Management of root knot nematode on tomato through application of fungal antagonists, Acremonium strictum and Trichoderma harzianum.

Management of root knot nematode disease infecting tomato, by the use of fungal bioagents Acremonium strictum and Trichoderma harzianum isolated from egg masses of M. incognita infecting tomato has been carried out. The rhizosphere and rhizoplane of root knot nematode infested tomato revealed consistent association of Acremonium strictum. In the present study A. strictum and other fungal bioagents viz. Aspergillus niger, Paecilomyces lilacinus, Rhizoctonia solani and Trichoderma harzianum isolated earlier from egg-masses of M. incognita, identified and maintained have been investigated through in-vitro and in-vivo trials for their potentiality against M. incognita. Out of the above, isolated mycoflora A. niger was identified to be toxic against M. incognita while A. strictum and T. harzianum was found to possess both egg parasitic or opportunistic and toxic properties. A field trial with all the above fungal bioagents both alone and together showed significant promising performance by the dual treatment of A. strictum and T. harzianum in improving the health of the tomato plant with a remarkable reduction in M. incognita population.

Inheritance and QTL analysis of durable resistance to stripe and leaf rusts in an Australian cultivar, Triticum aestivum 'Cook'.

An F4-derived F6 recombinant inbred line population (n = 148) of a cross between the durable stripe (yellow) rust (caused by Puccinia striiformis) and leaf (brown) rust (caused by Puccinia triticina) resistant cultivar, Triticum aestivum 'Cook', and susceptible genotype Avocet-YrA was phenotyped at several locations in Canada and Mexico under artificial epidemics of leaf or stripe rusts and genotyped using amplified fragment length polymorphism (AFLP) and microsatellite markers. Durable adult plant resistance to stripe and leaf rusts in 'Cook' is inherited quantitatively and was based on the additive interaction of linked and (or) pleiotropic slow-rusting genes Lr34 and Yr18 and the temperature-sensitive stripe rust resistance gene, YrCK, with additional genetic factors. Identified QTLs accounted for 18% to 31% of the phenotypic variation in leaf and stripe rust reactions, respectively. In accordance with the high phenotypic associations between leaf and stripe rust resistance, some of the identified QTLs appeared to be linked and (or) pleiotropic for both rusts across tests. Although a QTL was identified on chromosome 7D with significant effects on both rusts at some testing locations, it was not possible to refine the location of Lr34 or Yr18 because of the scarcity of markers in this region. The temperature-sensitive stripe rust resistance response, conditioned by the YrCK gene, significantly contributed to overall resistance to both rusts, indicating that this gene also had pleiotropic effects.

Genetic Analysis of Adult-Plant Resistance to Leaf Rust in Five Spring Wheat Genotypes.

Inheritance of adult-plant resistance to leaf rust, caused by Puccinia triticina, was studied in the progeny of a one-way diallel cross involving five CIMMYT-derived adult-plant resistant wheat (Triticum aestivum) genotypes and a susceptible wheat 'Avocet-YrA'. F1 progenies, F2 populations, F2-derived F3, and F4-derived F5 lines were field evaluated under artificial epidemics with leaf rust race MCJ/SP. Adult-plant resistance to leaf rust was incompletely dominant in crosses with the susceptible parent and was found to be controlled by additive interactions of Lr34 with at least two to three additional genes. Transgressive segregation giving rise to plants or lines with higher and lower levels of resistance than the parents was observed in all F2 and F5 derivatives of the resistant-parent intercrosses and suggested that, apart from Lr34, some of the other additive genes were nonallelic. Although specific combining ability was significant in some generations, general combining ability was found to be the major component of variation. Among generations, the estimates of the narrow-sense heritability of adult-plant resistance to leaf rust ranged from 0.67 to 0.97.

Distribution of Pathotypes of Rhynchosporium secalis and Cultivar Reaction on Barley in Alberta.

