Inheritance of Anthracnose Resistance in the Common Bean Differential Cultivar G 2333 and Identification of a New Molecular Marker Linked to the Co-42 gene

The inheritance of anthracnose resistance of the common bean ( Phaseolus vulgaris L.) differential cultivar G 2333 to Colletotrichum lindemuthianum races 73 and 89 was studied in crosses with the susceptible cultivar Rudá. The segregation ratios of 15 : 1 in the F₂ and 3 : 1 in the backcrosses to Rudá indicate that for each of the races tested there are two independent resistance loci in G 2333. A random amplified polymorphic DNA (RAPD) molecular marker (OPH18_1200C) linked in resistance to race 73 was identified in a BC₃F_2:3 population derived from crosses between Rudá and G 2333. A RAPD molecular marker OPAS13_950C, previously identified as linked to gene Co-4² , was also amplified in this population. Co-segregation analyses showed that these two markers are located at 5.6 (OPH18_1200C) and 11.2 (OPAS13_950C) cM of the Co-4² gene. These markers were not present in BC₁F_2:3 plants resistant to race 89 indicating that this population carries a different resistance gene. DNA amplification of BC₁F_2:3 plants with RAPD molecular marker OPAB_450C, previously identified as linked to gene Co-5 , indicated that this gene is present in this population.     

An SNP in the goat CSN2 promoter region is associated with the absence of beta-casein in milk

So far, at least eight alleles in the goat CSN2 locus have been associated with the level of β -casein expression in milk. Alleles CSN2 ^A , CSN2 ^{A 1}, CSN2 ^B , CSN2 ^C , CSN2 ^D and CSN2 ^E have been associated with normal content (allele effects of about 5 g of β -casein per litre), whereas the CSN2 ⁰ and CSN2 ⁰¹ alleles have been associated with non-detectable levels of β -casein. Most of these alleles have been characterized genetically. Herein, we report the identification of a previously unreported SNP in the goat CSN2 promoter region ( AJ011018 :g.1311T>C), which is associated with the absence of β -casein in the milk. Furthermore, we developed a PCR-based method that allows detection of this mutation.     

Detection of Clostridium botulinum types A, B, E and F in foods by PCR and DNA probe

A PCR procedure was developed for the detection of Clostridium botulinum in foods. PCR products were detected in agarose gels and by Southern hybridization. The sensitivity of PCR was tested in broth cultures and in canned asparagus, dry cured ham and honey. The sensitivity of the method in broth was high (2·1–8·1 cfu ml⁻¹) for types A and B, but rather low (10⁴ cfu ml⁻¹) for types E and F. However, after enrichment at 37°C for 18 h, it was possible to detect Cl. botulinum types A, B, E and F in food samples at initial levels of about 1 cfu 10 g⁻¹ of food. This PCR detection protocol provides a sensitive and relatively rapid technique for the routine detection of Cl. botulinum in foods.     

OBSERVATIONS ON THE MICROFLORA OF MINCED CHICKEN MEAT IRRADIATED WITH 4 MeV CATHODE RAYS

SUMMARY: Experiments are described in which minced chicken meat, packed anaerobically, was irradiated at room temperature and in the frozen state with a wide range of doses of 4 MeV cathode rays. Sterility was achieved in 14 out of 15 samples which had received 2 × 10⁶ rads or more. Doses of 0·5 and 1·0 × 10⁶ rads allowed survival of a few bacteria/g, usually spore formers. Bacterial counts indicated an approximately logarithmic decrease in numbers at lower doses, while freezing reduced the bactericidal effect.
The storage life at 5° was prolonged only slightly by doses of 5 × 10⁴ and 10 × 10⁴ rads, and highly variable results were obtained with 17·5 × 10⁴ rads. A dose of 25 × 10⁴ rads, however, increased the storage life very considerably. The types of bacteria present initially, and after irradiation with low doses and storage at 5°, were studied. After storage for 12 days or more various types of nonsporing Gram-positive rods were predominant in almost all samples, both control and irradiated. Streptococci were also important where irradiation with 17·5 × 10⁴ and 25 × 10⁴ rads was followed by long storage.     

