Quantification of Mycelium of Botrytis spp. and the Antagonist Ulocladium atrum in Necrotic Leaf Tissue of Cyclamen and Lily by Fluorescence Microscopy and Image Analysis

ABSTRACT A technique was developed to localize and quantify the internal mycelial colonization of necrotic leaf tissue of cyclamen (Cyclamen persicum) or lily (Lilium) by pathogenic Botrytis spp. and the antagonist Ulocladium atrum. This technique allows investigation of competitive substrate colonization by both fungi, which is a key process for biological control of Botrytis spp. by U. atrum. A combination of differential fluorescent labeling and image analysis was applied on cryostat sections of necrotic leaf tissue. Botrytis mycelium was labeled specifically by indirect immunofluorescence using a monoclonal antibody specific for Botrytis spp. And an antimouse fluorescein conjugate. Wheat germ agglutinin conjugated to the fluorochrome TRITC was used to label mycelium of both fungi. Image analysis was used to measure the relative surface area of the cryostat section covered by fluorescing hyphae of Botrytis spp. and by fluorescing hyphae of both fungi. A mathematical conversion was derived and used to calculate the relative mycelial volume of each fungal species in the necrotic tissue based on the measured relative surface areas. Temporal aspects of substrate colonization were studied in a short time series. An analysis of components of variance provided insight into spatial colonization patterns for the fungal species involved and allowed the design of efficient sampling strategies for future experiments.     

Soybean Brown Stem Rot, Phytophthora sojae, and Heterodera glycines Affected by Soil Texture and Tillage Relations

ABSTRACT Investigations were conducted to determine whether the effects of tillage practices on the prevalence of brown stem rot of soybean (caused by Phialophora gregata), Heterodera glycines, and Phytophthora sojae were confounded by soil texture in samples collected in the fall of 1995 and 1996. Soil and soybean stem samples, along with tillage information, were collected from 1,462 randomly selected fields in Illinois, Iowa, Minnesota, Missouri, and Ohio in collaboration with the National Agricultural Statistics Service. The incidence of brown stem rot was determined from 20 soybean stem pieces collected from each field in a zigzag pattern. The detection frequency of P. sojae (expressed as percent leaf disks colonized) and population densities of H. glycines were determined from soil cores also collected in a zigzag pattern. The soil samples were grouped into various textural classes, and the effect of soil texture and tillage relations on the activities of each pathogen were determined. Both tillage and soil texture affected the incidence of brown stem rot; however, there was no interaction between tillage and soil texture. Conservation tillage had a greater (P < 0.05) incidence of brown stem rot in clay loam and silty clay loam than did conventional tillage. The detection frequency of P. sojae was not affected by tillage, but a tillage x texture interaction (P = 0.013) indicated that the effect of tillage depended on soil texture. There was a greater (P < 0.05) detection frequency of P. sojae in conservation tillage than in conventional tillage in silt loam and loam soils. However, in sandy loam, the detection frequency of P. sojae was greater (P = 0.0099) in conventional tillage than in conservation tillage. Population densities of H. glycines were significantly affected by both tillage and soil texture, but overall, there was no tillage x texture interaction. There was an inverse relationship between population densities of H. glycines and percent clay (r = -0.81, P = 0.01) in no-till fields, but little or no change in nematode densities was observed with increasing clay content in tilled fields. Population densities of H. glycines were less (P < 0.05) in no-till fields than in tilled fields in silty clay loam and clay soils. There was no difference in H. glycines densities between the tillage categories in soils sandier than silty clay loam or clay. The findings emphasize the need for cautious interpretation of the effects of tillage practices on diseases and pathogens in the absence of information on soil texture.     

trans-Dominant Inhibition of Geminiviral DNA Replication by Bean Golden Mosaic Geminivirus rep Gene Mutants

ABSTRACT Geminiviruses are a group of single-stranded DNA viruses that cause major losses on a number of important crops throughout the world. Bean golden mosaic virus (BGMV) is a typical bipartite, whitefly-transmitted geminivirus that causes a severe disease on beans (Phaseolus vulgaris) in the Western Hemisphere. The lack of natural resistance to geminiviruses has led to attempts to engineer resistance, particularly through the use of pathogen-derived resistance strategies. The rep gene contains several conserved domains including nucleoside triphosphate (NTP)-binding and DNA-nicking domains and is the only geminiviral gene necessary for replication. Previous analysis by our group and others has demonstrated that the NTP-binding and DNA-nicking domains are necessary for geminiviral DNA replication. The ability of the rep gene and rep gene mutants to interfere with geminiviral DNA replication, when expressed in trans, was examined using a transient assay in a tobacco suspension cell culture system. Wild-type (wt) and mutant rep genes were cloned into plasmids under the control of the cauliflower mosaic virus 35S promoter for in planta expression and coinoculated into tobacco cells with infectious clones of various geminiviruses. The wt rep gene from BGMV-GA was able to support replication of BGMV-GA DNA-B. Several different rep gene mutants, with function-abolishing mutations in the NTP-binding or DNA-nicking domains, were potent trans-dominant inhibitors of geminiviral DNA replication.     

