The interaction of Lolium latent virus major coat protein with ankyrin repeat protein NbANKr redirects it to chloroplasts and modulates virus infection.

The Lolium latent virus (LoLV) major coat protein sequence contains a typical chloroplast transit peptide (cTP) domain. In infected Nicotiana benthamiana leaf tissue, LoLV coat proteins can be detected at the chloroplast. In transient expression, several N-terminal deletions of the CP sequence, increasing in length, result in disruption of the domain functionality, markedly affecting intracellular localization. A yeast two-hybrid-based study using LoLV CP as bait identified several potentially interacting Arabidopsis host proteins, most of them with chloroplast-linked pathways. One of them, an ankyrin repeat protein, was studied in detail. The N. benthamiana homologue (NbANKr) targets chloroplasts, is able to co-localize with LoLV CP at chloroplast membranes in transient expression and shows a robust interaction with LoLV CP in vivo by BiFC, which has been confirmed by yeast two-hybrid data. Silencing NbANKr genes in N. benthamiana plants, prior to challenging with LoLV by mechanical inoculation, affects LoLV infection, significantly reducing the level of viral RNA in young leaves, compared to levels in control plants, and suggesting an inhibition of virus movement. Silencing of NbANKr has no obvious effect on plant phenotype, but is able to interfere with LoLV infection, opening the way for a new strategy for virus infection control.

A contribution to the knowledge of Quadraseta brasiliensis Goff and Gettinger, 1989 (Trombidiformes: Trombiculidae), with description of the deutonymph instar

In the Neotropical region the genus Quadraseta Brennan, 1970, includes 14 species, with ectoparasitic habits during the larval stage. Quadraseta brasiliensis Goff and Gettinger, 1989, was described from larvae collected on the rodent Hylaeamys megacephalus (Fisher), cited as Oryzomys capito (Olfers). According to these authors, the holotype was deposited in the Museu de Zoologia da Universidade de Sao Paulo and the paratypes were deposited in three other collections: Bernice Pauahi Bishop Museum, Sam Noble Oklahoma Museum of Natural History and United States National Museum of Natural History, however, no type specimens were found in any of these museums. Here we redescribe the larva, describe the deutonymph instar obtained from field-collected larvae, and report new hosts and localities for this species in Brazil. In addition we provide sequences of the 18S ribosomal RNA gene for this species.

A contribution to the knowledge of Quadraseta brasiliensis Goff and Gettinger, 1989 (Trombidiformes: Trombiculidae), with description of the deutonymph instar

In the Neotropical region the genus Quadraseta Brennan, 1970, includes 14 species, with ectoparasitic habits during the larval stage. Quadraseta brasiliensis Goff and Gettinger, 1989, was described from larvae collected on the rodent Hylaeamys megacephalus (Fisher), cited as Oryzomys capito (Olfers). According to these authors, the holotype was deposited in the Museu de Zoologia da Universidade de Sao Paulo and the paratypes were deposited in three other collections: Bernice Pauahi Bishop Museum, Sam Noble Oklahoma Museum of Natural History and United States National Museum of Natural History, however, no type specimens were found in any of these museums. Here we redescribe the larva, describe the deutonymph instar obtained from field-collected larvae, and report new hosts and localities for this species in Brazil. In addition we provide sequences of the 18S ribosomal RNA gene for this species.

Lectin staining of the uterovaginal junction and sperm-storage tubule epithelia in broiler hens.

Mammalian sperm bind to terminal carbohydrates associated with glycoconjugates on the apical surface of oviduct epithelial cells in the caudal region of the oviduct and undergo cellular and molecular modifications associated with capacitation prior to ovulation. In contrast, chicken sperm are stored for up to 23 d in sperm-storage tubules (SST) localized in the uterovaginal junction (UVJ). Little is known of the cellular and molecular mechanisms that regulate sperm storage in and release from the SST. The purpose of this study was to identify glycoconjugates associated with the SST epithelial cell surface using lectins. Virgin hens and hens of higher and lower fertility in egg production for 6 to 16 wk were used in this study. Sections of UVJ mucosa containing SST were stained with fluorescent conjugated lectins and examined by confocal microscopy. Carbohydrate moieties associated with the UVJ and SST epithelia differed in their lectin binding patterns. No differences in the lectin binding patterns within the 2 epithelia were discernible between the virgin and younger and older hens. Minor differences were observed between the higher and lower fertility hens. Only lectins specific for galactose and N-acetylgalactosamine moieties were localized to the luminal surface of the SST. While resident sperm may be closely apposed to the SST epithelial cell apical microvilli, it is unlikely that sperm binding to the microvilli via terminal carbohydrates associated with glycoconjugates is a requisite for prolonged storage. However, the possibility of SST epithelial cell communication with resident sperm via shedding microvillous vesicles characterized by surface glycoconjugates with terminal galactose and N-acetylgalactosamine moieties is currently being investigated.

