Identification of additional sources of resistance to Puccinia striiformis f. sp. hordei (PSH) in a collection of barley genotypes adapted to the high input condition

A total of 336 barley genotypes consisting of released cultivars, advanced lines, differentials and local landraces from the ICARDA barley breeding programme were screened for seedling and adult‐plant resistances to barley stripe rust pathogen (Puccinia striiformis f. sp. hordei [PSH]). Seedling resistance tests were undertaken at Shimla, India by inoculating 336 barley genotypes with five prevalent PSH races [Q (5S0), 24 (0S0‐1), 57 (0S0), M (1S0) and G (4S0)] in India. Barley genotypes were also evaluated at the adult‐plant stage for stripe rust resistance at Durgapura (Rajasthan, India) in 2013 and 2014, and at Karnal (Haryana, India) in 2014 under artificial PSH infection in fields, using a mixture of the five races. Twelve barley genotypes (ARAMIR/COSSACK, Astrix, C8806, C9430, CLE 202, Gold, Gull, Isaria, Lechtaler, Piroline, Stirling, and Trumpf) were resistant to all five PSH races at the seedling and adult‐plant stages. Two of these genotypes, Astrix and Trumpf, were part of international differentials and reveal that five races were avirulent to genes Rps4 (yr4), rpsAst, rpsTr1 and rpsTr2. These genes were highly effective against PSH races prevalent in India. The virulence/avirulence formula reported in this study helped to determine the effectiveness of PSH resistance genes against Indian races. Forty‐five genotypes showed adult‐stage plant resistance (APR) in the field. The identified PSH resistant genotypes may possess novel resistance genes and might serve as potential donors of PSH resistance at seedling and APR in the future. Further research is needed to determine the nature of resistance genes through allelic studies and mapping of these genes.     

Epithelial oral mucosal cells: Do they behave differently when exposed to oral carcinogens?

Objective

To assess the level of maturation and proliferation of epithelial cells and the correlation with immunocytochemical expression of adhesion (E‐cadherin) and cell differentiation (involucrin) markers.

Methods

Cytopathological samples were obtained from four groups of patients: control (CG, n=30); alcohol/tobacco (ATG, n=31), leucoplakia (LG, n=31), and squamous cell carcinoma (SCCG, n=22). Cytopathological smears were collected from all groups for AgNOR, Papanicolaou and immunocytochemical staining.

Results

There was an increase in anucleated cells in ATG compared to CG and in LG compared to lesion‐free groups (P<.05). In addition, there was a higher rate of intermediate cells in lesion‐free groups than in LG (P=.001). When these findings were correlated with positive E‐cadherin expression, there was a smaller number of anucleated and intermediate cells (P<.05). The proliferation rate was higher in the SCCG than in the CG (P<.05) and in the ATG compared to LG (P<.05). Moreover, cell proliferation increased in the presence of positive E‐cadherin expression in the ATG and LG. No statistically significant results were obtained for involucrin analysis.

Conclusion

Cytopathology combined with quantitative techniques such as Papanicolaou, AgNOR, and immunocytochemical expression of E‐cadherin detects changes associated with oral carcinogenesis. The innovative approach used in this study allows assessing the expression of cell adhesion (E‐cadherin) and differentiation (involucrin) markers by means of oral mucosal cytopathology. The E‐cadherin imunocytochemical expression indicated changes associated with the oral carcinogenesis process. An increase in cell proliferation rate in oral squamous cell carcinoma group was associated with the lower immunoexpression of E‐cadherin. Cytopathology combined with quantitative techniques and immunocytochemical expression of E‐cadherin may detect early alterations associated with oral carcinogenesis.     

