV, Campbell and CM4-184.

The Weedy Rice Complex in Costa Rica. I. Morphological Study of Relationships Between Commercial Rice Varieties, Wild Oryza Relatives and Weedy Types

Weedy rice is a complex of Oryza morphotypes widely distributed in commercial rice fields, which interfere with rice cultivation, seed production, industrial processing and commercialization of this crop in several countries. The objective of this study was to characterize the weedy rice complex of Costa Rica by comparing it with the cultivated and wild rice species found in the country. A collection of weedy rice accessions, representative of the morphotypes found in the country, was established and characterized. Their morphometric relationships were established by comparing 27 morphological traits with commercial rice cultivars, landraces and wild Oryza species and by performing a multivariate analysis. Twenty-one weedy rice morphotypes were identified among 735 weedy accessions by using a three-digit code based on seed characters. Three principal components (PCs) explained 66.25% of the variation observed. The first PC accounted for 36.21% of the variation and separated CCDD genome type Oryza latifolia and O. grandiglumis from AA genome species O. sativa, O. glumaepatula, O. rufipogon and O. glaberrima. The second (18.9%) and third (11.14%) PCs separated the weedy morphotype groups from the AA genome species O.sativa, O. glaberrima and O. rufipogon. The weedy morphotypes were scattered between the indica commercial rice varieties, the cluster landraces–glaberrima and O. rufipogon. Additionally, a group of morphotypes showed intermediate characteristics between O. sativa and O. rufipogon, suggesting that hybridization could have taken place in the past between these species. None of the morphotypes collected in Costa Rica clustered with the allotetraploids CCDD species or O. glumaepatula.

Sequence analysis of rice hoja blanca virus RNA 3

RNA 3 of rice hoja blanca tenuivirus (RHBV) has 2299 nucleotides and resembles RNA 3 of other tenuiviruses such as maize stripe (MStV) and rice stripe (RStV) viruses in potentially coding with an ambisense strategy for two proteins. Both the viral-sense protein of 23K and the complementary-sense protein of 35K have about 46% amino acid identity with the analogous proteins encoded by RNA 3 of MStV and RStV. As the proteins of MStV and RStV have about 65% identity between themselves, RHBV cannot be a South and Central American strain of the Asian RStV. The intergenic region resembles those of other tenuiviruses, being rich in A and U residues, but its predicted folding pattern is unlike those of other tenuiviruses. Instead, the predicted folding of the intergenic region was indistinguishable from that of the coding regions and there was no evidence for a distinct hairpin-loop structure. The significance to the evolution of tenuiviruses of the similarities that the two proteins have with their analogues in other tenuiviruses is discussed.

Parasporal body formation via overexpression of the Cry10Aa toxin of Bacillus thuringiensis subsp. israelensis, and Cry10Aa-Cyt1Aa synergism.

ABSTRACT Bacillus thuringiensis subsp. israelensis is the most widely used microbial control agent against mosquitoes and blackflies. Its insecticidal success is based on an arsenal of toxins, such as Cry4A, Cry4B, Cry11A, and Cyt1A, harbored in the parasporal crystal of the bacterium. A fifth toxin, Cry10Aa, is synthesized at very low levels; previous attempts to clone and express Cry10Aa were limited, and no parasporal body was formed. By using a new strategy, the whole Cry10A operon was cloned in the pSTAB vector, where both open reading frames ORF1 and ORF2 (and the gap between the two) were located, under the control of the cyt1A operon and the STAB-SD stabilizer sequence characteristic of this vector. Once the acrystalliferous mutant 4Q7 of B. thuringiensis subsp. israelensis was transformed with this construct, parasporal bodies were observed by phase-contrast microscopy and transmission electron microscopy. Discrete, ca. 0.9-μm amorphous parasporal bodies were observed in the mature sporangia, which were readily purified by gradient centrifugation once autolysis had occurred. Pure parasporal bodies showed two major bands of ca. 68 and 56 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. These bands were further characterized by N-terminal sequencing of tryptic fragments using matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis, which identified both bands as the products of ORF1 and ORF2, respectively. Bioassays against fourth-instar larvae of Aedes aegypti of spore-crystal complex and pure crystals of Cry10Aa gave estimated 50% lethal concentrations of 2,061 ng/ml and 239 ng/ml, respectively. Additionally, synergism was clearly detected between Cry10A and Cyt1A, as the synergistic levels (potentiation rates) were estimated at 13.3 for the mixture of Cyt1A crystals and Cry10Aa spore-crystal complex and 12.6 for the combination of Cyt1A and Cry10Aa pure crystals.