Evidence for Systemic Infection by Puccinia horiana, Causal Agent of Chrysanthemum White Rust, in Chrysanthemum.

Puccinia horiana, causal agent of the disease commonly known as chrysanthemum white rust (CWR), is a quarantine-significant fungal pathogen of chrysanthemum in the United States and indigenous to Asia. The pathogen was believed to have been eradicated in the United States but recently reappeared on several occasions in northeastern United States. The objective of the study presented here was to determine whether P. horiana could systemically infect chrysanthemum plants, thus providing a means of survival through winters. Scanning and transmission electron microscopy revealed the development of P. horiana on the surface and within leaves, stems, or crowns of inoculated chrysanthemum plants artificially exposed to northeastern U.S. winter temperatures. P. horiana penetrated leaves directly through the cuticle and then colonized the mesophyll tissue both inter- and intracellularly. An electron-dense material formed at the interface between fungal and host mesophyll cells, suggesting that the pathogen adhered to the plant cells. P. horiana appeared to penetrate mesophyll cell walls by enzymatic digestion, as indicated by the absence of deformation lines in host cell walls at penetration sites. The fungus was common in vascular tissue within the infected crown, often nearly replacing the entire contents of tracheid cell walls. P. horiana frequently passed from one tracheid cell to an adjacent tracheid cell by penetration either through pit pairs or nonpitted areas of the cell walls. Individual, presumed, fungal cells in mature tracheid cells of the crown and stems arising from infected crowns suggested that the pathogen might have been moving at least partially by means of the transpiration stream. The demonstration that chrysanthemum plants can be systemically infected by P. horiana suggests that additional disease control measures are required to effectively control CWR.

Travelling with tea: a Tuckerella's tale.

Tuckerella japonica Ehara appears strongly associated with tea (Camellia sinensis (L.) Kuntze, Theaceae) and, due to certain cultural practices in tea production, has in fact become a world traveller, accompanying the greatly coveted tea plant as it spread across the planet. The history of tea production and culture, and its arrival in the USA, provides the backdrop for this traveller's tale. Tuckerella japonica is morphologically similar to T. flabellifera Miller, described from Tasmania in Australia from Bedfordia salicina (Labill.) D.G. (Asteraceae). These two species have historically been misidentified as each other, creating inaccuracies in the collection records. The implications of this in terms of host plant lists and world distribution are discussed further, along with their morphological separation. The male and immature stages of T. japonica are described for the first time. Tuckerella xinglongensis Lin and Fu, from tea in China, is considered a junior synonym of T. japonica. The loss of the ancestral prostigmatan condition of three nymphal stages during ontogeny is confirmed for males of T. flabellifera, which do not retain a tritonymphal stage.

External mouthpart morphology in the Tenuipalpidae (Tetranychoidea): Raoiella a case study.

The use of low-temperature scanning electron microscopy (LTSEM) to study external mouthpart morphology in the Tenuipalpidae, in particular the genus Raoiella, has brought some aspects of the mechanics of feeding in this group into question. In addition, an LTSEM study on the specialized feeding behaviour of Raoiella indica Hirst (Tetranychoidea: Tenuipalpidae) revealed host plant use in this species could be affected by stomatal complex morphology.

Description of Parasitorhabditis frontali n. sp. (Nemata: Rhabditida) from Dendroctonus frontalis Zimmermann (Coleoptera: Scolytidae).