Argyrophilic nucleolus-associated chromatin in epidermal cells ofVicia faba induced by cold treatment

Summary The effect of growth temperature on the structural organization of the nucleolus in the epidermal cells ofVicia faba was studied with the silver staining technique at the light and electron microscopic levels. In plants grown at 22 °C, the nucleoli of the epidermal cells were poorly developed, most were less than 3 m in diameter and they occasionally carried minute fluff-like or particle-like accessories. When the plants were transferred into 5 °C incubator, moderately silver-impregnated spherules (MIS) with diameters of about 1.0 to 1.5 m were discerned on the surfaces of the nucleoli. The incidence of nucleoli with the MIS rapidly increased within a few days and thereafter increased little by little up to 40 days at which time 90% of the nucleoli carried the MIS. The unfused nucleoli usually had a single MIS but most of the fused nucleoli had two MIS; in other words, most cells had two MIS per cell. On the other hand, when the plants grown for a prolonged time at 5 °C were transferred back into the 22 °C incubator, the proportion of nucleoli with the MIS drastically decreased within a day. Silver staining at the electron microscopic level revealed that the MIS exactly corresponded with the compact block of nucleolus-associated chromatin, since this compact chromatin block was significantly covered with silver grains while other chromatin was not. The present findings suggest that growth at low temperature allows incorporation of the argyrophilic nucleolar substance into the compact block of nucleolus-associated chromatin, resulting in the appearance of the MIS.     

An epidermal proliferative unit-like structure in the epithelium of mouse bladder observed by backscattered electron imaging

Summary The simultaneous use of a silver-staining technique, backscattered electron imaging and stereo-tilts has made it possible to visualize the spatial distribution of cell nuclei in the stretched epithelium of the bladder of mice. This study has led to the observation that a structural organization resembling the epidermal proliferative unit, previously found in the skin exists also in bladder epithelium. However, the proliferative unit in the bladder was different in that it contained a higher number of cells per unit, and an absence of columns of inactive squamous cells. These findings may indicate that epidermal proliferative unit-like structures represent a common form of organization in some epithelia.     

Genome Instability in interspecific cell hybrids II. Aminopterin resistance and gene amplification in lines arising from fusions of cells from divergent mammalian species

In order to investigate instances of genetic instability in divergent cell hybrids, we studied several RAT-resistant colonies recovered from fusions between HPRT or TK-deficient rodent cells and marsupial or monotreme cells. Most of these colonies proved to lack HPRT or TK activity and to have survived by acquiring resistance to aminopterin; such aminopterin-resistant lines were never recovered from parent cells subjected to HAT selection. Two of the aminopterin-resistant hybrids over-produced DHFR, and possessed either double minutes or an abnormally banded region, the cytological manifestations of gene amplification. Selection in higher aminopterin concentrations yielded a highly resistant line with 100X wild-type DHFR activity and a large homogeneously staining region. We suggest that interspecific cell hybrids are predisposed to gene amplification and may also show many other types of genetic and chromosomal instability, possibly thein vitro equivalent of the “genomic shock” phenomena described for interstrain or interspecies hybrids of plants or animals. This paper, no. II in a series by these authors, reached the Editorial Office on the date given, although it had been mailed earlier than paper no. III; the latter paper also appears in this number — Eds.     

The staining of fish otoliths for age determination

Attempts were made to find methods for the staining of fish otoliths which would give results comparable to those of the Christensen burning technique. A variety of different histological stains and otoliths of different species were experimented with. Otoliths of sole, turbot, brill and scad gave best results when sectioned and stained in acidified Neutral Red, whereas those of cod, hake, whiting, plaice and grey mullet showed up the annuli better when dyed in aqueous Aniline Blue or Toluidine Blue and then sectioned. Small, translucent otoliths such as those of pelagic species may be enhanced by staining in Eosin Y.     

Micropropagation of yellow cedar (Chamaecyparis nootkatensis)

Mature yellow cedar (Chamaecyparis nootkatensis (D. Don) Spach) embroys were exposed to a range of N⁶-benzyladenine concentrations in a variety of culture media generally used for conifer caulogenesis. All seven media supported the induction of adventitious shoots but Schenk & Hildebrandt medium was the best. The best cytokinin level of N⁶-benzyladenine was 0.35 mg 1^-1. This resulted in an average of 4–5 large adventitious shoots per explant. Shoots arose primarily from the cotyledons regardless of whether they were in contact with the medium or not. Embryos from seeds stratified four weeks at 21°C and eight weeks at 5°C were more caulogenic than unstratified controls. An additional four weeks at 5°C caused a change in the pattern of shoot induction in that shoots arose from the hypocotyl as well as the cotyledons. Shoots elongated on basal Schenk & Hildebrandt medium. The best rooting response was obtained under non-sterile greenhouse conditions where approximately 60% of the shoots formed roots. Over a 12-month period the average shoot height ranged between 10–13.9 cm with a stem diameter of 2.29–2.68 mm. These propagules are still being grown under forest nursery conditions.     