Tolerance of parr of Arctic charr, Salvelinus alpinus, to reduced dissolved oxygen concentrations

One year old pan Item Windermere (north west England) tolerated lower incipient lethal levels of oxygen [18 20 mg 1 ¹: 15 17% air saturation value (ASV)] at lower (5, 10°C) than at higher (15, 20°C) an elimation temperatures (2·2 2·4 mg 1 ¹ 22 25% ASV). Values were not significantly different for two races of charr in the lake and are amongst the lowest recorded for salmenid species.     

Modulation of 45Ca2+ Influx into Cells Stably Expressing Recombinant Human NMDA Receptors by Ligands Acting at Distinct Recognition Sites

Abstract: A ⁴⁵Ca²⁺ influx assay has been used to investigate the pharmacology of stably expressed recombinant human NR1a/NR2A and NR1a/NR2B N -methyl- d -aspartate (NMDA) receptors. Inhibition of glutamate-stimulated ⁴⁵Ca²⁺ influx by six glycine-site antagonists and inhibition of glycine-stimulated ⁴⁵Ca²⁺ influx by five glutamate-site antagonists revealed no significant differences between affinity values obtained for NR1a/NR2A and NR1a/NR2B receptors. The polyamine site agonist spermine showed differential modulation of glutamate- and glycine-stimulated ⁴⁵Ca²⁺ influx for recombinant NMDA receptors, inhibiting and stimulating ⁴⁵Ca²⁺ influx into cells expressing NR1a/NR2A receptors (IC₅₀ = 408 µ M ) and NR1a/NR2B receptors (EC₅₀ = 37.3 µ M ), respectively. The antagonist ifenprodil was selective for NR1a/NR2B receptors (IC₅₀ = 0.099 µ M ) compared with NR1a/NR2A receptors (IC₅₀ = 164 µ M ). The effects of putative polyamine site antagonists, redox agents, ethanol, and Mg²⁺ and Zn²⁺ ions were also compared between NR1a/NR2A and NR1a/NR2B receptors. This study demonstrates the use of ⁴⁵Ca²⁺ influx as a method for investigating the pharmacology of the numerous modulatory sites that regulate the function of recombinant human NMDA receptors stably expressed in L(tk-) cells.     

STUDIES ON PLANT CUTICLE: THE COMPOSITION OF THE CUTICLE OF APPLE LEAVES AND FRUITS

The levels of deposition of the waxy and cutin components of the cuticles of the mature leaves and developing fruits of the Merton Worcester and Cox's Orange Pippin varieties of apple over a period of 4 months are reported. The waxy materials on the leaves remain approximately constant at 20 μg./cm.², and the cutin at about 100 μg./cm.² of surface. The waxy and cutin deposits on the fruits increase markedly during the period of development, the wax and oil present on the surface and embedded within the cutin rising to 500 μg./cm.² and the cutin to a little under 1000 μg./cm.² of surface. Phenolic and acidic substances in the cuticles of the fruits are determined, and the possibility of their occurrence in surface deposits on the leaves is discussed.
An examination is made of the effect of nutrient deficiency upon the formation of waxy and phenolic substances in the leaves. Nitrogen deficiency in Worcester Pearmain leaves leads to a reduced deposition of waxy materials, and nitrogen, phosphorus and potassium deficiency results in higher contents of phenolic compounds. The separation and nature of the waxy covering on leaves, the effect of environmental conditions on its formation, and its importance in relation to the application of crop protection chemicals are discussed.     

A Rapid Tomato Seedling Assay for Verticillium Wilt

A rapid (10-min) tomato seedling assay was developed for determining the wilt-capacity of cell-free culture filtrates of race 1 and race 2 isolates of Verticiilium dahliae. The assay also rapidly determined the differences in wilt resistance between tomato cultivars. Rapidity was attained by manipulating incubation conditions to promote rapid wilting. These included inducing elongated stems in the susceptible cv. Bonny Best seedlings by growth of plants in subdued light (2.2 × 10² lux) and by concentrating two-fold the cell-free culture filtrates of the pathogen. Further, rapid uptake of the wilt factors in the culture filtrates was facilitated during incubation by increasing transpiration with bright light (23.8 × 10³ lux), a wind stream (125–150 metres/min) across the assay seedlings, low relative humidity (28%) and a relatively high assay temperature (30 C). When necessary, these conditions were altered to extend the assay times. This assay system was used to determine optimal incubation time, temperature and medium for obtaining culture filtrates with increased wilt capacities. The assay also determined the relative wilt capacities of races 1 and 2 and the comparative resistance of four tomato cultivars to wilt caused by races 1 and 2.     