A new Parasitorhabditis species with males and females was discovered from the southern pine beetle Dendroctonus frontalis and its galleries in loblolly pine, Pinus taeda, growing in Mississippi. Females of the new species have a cupola-shaped tail with a small spike; males possess a 2 + (3+2) + 3 ray pattern on the tail fan with ray 10 reaching the margin, and a distinctive stomatal tooth. Parasitorhabditis frontali n. sp. has some similarities to P. hylurgi Massey, 1974 from Hylurgops pinifex in New York, USA, P. terebranus Massey, 1974 from D. terebrans (Olivier, 1795) in Texas USA, P. ligniperdae Fuchs, 1915 from Hylergops ligniperda (Fabricius, 1787) and P. dendroctoni Rühm, 1956 from D. micans (Kugelann, 1794) in Europe, P. ateri Fuchs, 1915 isolated from the beetle Hylastes ater (Paykull, 1800) in Germany, and P. malii Devdariani and Kakulia,1970 from Scolytus mali (Bechstein, 1805) within the republic of Georgia. Morphometrics for 44 species of Parasitorhabditis are provided to update older keys. Parasitorhabditis frontali n. sp. was initially grown on Malt Extract (ME) agar with its own microbial contaminants that included a bacterium and fungus. The nematode also grew and reproduced after slices of ME agar with nematodes and microbial contaminants were transferred to water agar. It was killed by E. coli on NGM agar plates commonly used to raise other Rhabditida. Drawings of diagnostic anatomy and low-temperature SEM images of bodies, heads, and tails are provided for cultured specimens from pine beetle frass.

Constitutive heterochromatin DNA polymorphisms in diploid Medicago sativa ssp. falcata.

A Giemsa C-banding technique was used to study the amount and location of constitutive heterochromatin in diploid (2n = 2x = 16) Medicago sativa ssp. falcata (L.) Arcangeli. Most accessions had the standard C-banding pattern with centromeric bands on all the chromosomes and a prominent heterochromatic band at the nucleolar organizer regions (NOR) of the satellited (SAT) chromosomes. However, we observed in various accessions that constitutive heterochromatic C-bands can exist at the telomeric ends of all the chromosomes. Interstitial bands occurred on the short arms of all chromosomes except for chromosome 3 and on the long arms of chromosomes 1, 2, 3, and 6, only. Rearranged chromosomes such as isochromosomes have been observed for the short arms of chromosomes 2 and 6. This is the first report on the existence of C-banding polymorphisms and the detection of putative isochromsomes in the chromosomes of diploid ssp. falcata which could have contributed to the variation observed in cultivated alfalfa.

Karyotypic analysis of N-banded chromosomes of diploid alfalfa: Medicago sativa ssp. caerulea and ssp. falcata and their hybrid.

Chromosomes of two diploid (2n = 2x = 16) subspecies of Medicago sativa, ssp. caerulea and ssp. falcata, and their hybrid were studied using an N-banding technique. This study was undertaken to develop an N-banded karyotype of ssp. caerulea and ssp. falcata, and determine if the same technique could be used to identify parental chromosomes in hybrids. The chromosomes of ssp. falcata have only centromeric N bands and thus individual chromosomes could not be identified. However, chromosome-specific bands were observed in ssp. caerulea enabling the identification of each of the eight pairs of chromosomes and the development of an idiogram. All chromosomes have a centromeric band and a telomeric band in the short arm. All of the chromosomes, except chromosomes 7 and 8, have interstitial bands in their long arms and chromosome 5 has two faint interstitial bands in its long arm. The satellited chromosome is easily distinguished due to the secondary constriction and characteristic band at the nucleolar organizer region. The differences in banding patterns between these subspecies makes it possible to distinguish chromosomes from each other in their hybrids. This is the first report of the use of N banding to identify the diploid chromosomes of M. sativa ssp. caerulea and ssp. falcata.

Simple sequence repeat DNA markers in alfalfa and perennial and annual Medicago species.

Simple sequence repeat (SSR) or microsatellite DNA markers have been shown to function well in plant and mammalian species for genetic map construction and genotype identification. The objectives of the work reported here were to search GenBank for the presence of SSR-containing sequences from the genus Medicago, to assess the presence and frequency of SSR DNA in the alfalfa (Medicago sativa (L.) L. &L.) genome, and to examine the function of selected markers in a spectrum of perennial and annual Medicago species. The screening of an alfalfa genomic DNA library and sequencing of clones putatively containing SSRs indicated approximately 19 000 (AT)n + (CT)n + (CA)n + (ATT)n SSRs in the tetraploid genome. Inheritance was consistent with Mendelian expectations at four selected SSR loci with different core motifs. Additionally, genotypes of a range of Medicago species, including 10 perennial subspecies of the M. sativa complex and other perennial and annual Medicago species, were analyzed at each of the loci to ascertain the presence, number, and size of SSR alleles at each locus in each genotype. These studies indicate that SSR markers can function in alfalfa for the construction of genetic maps and will also be useful in a range of Medicago species for purposes of assessing genetic relatedness and taxonomic relationships, and for genotype identification.

