Characterization of mutations in the two-component histidine kinase gene that confer fludioxonil resistance and osmotic sensitivity in the os-1 mutants of Neurospora crassa

Osmotic-sensitive (os-1) mutant alleles in Neurospora crassa exhibit resistance to dicarboximides, aromatic hydrocarbons and phenylpyrroles. We have previously reported that the os-1 mutants can be classified into two groups based on their resistance to fungicides and osmotic stress: type I, which are highly resistant to iprodione and fludioxonil but moderately sensitive to osmotic stress, and type II, which are highly sensitive to osmotic stress but moderately resistant to fungicides. To explain the mechanism of resistance to these fungicides, we cloned and sequenced the mutant os-1 genes that encode putative osmo-sensing histidine kinase. Within the os-1 gene product (Os1p), the type I strains, NM233t and Y256M209, carried a stop codon at amino acid position 308 and a frameshift at amino acid position 294, respectively. These mutation sites were located on the upstream of histidine kinase and the response regulator domains of Os1p, strongly suggesting that type I strains are null mutants. The null mutants, NM233t and Y256M209, were highly resistant to iprodione and fludioxonil; thus Os1p is essential for these fungicides to express their antifungal activity. The amino acid changes in Os1p, 625Pro from Leu, 578Val from Ala, and 580Arg from Gly were found in the type II strains, M16, M155-1 and P5990, respectively. Os1p is novel in having six tandem repeats of 90 amino acids in the N terminal. Each amino acid change of the type II strains was located on the fifth unit of six tandem repeats. Type II strains with single amino acid changes were more sensitive to osmotic stress than the null mutants (type I), indicating that the amino acid repeats of Os1p were responsible for an important function in osmo-regulation.     

Genetic diversity and mixed infections of begomoviruses infecting tomato, pepper and cucurbit crops in Nicaragua

Begomoviruses were detected in Nicaraguan fields of tomato ( Lycopersicon esculentum ) and adjacently growing plants of pepper ( Capsicum annuum ), chilli pepper ( C . baccatum ), cushaw ( Cucurbita argyrosperma ) and Mexican fireplant ( Euphorbia heterophylla ) using polymerase chain reaction (PCR) and universal begomovirus primers. All tomato and Mexican fireplant plants showing symptoms were infected with begomoviruses, while only 30–46% of the pepper, chilli pepper and cushaw plants showing symptoms tested virus-positive. No begomoviruses were found in potato. The virus species were provisionally identified by sequencing 533 bp of the viral coat protein gene ( AV1 ). Tomato severe leaf curl virus (ToSLCV), Tomato leaf curl Sinaloa virus (ToLCSinV) and Pepper golden mosaic virus (PepGMV) were found to infect both tomato and pepper. A new provisional species designated Tomato leaf curl Las Playitas virus (ToLCLPV) was detected in a tomato plant. Squash yellow mottle virus (SYMoV) and PepGMV were found in cucurbits, the latter for the first time in this host. Euphorbia mosaic virus (EuMV) was detected in Mexican fireplant. Sequencing of a larger number of PCR-amplified clones from selected plants revealed intraspecific viral sequence variability, and also multiple begomovirus infections which could represent up to three species in a single tomato or cushaw plant. Phylogenetic grouping of virus sequences did not correlate with the host of origin.     

Leishmaniasis in Canis familiaris – a Report About Microvascular and Cellular Architecture of the Infected Spleen

Leishmaniases are a globally widespread group of parasitic diseases from the group of anthropozoonoses. They are caused by an intracellular protozoan parasite that causes a wide spectrum of diseases in humans and dogs worldwide. In fact, the dog is considered one of the most important reservoir. The spleen is one of the several haematopoietic and immunocompetent organs involved. As this organ is a blood filter, the authors looked for the expression of this infection over the microvascular environment and modifications of the spleen cell population related to immunological responses to this parasitic condition.
Tools:  Scanning electronic microscopy over intact tissue and corrosion casts, transmission electronic microscopy, histology and immunohistochemistry (MAC 387 and CD3).
Results:  Our results show three important modifications concerning the microvascular architecture of the spleen when compared to the normal pattern. A striking scarcity of the sinusoidal system sheath that surrounds the central arteries of the white pulp, a huge development of pulp venules and veins, differentiation of typical features of high endothelial venular cells.
Remarks:  According to our results it seems that independent of the virulence of the parasite involved and the kind of immunity prevalent in a particular host, the spleen develops blood dynamic conditions that allows a sluggish blood flow so that cells involved in immunological processes can proliferate and differentiate and it also contributes to the trapping of lymphocytes to the area through the differentiation of typical characteristics of HEV endothelial cells.     