Distribution of transition metal ions in multiple forms of Methanosarcina hydrogenase

There were significant levels of in vitro hydrogenase activity in Methanosarcina strains. The multiple forms of hydrogenase were observed in cell free extracts of cells grown on methanol. Strains having poor growth on H₂ : CO₂ had four forms while strains having normal growth on all substrates contained two forms of hydrogenase. These multiple forms differ in their charges as well as in their composition of transition metal ions. The strain having normal growth showed higher incorporation of ⁶³Ni²⁺ and ⁶⁵Zn²⁺. Both hydrogenases, A and D, of strain P₃ had methylviologen and F₄₂₀-reducing activity and contained Zn²⁺ and Co²⁺ respectively. Hydrogenases A and D of strains P₁ and P₄ also had similar characteristics whereas hydrogenases B and C had only methylviologen reducing activity.     

Efflux of ions and ATP depletion induced by pediocin PA-1 are concomitant with cell death in Listeria monocytogenes Scott A

Domination of Carnobacterium divergens LV13 by a bacteriocin-producing (bac⁺) organism Carnobacterium piscicola LV17 was dependent on the level of inoculum of the producer strain and its bacteriocin production. When C. piscicola LV17 was grown in APT broth from an initial inoculum of α-10⁴ cfu ml^-1, bacteriocin was not produced (bac^-) although maximum population was reached. The culture remained bac^- during subsequent inoculation at 10²-10⁷ cfu ml^-1 unless it was first grown on solid medium or if heat-treated supernatant fluids from a bac⁺ culture of C. piscicola LV17, LV17A or LV17B were added to the culture prior to the stationary phase of growth. Use of purified carnobacteriocins from C. piscicola LV17A and LV17B confirmed their role in regulation of the bac⁺ phenotype. The need for induction might account in part for differences in bacteriocin production by cultures in liquid and on solid growth media.     

Improvement in the growth performance of white shrimp, Litopenaeus vannamei, by a protease-producing probiotic, Bacillus subtilis E20, from natto

Aims:  To isolate and identify a benefic bacterium, Bacillus subtilis E20, from natto (fermented soybeans), and incorporate it into shrimp feed to promote shrimp growth performance.
Methods and Results:  A protease-producing bacterium, E20, isolated from natto was identified as B. subtilis by an API 50 CHB kit and the 16S rDNA sequence. B. subtilis E20 was able to grow at a broad range of temperatures (10–50°C), pH values (5–10), and NaCl levels (0–9%). The best culture conditions for B. subtilis E20 to produce the protease were 40°C, a pH of 6–8 and 0% NaCl. No shrimp died after being injected with B. subtilis E20 [up to 10⁹ colony-forming units (CFU) per shrimp]. Bacillus subtilis E20 was incorporated in diets at the levels of 0 (control), 10⁶, 10⁷, and 10⁸ CFU kg⁻¹ for shrimp grow-out culture, and results showed that after feeding on B. subtilis E20-containing diets (10⁸ CFU kg⁻¹ of diet), shrimp had excellent growth performance and production compared to the control because protease activities in the digestive tract were improved by B. subtilis E20.
Conclusions:  Bacillus subtilis E20 isolated from natto is a great protease producer and is able to improve shrimp growth performance through increasing the digestibility of food.
Significance and Impact of the Study:  Results suggest that B. subtilis E20 is a potential candidate for use as a probiotic to improve shrimp growth performance, and consequently reduce feed costs.     