In situ immunogold labeling analysis of the rice hoja blanca virus nucleoprotein and major noncapsid protein.

Ribonucleoprotein particles (RNPs) of rice hoja blanca virus (RHBV) were purified and used for electron microscopic analysis and antibody production. Antibodies made to RNPs specifically decorated purified RNPs. The RNPs typically showed characteristic tenuivirus morphologies. They were approximately 8 nm in diameter, mostly circular in nature, and exhibited branching and a high degree of superhelicity. When the RNP antibodies were used for in situ immunogold labeling analysis of RHBV-infected tissues, no specific structures were identified, but gold particles were distributed throughout the cytosol of RHBV-infected but not healthy plants. However, amorphous semi-electron opaque inclusion bodies (ASO-IBs) were abundant in cells of RHBV-infected plants. While the ASO-IBs were not labeled with the anti-RNP antiserum, they were specifically labeled with antibodies to the RHBV major noncapsid protein (NCP) and with antibodies to the NCP of another tenuivirus, maize stripe virus.

Sequence of rice hoja blanca tenuivirus RNA-2.

The sequence of rice hoja blanca tenuivirus RNA-2 is analysed and compared to its counterpart in rice stripe tenuivirus. The RNA encodes two proteins, in an ambisense arrangement. The 94 kD pc2, located in the complementary sense RNA, has several features typical of viral membrane (glyco)proteins, and also has regions of local homology to the glycoproteins of the Phleboviruses (Bunyaviridae). The 23 kD pv2 lies in the viral sense RNA and has two small conserved domains that are almost exclusively found in retro-viral membrane glycoproteins. Its genome location is analogous to the NSm protein of several of the Bunyaviridae species, which is thought to have a membrane-related function. The two open reading frames are separated by a large intergenic region which, in common with the other tenuivirus ambisense RNA segments, has a short region that is highly conserved between RStV and RHBV. The significance of these results with respect to the virus structure and gene expression is discussed.

Phylogenetic Placement of a Novel Tenuivirus from the Grass Urochloa plantaginea

Evidence is presented that a tenuivirus recovered from the grass Urochloa plantaginea is probably a novel tenuivirus species, to be called Urochloa hoja blanca virus (UHBV). It is related to both Echinochloa hoja blanca virus (EHBV) and Rice hoja blanca virus (RHBV), and these three form a group distinct from Maize stripe virus (MStV) and Rice stripe virus (RStV). Phylogenetic analysis of the sequence data for RNA-3 and RNA-4 of these viruses supports the hypothesis that EHBV and UHBV may have evolved from an ancestral form of RHBV, precipitated by the introduction of Echinochloa colona and Urochloa plantaginea to America.

Expression of cauliflower mosaic virus ORF II in a baculovirus system.

The cauliflower mosaic virus ORF II encoding the aphid transmission factor (ATF) was mutagenized to introduce a BamHI restriction site upstream from the initiation codon and then cloned into an eukaryotic viral expression vector (Autographa californica nuclear polyhedrosis virus). All recombinant viruses tested in Spodoptera frugiperda (SF21) cells expressed a protein of about 18 kD which comigrated in PAGE with ATF from infected plants. Western blotting using an oligopeptide antiserum to ATF confirmed the identity of the 18-kD protein from infected cells as the product of the ORF II sequences (P18). Subcellular fractionation of cells infected with the recombinant AcMNPV demonstrated that the expressed P18 accumulated intracellularly in an insoluble form. Antiserum was produced in rabbit against the partially purified P18 expressed in SF21 cells. When used to immunogold label ultrathin sections of cauliflower mosaic virus (CaMV)-infected turnip tissue, this antiserum was shown to be highly specific, labelling only the electronlucent inclusion bodies (containing P18) and not other plant cellular components.

Costa Rica: revealing data on public perception of GM crops

This work was supported by The Rockefeller Foundation, the Costa Rica–United States Foundation for Cooperation (CR–USA) and the Vicerrectoria de Investigacion (UCR). The fieldwork was facilitated and coordinated by Nora Garita and Jorge Poltronieri (Proyecto Estructura de la Opinion Publica, UCR). We thank Frank McNeil for the critical review of the manuscript.