Karyotypic analysis of C-banded chromosomes of diploid alfalfa: Medicago sativa ssp. caerulea and ssp. falcata and their hybrid.

Chromosomes of two diploid (2n = 2x = 16) subspecies of Medicago sativa ssp. caerulea and ssp. falcata and their hybrid were studied by C-banding. This study was undertaken to improve the C-banding technique for alfalfa chromosomes, develop a C-banded karyotype of the ssp. caerulea and ssp. falcata, and determine if the same C-banding technique could be used to identify parental chromosomes in hybrids. The chromosomes of ssp. falcata have only centromeric bands and thus individual chromosomes could not be identified. One accession of ssp. falcata displayed an interstitial band in the middle of the long arm on the satellite chromosome. However, chromosome-specific bands were observed in ssp. caerulea enabling the identification of each of the eight pairs of chromosomes and the development of a idiogram. All chromosomes had centromeric bands and a terminal band in the short arm except the satellite chromosome (chromosome 8). Interstitial bands were also observed in the short arms, with the exception of chromosome 7. Chromosomes 1, 2, 3, and 8 each had one prominent interstitial band in their long arm. The satellited chromosome is easy to identify because of the presence of the secondary constriction, two bands located on either side of the nucleolar organizer region, and a large terminal band on its long arm. The differences in banding patterns between these subspecies allowed the identification of parental chromosomes in hybrid cells.

Methods of developing a core collection of annual Medicago species.

A core collection is a subset of a large germplasm collection that contains accessions chosen to represent the genetic variability of the germplasm collection. The purpose of the core collection is to improve management and use of a germplasm collection. Core collections are usually assembled by grouping accessions and selecting from within these groups. The objective of this study was to compare 11 methods of assembling a core collection of the U.S. National collection of annual Medicago species. These methods differed in their use of passport and evaluation data as well as their selection strategy. Another objective was to compare core collections with sample sizes of 5%, 10% and 17% of the germplasm collection. Core collections assembled with evaluation data and cluster analysis better represented the germplasm collection than core collections assembled based solely on passport data and random selection of accessions, The Relative Diversity and the logarithm methods generated better core collections than the proportional method. The 5% and 10% sample size core collection were judged insufficient to represent the germplasm collection.

Influence of Zinnia angustifolia HBK genotype on embryonic and vegetative development of Z. angustifolia x Z. elegans Jacq. interspecific hybrids.

Interspecific crosses between Zinnia angustifolia clones (maternal parents) and Z. elegans lines (paternal parents) were performed to investigate postzygotic barriers among Z. angustifolia X Z. elegans hybrids and to determine influence of parental genotype on embryonic and vegetative development of interspecific hybrids. Variation in percentage of emerged seedlings (PES) and percentage of morphologically normal hybrids (PNH) was attributable to Z. angustifolia clones with minor or no effect attributable to Z. elegans lines. Heterogeneity in PES values among Z. angustifolia clones was due to differences in amount of hybrid embryo breakdown and ungerminable seed. Cytological observations of normal and abnormal interspecific hybrids revealed similar chromosome numbers (2n=23) but indicated a low mitotic index for abnormal hybrids. Genetic analysis of PES and PNH suggested control by multiple genes inherited from the Z. angustifolia genome. Adequate sampling of the Z. angustifolia gene pool would permit exploitation of genetic variability present within the species and allow improvements in PES and PNH for interspecific hybrids.

Regeneration of peach plants from callus derived from immature embryos.

Peach plants were repeatedly regenerated from immature embryos but not from callus derived from mature embryos. A white, nodular, highly regenerative callus was obtained when friable, primary callus from immature embryos was transferred from medium containing 4.5 µM 2,4-dichlorophenoxyacetic acid and 0.44 µM benzyladenine (BA) to media containing 0.27 µM α-naphthaleneacetic acid (NAA) and 2.2 µM BA. This callus retained its morphogenetic potential for a minimum of three subcultures. Green nodular callus, that lacked regenerative capacity, was produced from primary callus derived from mature embryos. Maximum regeneration of shoots occurred when highly regenerative callus was transferred to a medium in which the NAA concentration was reduced five times and the BA concentration was increased two times. Regenerated shoots were rooted in the dark on a medium containing 28.5 µM indoleacetic acid. Cytogenetic analysis of regenerated plants indicated that all plants were diploid, 2n = 2x = 16. Phenotypic evaluation of regenerated plants, grown under field conditions, is now in progress.