Tracheobronchial washings from seven vertebrate species as growth media for the four species of Bordetella

Abstract The four species of Bordetella differed in their ability to grow at 37°C in membrane-filtered tracheobronchial washings (TBW) from seven vertebrate species, including their natural hosts. From washed inocula of approximately 2×10³ colony-forming units per ml (cfu ml⁻¹), Bordetella bronchiseptica and B. avium grew much better than the other two bordetellae and yielded stationary-phase cultures containing 10⁸−10⁹ cfu ml⁻¹ in most of the TBW samples. These counts were only moderately lower than those attained in CL medium which contains about a 450-times higher concentration of amino acids. B. bronchiseptica and B. avium also grew to a limited extent in phosphate-buffered saline without nutrient supplements. B. parapertussis grew in TBW from man, sheep, rabbit, mouse and chicken, but not in TBW from a dog and a horse or in PBS. B. pertussis grew well in CL medium, but not in PBS or in any of 13 samples of TBW from the seven vertebrate species, which included three samples of lung lavage fluid from human patients. Analysis of the TBW samples for known Bordetella nutrients revealed concentrations of amino acids and nicotinic acid averaging 0.35 mM and 0.56 μg ml⁻ respectively.     

Population Dynamics of Pseudomonas syringae pv. tomato Strains on Tomato Cultivars Rio Grande and Rio Grande-Pto under Field Conditions

We examined the effects of the Pto resistance locus on the population dynamics of Pseudomonas syringae pv. tomato (Pst) strains in field experiments with the nearly isogenic tomato lines Rio Grande (RG, susceptible to Pst races 0 and 1) and Rio Grande-Pto (RG-Pto, resistant to Pst race 0, susceptible to Pst race 1). Pst strain SM78-1Sm^r (race 0) grew well under field conditions and caused ample bacterial speck disease on susceptible RG plants. In contrast, strain DC3000 failed to establish large populations when inoculated onto field grown RG plants. Mean population sizes of SM78-1Sm^r were 4–5 orders of magnitude larger on RG than RG-Pto plants indicating that RG-Pto plants were highly effective in attenuating pathogen population development. Most of the sampled leaflets from RG-Pto field plots harboured small numbers of SM78-1Sm^r. However, population sizes SM78-1Sm^r as large as 10⁵–10⁶ CFU were found on a few leaflets. Bacteria isolated from these leaflets had phenotypes characteristic of Pst race 1 strains. In growth chamber plant assays, the bacterial strains grew well and caused typical speck lesions on RG-Pto plants. The strains appeared to be race-shift mutants of SM SM78-1Sm^r. Interestingly, results from DNA hybridization experiments demonstrated that the race-shift mutants were deleted for the avirulence gene, avrPto but not for avrPtoB.     

Effect of high-temperature, short-time (HTST) pasteurization on milk containing low numbers of Mycobacterium paratuberculosis

The efficacy of high-temperature, short-time (HTST) pasteurization (72 °C/15 s) when low numbers (≤ 10³ cfu ml ⁻¹ ) of Mycobacterium paratuberculosis are present in milk was investigated. Raw cows' milk spiked with Myco. paratuberculosis (10³ cfu ml⁻¹, 10² cfu ml⁻¹, 10 cfu ml⁻¹, and 10 cfu 50 ml⁻¹) was subjected to HTST pasteurization using laboratory pasteurizing units. Ten bovine strains of Myco. paratuberculosis were tested in triplicate. Culture in BACTEC Middlebrook 12B radiometric medium detected acid-fast survivors in 14·8% and 10% of HTST-pasteurized milk samples at the 10³ and 10² cfu ml⁻¹ inoculum levels, respectively, whereas conventional culture on Herrold's egg yolk medium containing mycobactin J detected acid-fast survivors in only 3·7% and 6·7% of the same milk samples. IS900-based PCR confirmed that these acid-fast survivors were Myco. paratuberculosis . No viable Myco. paratuberculosis were isolated from HTST-pasteurized milk initially containing either 10 cfu ml⁻¹ or 10 cfu 50 ml⁻¹.     