Forty-four barley accessions and commercial cultivars with different levels of resistance to scald caused by Rhynchosporium secalis were evaluated for scald reaction from 1997 to 1999 at various sites in Alberta. The accessions Hudson, Atlas, Atlas 46, Atlas 68, Abyssinian, and Kitchin that have the major resistance genes were resistant to pathotypes of R. secalis at all sites. Although scald levels were low for these accessions, they were significantly different among years. Pathotypes of R. secalis and environmental conditions affected diseases levels on 32 commercial cultivars, resulting in significantly different scald reactions among sites and seasons. Resistance in commercial cultivars, AC Stacy, Kasota, and Seebe, held up at most sites with the majority of cultivars being intermediate to moderately susceptible. Cultivars that were previously considered resistant were intermediate in reaction and became increasingly susceptible at some sites from 1997 to 1999. Pathogen virulence was more diverse at the sites where the cultivars became increasingly susceptible compared with sites where the same cultivars were resistant. Scald reactions of the commercial cultivars depended on location, which reflected the presence of different pathotypes, as well as variation in environmental conditions. Consequently, scald management via cultivar choice will be dependent on location.

Detection of aster yellows phytoplasma in false flax based on PCR and RFLP.

False flax (Camelina sativa L.) plants were found to be infected with a yellows-type disease caused by a phytoplasma in experimental plots at the Edmonton Research station. Alberta, Canada. Typical phytoplasmas were detected in the phloem cells in ultrathin sections from leaf midrib tissues examined by electron microscopy. These observations were supported by polymerase chain reaction (PCR) using two primer pairs, R16 F2n/R2 and R16(1)F1/R1, derived from phytoplasma rDNA sequences. Aster yellows (AY) and potato witches'-broom (PWB) phytoplasma DNA samples served as controls and were used to study group relatedness. In a direct PCR assay, DNA amplification with universal primer pair R16F2n/R2 gave the expected PCR products of 1.2 kb. Based on a nested-PCR assay using the latter PCR products as templates, and a specific primer pair, R16(1)F1/R1, designed on the basis of AY phytoplasma rDNA sequences, a PCR product of 1.1 kb was obtained from each phytoplasma-infected false flax and AY sample, but not from PWB phytoplasma and healthy controls. DNA amplification with specific primer pair R16(1)F1/R1 and restriction fragment length polymorphism indicated the presence of AY phytoplasma in the infected false flax sample. This is the first reported characterization of AY phytoplasma in false flax.

A PCR-Based Assay to Detect Rhynchosporium secalis in Barley Seed.

A polymerase chain reaction (PCR)-based diagnostic assay was developed to detect Rhynchosporium secalis, the barley scald fungus, in barley seed. Species-specific primers were designed based on sequence data of a region consisting of the 5.8S RNA gene and internal transcribed spacers 1 and 2 of R. secalis. The sequenced regions showed 100% homology between the two R. secalis isolates and 93% homology between R. secalis and R. orthosporum. Five sets of synthesized oligonucleotide primers were tested for their specificity using 29 isolates of R. secalis of diverse geographic origins and from different barley cultivars. In addition, DNA extracts from 22 species of microbes either taxonomically related to or from the same niche as R. secalis were tested as negative controls. Among five sets of primers, a primer set, RS8 and RS9, was selected for use in detecting R. secalis because it amplified a 264-bp fragment from the DNA of all R. secalis isolates but not the DNA from other species used for validation of the specificity of this primer set. This primer set was also used to detect R. secalis in barley seed and successfully amplified the predicted size of the DNA fragment in the infected material. PCR detection of as little as 1 to 10 pg of R. secalis DNA was possible. The method described here requires 1 day for completion, compared to 10 days required for the cultural method.

Histopathological Study of Barley Cultivars Resistant and Susceptible to Rhynchosporium secalis.