Sexual Recombination in Gibberella zeae

ABSTRACT We developed a method for inducing sexual outcrosses in the homothallic Ascomycete fungus Gibberella zeae (anamorph: Fusarium graminearum). Strains were marked with different nitrate nonutilizing (nit) mutations, and vegetative compatibility groups served as additional markers in some crosses. Strains with complementary nit mutations were cocultured on carrot agar plates. Ascospores from individual perithecia were plated on a minimal medium (MM) containing nitrate as the sole nitrogen source. Crosses between different nit mutants segregated in expected ratios (3:1 nit(-):nit(+)) from heterozygous perithecia. Analysis of vegetative compatibility groups of progeny of two crosses indicated two and three vegetative incompatibility (vic) genes segregating, respectively. For rapid testing of sexual recombination between nit mutants, perithecia were inverted over MM to deposit actively discharged ascospores. Development of proto-trophic wild-type colonies was taken as evidence of sexual recombination. Strains of G. zeae group 2 from Japan, Nepal, and South Africa, and from Indiana, Kansas, and Ohio in the United States were sexually interfertile. Four group 1 strains were not interfertile among themselves or with seven group 2 strains. Attempts to cross G. zeae with representatives of F. acuminatum, F. avenaceum, F. culmorum, F. crookwellense, F. oxysporum, and three mating populations of G. fujikuroi were not successful.     

Antibody response in goats experimentally infected with Toxoplasma gondii

Serum samples of five goats inoculated with Toxoplasma gondii were analyzed using the enzyme linked immunosorbent assay (ELISA), indirect hemagglutination (IHA) and western blotting (WB). Antibodies detected by ELISA peaked between 19 and 62 days after inoculation and persisted throughout the experiment with no association to parasitaemia. Using WB, the main antigens detected had molecular weights of approximately 68, 62, 50, 48, 42, 34, 28, 26, 22 and 19 kDa. Antibody titers of between 1:256 and 1:32000 were observed using IHA, with a significant drop in activity after treatment with 2-mercapto-ethanol between days 12 and 48. This coincided with the parasitaemic period that occurs between 5 and 64 days after inoculation.     

Seasonal dynamics of the free-living phase of Anocentor nitens at Pedro Leopoldo, Minas Gerais, Brazil

An understanding of seasonal dynamics in the free-living phase of the tropical horse tick, Anocentor nitens, is vital for effective control measures. This study examines seasonal changes in the oviposition and incubation periods, eclosion rates, reproductive success, and larval survival in the free-living phase during the period June 1995-July 1997. Ten engorged female ticks, placed individually in wire-gauze tubes, were exposed in a field of Brachiaria decumbens fortnightly each month, and monitored to measure the duration of the various reproductive parameters, and success. Larval abundance was estimated indirectly, through the percentage of females with viable progeny, and directly by using a flagging method. Duration of the free-living cycle ranged from 12.3 weeks (October and December) to 23.5 weeks (March). The cycle was prolonged in cold and dry months. Larval survival was longer from February to July, and reduced from August to February. Oviposition was recorded in all months, and the egg production index ranged from 26.7 to 47.1%. The eclosion rate ranged from 0.1 to 76.6% in different months. Monthly estimates of larval abundance in the pasture were similar for the two methods used, being higher in months with higher temperatures, relative humidity and rainfall.     

Ultrastructural identification of small granule prolactin (PRL) cells in goat adenohypophysis by the colloidal-gold method

We describe ultrastructural characteristics of PRL cells with small secretory granules, immunostained with ovine antiserum, in adult Murciano-Granadina milking goats during anoestrus, the last third of gestation and lactation. This cell subtype is considered to comprise a stable population that decreases numerically during gestation and lactation, and that may change during these stages to show large granules.     

Vaccination of ostriches (Struthio camelus, Linnaeus, 1758) against Newcastle disease: evidence for vaccine compatibility and seroconversion after vaccinations using the hemagglutination inhibition and virus neutralization tests

Newcastle disease (ND) remains to be the worldwide most important infectious disease of poultry. This epizootic is in Germany and many other countries a notifiable disease. Prophylactic vaccination is the major tool for the control of ND in poultry and other birds. Eighty-three ostriches (Struthio camelus) which were kept on farms in Germany were checked for the presence of NDV-specific antibodies. Some of these birds are said to be vaccinated against Newcastle disease. Only some of these ostriches contained antibodies which were measurable in haemagglutination inhibition and virus neutralisation tests. Twenty-three previously unvaccinated ostriches were vaccinated with commercially available vaccines. Both the LaSota live and inactivated oil emulsion vaccines were well tolerated following conjunctival or subcutaneous application, respectively. Neither local nor systemic side reactions were observed. After the vaccinations high antibody titres were detected in hemagglutination inhibition and virus neutralisation tests. A strong correlation between both established methods (r = 0.92; < 0.001) were noted.     

Trichinella antigens: a review

This paper presents a review of the Trichinella antigens within the context of species variation. As with other parasites, Trichinella antigens can be classified according to their localisation as surface, excretory/secretory (ES) and somatic components. Surface antigens are mainly constituents of the outer cuticle although secretions from inner parts of the body wall as well as from the oesophagus can temporarily accumulate in the surface. ES antigens come mainly from the excretory granules of the stichosome, whereas somatic constitutive antigens come from the internal parts of the worms. ES products are considered very important from an immunological point of view as they are easily targeted by the immune system, whereas parasite death is required for exposure of somatic products. Some of the antigenic components have been characterised chemically. Phosphorylcholine is an important hapten that modulates the immune responses in Trichinella infections. Glycoproteins are the major components of surface and excretory/secretory products. A 43-kDa glycoprotein has been regarded as a good candidate for diagnosis and vaccination purposes. Recently some glycans have received special attention either as relevant epitopes or as parasite evasion strategies.