Yield, biological activity, and field performance of a wild-type Helicoverpa nucleopolyhedrovirus produced in H. zea cell cultures

This paper reports studies of the in vitro production of a virus from Helicoverpa armigera (HaSNPV) and its possible use as a specific Helicoverpa/Heliothis larvicide. Growth kinetics of Helicoverpa zea (H. zea) cells and virus occlusion body yields were compared in three SF900II-based media, namely, SF900II (serum-free), SF900II + 1% serum, or SF900II + 10% serum. Viable cell densities were usually higher in the media supplemented with serum than in the serum-free medium; however, in the serum-free medium, cell diameters were 1.7 times greater (i.e., individual cell volumes were five times larger). Both volumetric production of virus occlusion bodies and production per cell were higher in the serum-free medium than in the media supplemented with serum. However, the infectivity of the occlusion bodies from the serum-free medium was less than that with those from the medium supplemented with 10% serum, when compared in bioassays employing newly hatched larvae. The infectivity of the in vitro produced occlusion bodies was also less than that of in vivo produced occlusion bodies in a commercially available virus product, GemStar. High levels of infection of H. armigera larvae obtained in a preliminary field assessment on preflowering tomatoes using the in vitro produced occlusion bodies indicated the suitability of the in vitro process for biopesticide production.     

Integration of the cytogenetic and genetic linkage maps of Brassica oleracea

We have assigned all nine linkage groups of a Brassica oleracea genetic map to each of the nine chromosomes of the karyotype derived from mitotic metaphase spreads of the B. oleracea var. alboglabra line A12DHd using FISH. The majority of probes were BACs, with A12DHd DNA inserts, which give clear, reliable FISH signals. We have added nine markers to the existing integrated linkage map, distributed over six linkage groups. BACs were definitively assigned to linkage map positions through development of locus-specific PCR assays. Integration of the cytogenetic and genetic linkage maps was achieved with 22 probes representing 19 loci. Four chromosomes (2, 4, 7, and 9) are in the same orientation as their respective linkage groups (O4, O7, O8, and O6) whereas four chromosomes (1, 3, 5, and 8) and linkage groups (O3, O9, O2, and O1) are in the opposite orientation. The remaining chromosome (6) is probably in the opposite orientation. The cytogenetic map is an important resource for locating probes with unknown genetic map positions and is also being used to analyze the relationships between genetic and cytogenetic maps.     

EST-derived SSR markers from defined regions of the wheat genome to identify Lophopyrum elongatum specific loci.

Lophopyrum elongatum, a close relative of wheat, provides a source of novel genes for wheat improvement. Molecular markers were developed to monitor the introgression of L. elongatum chromosome segments into hexaploid wheat. Existing simple sequence repeats (SSRs) derived from genomic libraries were initially screened for detecting L. elongatum loci in wheat, but only 6 of the 163 markers tested were successful. To increase detection of L. elongatum specific loci, 165 SSRs were identified from wheat expressed sequence tags (ESTs), where their chromosomal positions in wheat were known from deletion bin mapping. Detailed sequence analysis identified 41 SSRs within this group as potentially superior in their ability to detect L. elongatum loci. BLASTN alignments were used to position primers within regions of the ESTs that have sequence conservation with at least 1 similar EST from another cereal species. The targeting of primers in this manner enabled 14 L. elongatum markers from 41 wheat ESTs to be identified, whereas only 2 from 124 primers designed in random positions flanking SSRs detected L. elongatum loci. Addition and ditelosomic lines were used to assign all 22 markers to specific chromosome locations in L. elongatum. Nine of these SSR markers were assigned to homoeologous chromosome locations based on their similar position in hexaploid wheat. The remaining markers mapped to other L. elongatum chromosomes indicating a degree of chromosome rearrangements, paralogous sequences and (or) sequence variation between the 2 species. The EST-SSR markers were also used to screen other wheatgrass species indicating further chromosome rearrangements and (or) sequence variation between wheatgrass genomes. This study details methodologies for the generation of SSRs for detecting L. elongatum loci.     

Towards new sources of resistance to the currant-lettuce aphid (<Emphasis Type="Italic">Nasonovia ribisnigri</Emphasis>)

Domesticated lettuce varieties encompass much morphological variation across a range of crop type groups, with large collections of cultivars and landrace accessions maintained in genebanks. Additional variation not captured during domestication, present in ancestral wild relatives, represents a potentially rich source of alleles that can deliver to sustainable crop production. However, these large collections are difficult and costly to screen for many agronomically important traits. In this paper, we describe the generation of a diversity collection of 96 lettuce and wild species accessions that are amenable to routine phenotypic analysis and their genotypic characterization with a panel of 682 newly developed expressed sequence tag (EST)-linked KASP? single nucleotide polymorphism (SNP) markers that are anchored to the draft Lactuca sativa genome assembly. To exemplify the utility of these resources, we screened the collection for putative sources of resistance to currant-lettuce aphid (Nasonovia ribisnigri) and carried out association analyses to look for potential SNPs linked to resistance.     