ABSTRACT Differences in the penetration process by Rhynchosporium secalis were compared in resistant and susceptible barley cultivars at the seedling stage. Percent penetration and percent host cell wall alteration (HCWA) differed significantly among cultivars and isolates as revealed by light microscopy. Based on these two variables, the cultivars were statistically separated into two groups that corresponded to their disease reactions. The resistant cultivars, Johnston and CDC Guardian, showed 81.2 to 99.4% HCWA and 0.1 to 20.1% penetration at encounter sites, whereas the susceptible cultivars, Harrington, Argyle, and Manley, had 30.1 to 78.3% HCWA and 31.8 to 81.8% penetration. In the current study, cv. Leduc, which is susceptible at the seedling stage and resistant at the adult stage, showed the same percent HCWA and penetration as did susceptible cultivars. A significant negative correlation (P < 0.01) was found between percent penetration and percent HCWA for cultivars inoculated with two isolates of the pathogen. Isolate 1 was less virulent than isolate 2 with respect to percent penetration and induced significantly fewer HCWA. Scanning electron microscopy showed various shapes of fungal appressoria but no apparent difference in host reaction between resistant and susceptible cultivars. Transmission electron microscopy revealed interactions between the host and pathogen at various stages of penetration. The resistant cv. Johnston responded by producing appositions, as evidenced by a layer of compact osmiophilic material deposited on the inner side of the cell wall. Infection pegs produced by conidia were unable to penetrate the cuticle where an apposition had formed inside. When penetration occurred in the susceptible cv. Argyle, cytoplasmic aggregates and separation of the plasmalemma were visible from the host cell wall, but the layer of compact osmiophilic material was not always present. Data based on light microscopic observations suggested that HCWA may be one of the mechanisms responsible for resistance that is characterized as penetration prevention rather than as a slow rate of mycelial growth after successful penetration. HCWA occurred in response to attempted cuticle penetration, suggesting that HCWA may produce chemical barriers that help to prevent penetration.

Contact sites between mitochondrial membranes: comments.

Extensive contact sites between the two mitochondrial membranes are visualized particularly when tissues are prepared by freeze-substitution methods for electron microscopy. Such contacts rapidly diminish if animals are asphyxiated even for a relatively short period of 8 min. Thus contacts between the mitochondrial membranes are likely to be labile.

Evaluation of soil microorganisms with inhibitory activity against Rhizoctonia solani causal agent of the damping-off of canola.

Pre- and post-emergence damping-off of canola seedlings caused by Rhizoctonia solani is a serious disease in Western Canada. Other fungi such as Fusarium spp. and Pythium spp. are also related to seedling damping-off. To-day, the search of soil bacteria is becoming a tool to use microorganisms as potential biocontrol agents for several plant diseases. The purpose of this research was to detect bacteria to biologically control R. solani, Pythium spp., and Fusarium spp. Soil samples were collected throughout Alberta during 1987 to isolate bacteria. Canola seedlings were also used to obtain bacteria from the same samples. Plant pathogenic fungi were tested to detect the antagonistic activity of the isolates. Tests were made with coated canola seeds, amendments and fresh of freeze-dried cells. Three hundred forty-one bacterial cultures were isolated. Only 16 inhibited fungal growth: 7 showed the same effects against R. solani and 9 showed uneven effects. Some isolates showed a weak action to Pythium spp. and Fusarium spp. Three isolates showed inhibitory effect on R. solani and Pythium spp. Isolate F1 improved by about 50% the germination of canola seeds in inoculated pots when compared with the inoculated control. Coated seeds had low germination and emergence was below the inoculated control. The emergence of canola seedlings was very much improved when isolate 147 was delivered as an amendment in inoculated pots. Identification showed that 3 bacterial belonged to Bacillus spp., 4 to green fluorescent Pseudomonas spp. and 2 were Streptomyces spp.

Evaluation of soil microorganisms with inhibitory activity against rhizoctonia solani causal agent of the damping-off of canola