Hormonal and Metabolic Profiles of High-yielding Dairy Cows Prior to Ovarian Cyst formation or First Ovulation Post Partum

The present study aimed to investigate the pathogenesis of cystic ovarian disease (COD) in high-yielding dairy cows postpartum (pp). Hormonal and metabolic profiles during the first 3 weeks pp as well as during the final week prior to ovulation/cyst formation, were compared between dairy cows that developed either an ovulatory follicle (OV) or a cyst (CYST) < day 60 pp. Thirty-four lactations of 28 high-yielding (9500 kg/305 days) Holstein-Friesian dairy cows were studied. Ovaries of cows were scanned twice a week from day 10 pp on, until ovulation/cyst formation. Milk yield data, body condition scores and blood samples, for determination of oestradiol-17beta, insulin, beta-OH-butyrate and non-esterified fatty acids, were collected simultaneously. Milk samples for progesterone analysis were collected daily. Four lactations were excluded from further analysis because of irregular pp ovarian cyclicity, excluding COD. Eight lactations (26.7%) developed a cyst, while 22 lactations ovulated < days 60 pp. Ovulation and cyst formation occurred at similar times pp. Metabolic and hormonal profiles did not differ between CYST and OV lactations during the first 3 weeks pp. In the final week prior to cyst formation/ovulation, insulin concentrations were lower in CYST than in OV lactations while no differences were observed for any of the other parameters tested. In two lactations, cyst formation was preceded by suprabasal progesterone and increased oestradiol-17beta concentrations. These results suggest that cyst formation in high-yielding dairy cows pp is associated with lower insulin levels but not with other distinct hormonal and metabolic alterations. However from this study, we cannot exclude the involvement of subtle hormonal and metabolic changes in the pathogenesis of ovarian cysts. Suprabasal progesterone, and altered oestradiol-17beta concentrations, seem to play a minor role in cyst formation.     

Corneal endothelium of the Magellanic penguin (Spheniscus magellanicus) by scanning electron microscopy.

The corneal endothelium is essential for the maintenance of the corneal transparency. The aim of this study was to examine the morphology of the endothelial surface and perform morphometric analysis of the normal corneal endothelial cells of the Magellanic penguin (Spheniscus magellanicus) using scanning electron microscopy. The present work demonstrates that the corneal endothelium of the Magellanic penguin is similar to those described in other vertebrates.     

Cutting weeds with a CO2 laser

Stems of Chenopodium album . and Sinapis arvensis . and leaves of Lolium perenne . were cut with a CO₂ laser or with a pair of scissors. Treatments were carried out on greenhouse-grown pot plants at three different growth stages and at two heights. Plant dry matter was measured 2 to 5 weeks after treatment. The relationship between dry weight and laser energy was analysed using a non-linear dose–response regression model. The regression parameters differed significantly between the weed species. At all growth stages and heights S. arvensis was more difficult to cut with a CO₂ laser than C. album . When stems were cut below the meristems, 0.9 and 2.3 J mm⁻¹ of CO₂ laser energy dose was sufficient to reduce by 90% the biomass of C. album and S. arvensis respectively. Regrowth appeared when dicotyledonous plant stems were cut above meristems, indicating that it is important to cut close to the soil surface to obtain a significant effect. When cutting L. perenne plants with 2-true leaves at a height of 2 cm from the soil surface with a laser, the biomass decreased significantly compared with plants cut by scissors, indicating a delay in regrowth. This delay was not observed for the dicotyledonous plants nor for the other growth stages of L. perenne .     

Optimization of production of nucleopolyhedrovirus ofHelicoverpa armigera throughout larval stages

Studies were carried out to optimize production of nucleopolyhedrovirus of American bollworm,Helicoverpa armigera (Hübner), by treating larvae individually with an inoculum dose that allowed maximal larval growth and also gave the highest occlusion bodies (OB) yield/larva. The maximum virus yield of 12.2x 10⁹ OB/larva was obtained when 6-day-old larvae were fed individually with a dose of 1 x 10³ OB. Topical spiracular treatment of larvae as old as 8 days with 10 μ of 2x 10⁷ OB ml^-1 gave the highest yield, of 15.2x 10⁹ OB from 13-day-old larvae, of 12.8x 10⁸ OB from prepupae and of 1.49x 10⁸ OB from pupae at the time of their death. These studies showed that dietary inoculum is the best route for 6-day-old larvae and topical spiracular treatment is the best for 8-day-old larvae.     

The role ofPseudomonas spp. and competition for carbon,nitrogen and iron in the enhancement of appressorium formation byColletotrichum coccodes on velvetleaf

Colletotrichum coccodes is currently being investigated as a mycoherbicide against the weed velvetleaf (Abutilon theophrasti). Two isolates ofPseudomonas spp. (Ps2 and Ps5) reduced the percentage of germ tubes and increased appressorial formation ofC. coccodes on detached leaves of velvetleaf. A study was conducted to see whether this effect could be attributed to competition for nutrients or iron betweenC. coccodes andPseudomonas spp. Ps2 and Ps5 had no effect on early spore germination, but reduced the percentage of germ tubes at 24 and 30 h, compared to the nontreated control. This reduction was diminished by the addition of nutrients but not Fe³⁺. Ps2 and Ps5 stimulated the formation of dark-coloured appressoria without germ tubes (AWGT), but this stimulation was diminished by the addition of nutrients or Fe³⁺. Germ tube branching at 30 h was also inhibited by the bacteria, but was not diminished by the addition of nutrients or iron. EDTA stimulated conidial germination at 10 h, which was reduced by the addition of Fe³⁺. However, EDTA did not stimulate the formation of appressoria (AWGT). These results suggest that the reduction in the percentage of germ tubes and the increase in the percentage of appressoria induced by the bacteria may be due to the competition for carbon or nitrogen. Iron competition may also be involved in the stimulation of appressorial formation, but not in the reduction in germ tube percentage and branching. Phylloplane bacteria may compete for carbon, nitrogen and iron, limiting the saprophytic phase of the pathogen on the phylloplane and accelerating the development of the parasitic phase. This may enhance the field efficacy ofC. coccodes as a biocontrol agent against velvetleaf.