Aerenchymatous phellem in hypocotyl and roots enables O2 transport in Melilotus siculus

? Aerenchymatous phellem (secondary aerenchyma) has rarely been studied in roots. Its formation and role in internal aeration were evaluated for Melilotus siculus, an annual legume of wet saline land. ? Plants were grown for 21 d in aerated or stagnant (deoxygenated) agar solutions. Root porosity and maximum diameters were measured after 0, 7, 14 and 21 d of treatment. Phellem anatomy was studied and oxygen (O(2)) transport properties examined using methylene blue dye and root-sleeving O(2) electrodes. ? Interconnecting aerenchymatous phellem developed in hypocotyl, tap root and older laterals (but not in aerial shoots), with radial intercellular connections to steles. Porosity of main roots containing phellem was c. 25%; cross-sectional areas of this phellem were threefold greater for stagnant than for aerated treatments. Root radial O(2) loss was significantly reduced by complete hypocotyl submergence; values approached zero after disruption of hypocotyl phellem below the waterline or, after shoot excision, by covering hypocotyl phellem in nontoxic cream. ? Aerenchymatous phellem enables hypocotyl-to-root O(2) transport in M. siculus. Phellem increases radially under stagnant conditions, and will contribute to waterlogging tolerance by enhancing root aeration. It seems likely that with hypocotyl submerged, O(2) will diffuse via surface gas-films and internally from the shoot system.     

CSF1R-dependent macrophages control postnatal somatic growth and organ maturation

Homozygous mutation of the Csf1r locus (Csf1rko) in mice, rats and humans leads to multiple postnatal developmental abnormalities. To enable analysis of the mechanisms underlying the phenotypic impacts of Csf1r mutation, we bred a rat Csf1rko allele to the inbred dark agouti (DA) genetic background and to a Csf1r-mApple reporter transgene. The Csf1rko led to almost complete loss of embryonic macrophages and ablation of most adult tissue macrophage populations. We extended previous analysis of the Csf1rko phenotype to early postnatal development to reveal impacts on musculoskeletal development and proliferation and morphogenesis in multiple organs. Expression profiling of 3-week old wild-type (WT) and Csf1rko livers identified 2760 differentially expressed genes associated with the loss of macrophages, severe hypoplasia, delayed hepatocyte maturation, disrupted lipid metabolism and the IGF1/IGF binding protein system. Older Csf1rko rats developed severe hepatic steatosis. Consistent with the developmental delay in the liver Csf1rko rats had greatly-reduced circulating IGF1. Transfer of WT bone marrow (BM) cells at weaning without conditioning repopulated resident macrophages in all organs, including microglia in the brain, and reversed the mutant phenotypes enabling long term survival and fertility. WT BM transfer restored osteoclasts, eliminated osteopetrosis, restored bone marrow cellularity and architecture and reversed granulocytosis and B cell deficiency. Csf1rko rats had an elevated circulating CSF1 concentration which was rapidly reduced to WT levels following BM transfer. However, CD43^hi non-classical monocytes, absent in the Csf1rko, were not rescued and bone marrow progenitors remained unresponsive to CSF1. The results demonstrate that the Csf1rko phenotype is autonomous to BM-derived cells and indicate that BM contains a progenitor of tissue macrophages distinct from hematopoietic stem cells. The model provides a unique system in which to define the pathways of development of resident tissue macrophages and their local and systemic roles in growth and organ maturation.     

Kinetics of Colonization of Adult Queensland Fruit Flies (Bactrocera tryoni) by Dinitrogen-Fixing Alimentary Tract Bacteria

The average total population of bacteria remained constant in the alimentary tracts of adult laboratory-raised Queensland fruit flies (Bactrocera tryoni) although the insects had ingested large numbers of live bacteria as part of their diet. The mean number of bacteria (about 13 million) present in the gut of the insects from 12 to 55 days after emergence was not significantly modified when, at 5 days after emergence, the flies were fed antibiotic-resistant bacteria belonging to two species commonly isolated from the gut of field-collected B. tryoni. Flies were fed one marked dinitrogen-fixing strain each of either Klebsiella oxytoca or Enterobacter cloacae, and the gastrointestinal tracts of fed flies were shown to be colonized within 7 days by antibiotic-resistant isolates of K. oxytoca but not E. cloacae. The composition of the microbial population also appeared to be stable in that the distribution and frequency of bacterial taxa among individual flies exhibited similar patterns whether or not the flies had been bacteria fed. Isolates of either E. cloacae or K. oxytoca, constituting 70% of the total numbers, were usually dominant, with oxidative species including pseudomonads forming the balance of the population. Antibiotic-resistant bacteria could be spread from one cage of flies to the adjacent surfaces of a second cage within a few days and had reached a control group several meters distant by 3 weeks. Restriction of marked bacteria to the population of one in five flies sampled from the control group over the next 30 days suggested that the bacterial population in the gut of the insect was susceptible to alteration in the first week after emergence but that thereafter it entered a steady state and was less likely to be perturbed by the introduction of newly encountered strains. All populations sampled, including controls, included at least one isolate of the dinitrogen-fixing family Enterobacteriaceae; many were distinct from the marked strains fed to the flies. Nitrogenase activity detected by the acetylene reduction assay was associated with flies fed dinitrogen-fixing bacteria as well as with control groups given either no supplement or free access to a yeast hydrolysate preparation. Nitrogen fixed from the atmosphere may supplement the nutrition of the alimentary tract microbial population of B. tryoni. Transmission electron microscopy showed that the principal site of bacterial colonization in the abdominal alimentary tract was the lumen of the midgut inside the peritrophic membrane. No intracellular symbionts were seen in the gut tissues nor were bacteria found attached to the cuticular folds of the hindgut. The ultrastructure of the gut resembled that of other fly genera except that the intercellular spaces between rectal epithelial cells were more extensive, suggesting a role for unspecialized epithelium in water and solute uptake in B. tryoni.