THE NORMAL FLORA OF THE BOVINE RUMEN II. QUANTITATIVE BACTERIOLOGICAL STUDIES

SUMMARY: A simple medium and dilution count technique was evolved for determining the viable count of rumen bacteria. Material from three fistulated cows was examined and the viable population found to vary within the range 10⁷-10¹²/g; the most frequently occurring values were 10⁸-10¹⁰/g (94.0% of 350 counts). Observations were made of the homogeneity of rumen contents, of variations in count with time after sampling and between cows, and of variation from day to day, hour to hour, before and after feeding and on different diets. The variations in the viable count, even with gross changes in diet, were never greater than those occurring in a single animal on a fixed diet. Microscopic examinations showed Gram-positive cocci and rods to be the most frequently occurring types of bacteria, Gram-negative organisms of the coli-aerogenes type being present only in low numbers.     

Compatibility of the insect pathogenic fungus Beauveria bassiana with neem against sweetpotato whitefly, Bemisia tabaci, on eggplant

A study on the compatibility of the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) with neem was conducted against sweetpotato whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), on eggplant. Initially, three concentrations of B. bassiana (10⁶, 10⁷, and 10⁸ conidia ml⁻¹) and three concentrations of neem (0.25, 0.5, and 1.0%) were used as individual treatments against B. tabaci . The highest concentration of B. bassiana yielded the highest B. tabaci egg (25.2%) and nymph mortalities (73.0%), but this was not significantly different from the mortalities caused by the 10⁷ conidia ml⁻¹ suspension. Similarly, the highest concentration of neem yielded the highest egg (27.3%) and nymph mortalities (75.5%), which was also not significantly different from the 0.5% suspension. Therefore, 0.5% neem was used along with 10⁷ B. bassiana conidia ml⁻¹ suspension as an integrated pest management program against B. tabaci . The combination of B. bassiana and neem yielded the highest B. tabaci egg (29.5%) and nymph mortalities (97.2%), and the lowest LT₅₀ (2.08 day) value. Moreover, an integrated combination of B. bassiana with neem caused 27.6 and 20.5% more nymphal mortality than individual treatments of B. bassiana and neem, respectively, 7 days post-application. Thus, a combined application of an entomopathogenic fungus and a botanical insecticide may benefit from both, and it has proven effective for the control of B. tabaci on eggplant.     

Size-dependent mortality of red sea bream, Pagrus major, juveniles released with fluorescent otolith-tags in News Bay, Japan

Totals of 2^.67 x 10⁵ and 7^.56 x 10⁵ juvenile red sea bream of three size groups (10, 20 and 40 mm t.l.) marked with a fluorescent substance in the otolith were released in News Bay, Oita Prefecture, Japan, in July 1987 and June 1988, respectively; the aim was to estimate growth and mortality of different developmental stages.
Of fish released in 1987 and 1988, 10 618 and 4413, respectively were recaptured during those two years. Released fish remained in the bay until the end of summer, and afterwards migrated out towards open waters. Fish of the 40-mm group released in 1987 grew to over 200 mm t.l. in one year. Mean growth rate for 19 days after release was higher in the 40-mm group (0^.87 mm day⁻¹) than in the 20-mm group (0^.74mm day⁻¹). Survival rates over 19 days were 59^.0 and 10^.1 % for 40-mm and 20-mm fish, respectively, in 1987, and those over 30 days were 69^.2, 3^.3 and 0^.0% for 40-mm, 20-mm and 10-mm fish, respectively, in 1988.
Cannibalism was indicated by the presence of marked otoliths for 20-mm fish in the stomachs of a few 40-mm individuals recaptured 2 days after release. Size-dependent growth and size-selective mortality were both noted in juvenile red sea bream, i.e. the relative size differential between larger and smaller individuals was maintained in the period between marking and recapture, and mortality was inversely proportional to size.     