The innervation of the corpus cardiacum of Locusta migratoria: A neuroanatomical study with the use of Lucifer yellow

Summary Neural connections of the corpus cardiacum (CC) in the African locust, Locusta migratoria, were labelled with the fluorescent tracer Lucifer yellow. (1) Unilateral anterograde labelling of the nervus corporis cardiaci I revealed fluorescent fibres in the storage lobe of the CC (CCS). Some fluorescent fibres in the CCS closely approached the ipsilateral border of the glandular lobes of the CC (CCG). Fluorescent fibres also projected into the neuropile of the hypocerebral ganglion via the ipsilateral nervi cardiostomatogastrici I and II, and from there into the oesophageal nerves. (2) Unilateral anterograde labelling of the nervus corporis cardiaci II revealed fluorescent fibres in the CCS and in the ipsilateral CCG. Fluorescent fibres also projected via the ipsilateral nervus corporis allati I into the corpus allatum. (3) Unilateral retrograde labelling of the nervus corporis allati I revealed a distinct fluorescent nerve tract that runs through the CCS and into the nervus corporis cardiaci II. The tract arises from about eight cell bodies in the brain at the rostroventral side of the ipsilateral calyx of the mushroom body. (4) Labelling of the recurrent nerve revealed fluorescent fibres and some fluorescent cell bodies in the hypocerebral ganglion and, via the nervi cardiostomatogastrici I and II, also in the CCS. Fluorescent fibres were also present in the oesophageal nerves.     

Immunoassay Comparison of Haplosporidan Spores from Teredo navalis and Haplosporidium nelsoni Spores from Crassostrea virginica

Spores of a haplosporidan infecting Teredo navalis Linnaeus have been described as morphologically indistinguishable from spores of Haplosporidium nelsoni . To test the hypothesis that these organisms are conspecific, a colloidal gold immunoassay was used to compare antigenic characteristics of the spores from both hosts. Rabbit antibody to formalin-fixed spores from T. navalis was tested against paraffin sections of Crassostrea virginica infected with spores of H. nelsoni and against paraffin sections of infected 7". navalis . Application of primary antibody was followed by addition of affinity purified goat anti-rabbit IgG coaled on 5-nm colloidal gold particles. The reaction was enhanced by precipitation of metallic silver; a positive reaction appeared as a dark brown to black signal at the site of each antigen-antibody complex. Haplosporidium nelsoni spores did not react when assayed with the antibody made to spores from T. navalis . Spores from infected T. navalis tissue reacted positively with rabbit antibody. This result indicates that the spores from the 2 hosts are antigenicaliy distinct and suggests that they are different species.     

Detection of IgG and IgE antibodies to Aspergillus fumigatus in human sera by immunogold assay

An immunogold assay (IGA) was developed to detect IgG and IgE antibodies to Aspergillus fumigatus. Sixteen sera from patients with allergic bronchopulmonary aspergillosis (ABPA), aspergilloma, and normal controls were studied. All sera were also evaluated for antibodies against A. fumigatus by biotin-avidin linked enzyme immunosorbent assay (BALISA) and by agar gel double diffusion method. A. fumigatus specific IgG and IgE antibodies could be detected by IGA in all the patients' sera but not in the sera of normal controls. Both IgG and IgE antibodies to A. fumigatus could be demonstrated in all the sera by BALISA and normal controls showed only low levels of these antibodies. There was a positive correlation between the degree of reactivity detected by IGA, the BALISA titer and the precipitins by agar gel diffusion. It can be concluded that IGA is a reliable, sensitive and simple method capable of detecting both IgG and IgE antibodies against A. fumigatus in patient serum.