Pre-and post-emergence damping-off of canola seedlings caused by Rhizoctonia solani is a serious disease in Western Canada. Other fungi such as Fusarium spp. and Pythium spp. are also related to seedling damping-off. To-day, the search of soil bacteria is becoming a tool to use microorganisms as potential biocontrol agents for several plant diseases. The purpose of this research was to detect bacteria to biologically control R. solani, Pyrhium spp., and Fusarium spp. Soil samples were collected throughout Alberta during 1987 to isolate bacteria. Canola seedlings were also used to obtain bacteria from the same samples. Plant pathogenic fungi were tested to detect the antagonistic activity of the isolates. Tests were made with coated canola seeds, amendments and fresh of freeze-dried cells. Three hundred forty-one bacterial cultures were isolated. Only 16 inhibited fungal growth: 7 showed the same effects against R. solani and 9 showed uneven effects. Some isolates showed a weak action to Pythium spp. and Fusarium spp. three isolated showed inhibitory effect on R. solani and Pythium spp. isolate F1 improved by about 50% the germination of canola seeds in inoculated pots when compared with the inoculated control. Coated seeds had low germination and emergence was below the inoculated control. the emergence of vanola seedlings was very much improved when isolate 147 was delivered as an amendment in inoculated pots. Identification showed that 3 bacterial belonged to Bacillus spp., 4 to green fluorescent Pseudomonas spp. and 2 were Streptomyces spp

Microanalysis of the reaction product in Karnovsky and Roots histochemical localization of acetylcholinesterase.

X-ray energy dispersive microanalysis of the reaction product in Karnovsky and Roots histochemical localization of acetylcholinesterase indicated the presence of sulfur, iodine, copper, and iron. The reaction was run in vitro using purified acetylcholinesterase from the electric eel to confirm our previous results on similarly treated neuromuscular junction in situ.

Subunit structure of the connexons in hepatocyte gap junctions.

The connexons in the mouse hepatocyte gap junctions consistently reveal a subunit structure in rotary shadowed replicas of quick frozen freeze-fractured material. Quick freezing eliminates treatment of the material with chemical fixatives and cryoprotectants and hence captures the structures in their native state. Previous biophysical studies have indicated that the connexon in hepatocyte gap junction is composed of 6 subunits. In rotary shadowed replicas, some connexons have 6 subunits while the rest reveal less than 6 subunits. Visualization of less than 6 subunits may be due to local variations in the angle of shadowing because of an uneven plane of fracturing and/or breaking of some of the subunits during freeze-fracturing. Markham's rotation technique reveals that some connexons have a hexameric symmetry while others approach a tetrameric symmetry. Using models of connexons and tilting experiments with the replicas it is shown that these apparent deviations from the hexameric symmetry result from inclination of the fractured face relative to the plane of viewing.

Thiocholine methods for the demonstration of acetylcholinesterase in neuromuscular junctions.

The extensor digitorum longus muscles of rat were stained for the localization of acetylcholinesterase activity at the neuromuscular junctions. The modified methods of Koelle-Friedenwald and Karnovsky-Roots were used with acetylthiocholine iodide as the substrate. The merits and demerits of both these methods are discussed. TEM and SEM X-ray dispersive analyses of the muscle fibres treated histochemically by both the methods were also made in order to elucidate further the nature of the reaction products. Denervated muscles were subjected to similar treatment.

Multilamellar surface layer of the cell wall of Albugo candida and Phycomyces blakesleeanus.

The surface layer of the cell wall of the sporangia of Albugo candida and of the sporangiophores of Phycomyces blakesleeanus was composed of a series of lamellae. The evidence from freeze-fracture, freeze-etch, and single-stage replicas indicated that the lamellae are organized as bilayers, an organization associated with the presence of lipids. The role of these lamellae in dispersibility and resistance is discussed.

Functional characterization of types of plasma membranes in cerebral cortex.

The cerebral cortex of normal oxygenated and of asphyxiated mice has been studied by freeze-fracturing technique with a twofold purpose. First, to investigate changes, if any, in the molecular organization of the plasma membrane of any specific cell type(s) that could be correlated with permeability changes thought to take place as a consequence of asphyxiation. Secondly, to attempt characterization of plasma membranes on the basis of the organization of their fractured faces. The decrease in the extracellular material in asphyxiated cerebral cortex seen in electron micrographs of thin sections could not be correlated with change(s), if any, in the molecular organization of the plasma membrane of any particular cell type. Plasma membranes of various types could be characterized on the basis of the arrangement of particles on the fractured faces. Some of these types correspond to identifiable cell processes, while others have not yet been identified with certainty. Fusion of synaptic vesicles with the presynaptic membrane is mediated through clustering of 100-150 A membrane-associated particles.