SODIUM AND THE FLUX OF CALCIUM IONS IN ELECTRICALLY-STIMULATED CEREBRAL TISSUE

Abstract— The rate of efflux of ⁴⁵Ca²⁺ from slices of rat cerebral cortex was resolved into two exponential curves which were attributed to an extracellular component and an intracellular or bound component. Electrical stimulation increased efflux of ⁴⁵Ca²⁺ from the more stable pool and the time course for the redistribution of Na⁺ and K⁺ paralleled that for the increased efflux of Ca²⁺. This effect of stimulationwas dependent on the presence of Na⁺ in the incubation medium. Lack of Na⁺ in the medium during loading of the slices with ⁴⁵Ca²⁺ increased uptake but on subsequent transfer to a medium containing Na⁺, electrical pulses failed to increase the rate of efflux of ⁴⁵Ca²⁺. In unstimulated slices, the rate of efflux of ⁴⁵Ca²⁺ was dependent upon the concentration ratio of Na⁺ to Ca²⁺ in the incubation medium. Saxitoxin and tetrodotoxin inhibited the increased efflux of ⁴⁵Ca²⁺ that occurred during electrical stimulation but exerted no effect on Ca²⁺-Ca²⁺ exchange. Our results suggest that there is a Na⁺-dependent turnover of Ca²⁺ in brain slices which may involve changes in affinity at a common binding site. The possible involvement of such a Na⁺-Ca²⁺ interaction in the regulation of neurotransmitter function is discussed.     

Ca2+ UPTAKE BY SYNAPTOSOMES AND ITS EFFECT ON THE INHIBITION OF ACETYLCHOLINE RELEASE BY BOTULINUM TOXIN

Abstract— ⁴⁵Ca²⁺ uptake by cerebral cortex synaptosomes was determined by gel filtration, glass fibre disc filtration under suction and by centrifugation with EGTA present. The filtration methods gave comparable results which were higher than values obtained by the centrifugation method. Uptake was increased by 25mM-K⁺ at all times investigated. The accumulated ⁴⁵Ca²⁺ was bound within the synaptosome. ⁴⁵Ca²⁺-ionophore A23187 stimulated uptake only during the first min; levels of intra-synaptosomal ⁴⁵Ca²⁺ then returned to control values. A23187 also increased intra-synaptosomal Na⁺ and Cl⁻ contents. Botulinum toxin inhibits the K.⁺-stimulated release of [¹⁴C]ACh from synaptosomes but the ionophore released [¹⁴C]ACh from both normal and botulinum-treated preparations in a Ca²⁺-dependent manner. However, it also elicited Ca²⁺-dependent release of [choline. Increased extracellular Ca²⁺ (10 mM and 20 mM) released [¹⁴C]ACh (but not [¹⁴C]choline) from both normal and botulinum-treated synaptosomes. It is concluded that botulinum toxin interferes with the provision of Ca²⁺ essential for the mechanism of ACh release.     

CHOLINERGIC STIMULATION OF PHOSPHOLIPID LABELLING FROM [32P]ORTHOPHOSPHATE IN GUINEA-PIG CORTEX SYNAPTOSOMES IN VITRO: SUBSYNAPTOSOMAL LOCALIZATION

Abstract— Subsynaptosomal localization of stimulation of phospholipid labelling by cholinergic agents was investigated. Synaptosomes prepared from guinea-pig cortex were incubated with [³²P]orthophosphate in the presence or absence of 10⁻³ m carbamylcholine. Following incubation and osmotic shock, lysed synaptosomes were subjected to density gradient fractionation. Subsynaptosomal fractions were examined by electron microscopy and analysed for enzyme activities and ³²P-labelled lipids.
In the absence of carbamylcholine, labelled phosphatidate and phosphatidylinositol were recovered in layers and interfaces A, B, C and D formed over 0.9, 1.1, 1.2 and 1.3 m sucrose, with highest amounts of label in fractions C and D for both lipids. Carbamylcholine induced the greatest increment in these two labelled lipids in fractions A and B. This distribution correlated with the presence of acetylcholinesterase activity and membrane ghosts. No correlation was found among the four fractions between the induced increase in labelling and succinic dehydrogenase activity or with the abundance of mitochondria, synaptic vesicles, or cytoplasmic fragments identified by electron microscopy. In contrast with the increases seen in phosphatidylinositol and phosphatidate labelling, carbamylcholine caused a decrease in ³²P-labelling of the polyphosphoinositides, and this effect was seen primarily in the heavier subsynaptosomal fractions, C and D.