Solubilization of galactosyltransferase that synthesizes 1,4-beta-galactan side chains in pectic rhamnogalacturonan I

β -1,4-Galactan galactosyltransferase (GT) activity was solubilized from potato microsomal membranes in the presence of 78 m M 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonic acid. The solubilized GT activity transferred ¹⁴[C]galactose from UDP-¹⁴[C]galactose onto the acceptor-substrates composed of rhamnogalacturonan (RG) with short galactan chains (RG-A, approximately 1.2 MDa, mol% Gal/Rha = 0.7; RG-B, approximately 21 kDa, mol% Gal/Rha = 1.2). However, shorter RG containing short galactan chains (approximately 2 kDa and 1.2 kDa), RG oligomers without galactosyl-residues, galactan, and galactooligomers did not act as acceptor-substrates. Optimal pH for ¹⁴[C] incorporation onto RG-A and RG-B was around 5.6 and 7.5, respectively. The ¹⁴[C]-labelled products synthesized upon RG-A and RG-B could be digested with a RG specific lyase into smaller RG fragments. 1,4- β - Endog alactanase could not digest the former product, whereas the latter product was digested to ¹⁴[C]galactobiose and ¹⁴[C]galactose. This demonstrates that at least two GT activities were solubilized from potato microsomal membranes. One had optimal pH around 5.6 to transfer galactosyl residues onto RG-A, whereas the other had optimal pH around 7.5 to transfer galactosyl residues onto RG-B. Both synthesized galactan attached to the RG backbone of RG-A and RG-B, and the galactan synthesized onto the RG-B acceptor was 1,4- β -linked.     

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.     

A note on the attachment rate of suspended bacteria to submerged aquatic plants in a calcareous stream

The density of epiphytic bacteria on leaflets of laboratory-grown Apium nodiflorum , immersed in calcareous stream water, increased linearly with time over 10 h, both in the laboratory and in the field. This increase was probably due to attachment of suspended bacteria. The rate of attachment to leaflets of plants, immersed in a stream at different concentrations of suspended bacteria, was linearly related to the concentration of suspended bacteria. The attachment rate, relative to concentration, was 1.7 × 10⁴ bacteria/cm²/h for each 10⁵ bacteria/ml in the surrounding water.     

PHYSIOLOGICAL RACES OF PHYTOPHTHORA INFESTANS: A COMPARISON OF THE DIFFERENTIAL HOSTS AT THE PLANT BREEDING INSTITUTE, CAMBRIDGE, WITH THOSE OF THE SCOTTISH SOCIETY FOR RESEARCH IN PLANT BREEDING

At the Plant Breeding Institute, Cambridge, there have been recognized three physiological races of blight ( Phytophthora infestans ), A, B and C ; and at the Scottish Society for Research in Plant Breeding, Edinburgh, there have been used five races, A, B ¹, B ², C and D , obtained in the British Isles.
It is shown that the two Cambridge types of differential hosts, AbC and ABc (where A = resistant to race A, a = susceptible to race A , etc.), are Ab ¹ b ² CD and AB ¹ B ² cD respectively on the Scottish scheme, and that the Cambridge races A, B and C correspond to the Scottish races A, B ¹ and C respectively.
A number of blight isolates were tested on both the Cambridge and Scottish differential hosts. Isolates of race types A, B ¹, B ¹, C and D were found.
The identification and origin of physiological races of blight, and the breeding of blight-resistant potatoes, are discussed.     

Virulence systems of Pseudomonas syringae pv. tomato promote bacterial speck disease in tomato by targeting the jasmonate signaling pathway

Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000) causes bacterial speck disease on tomato. The pathogenicity of Pst DC3000 depends on both the type III secretion system that delivers virulence effector proteins into host cells and the phytotoxin coronatine (COR), which is thought to mimic the action of the plant hormone jasmonic acid (JA). We found that a JA-insensitive mutant (jai1) of tomato was unresponsive to COR and highly resistant to Pst DC3000, whereas host genotypes that are defective in JA biosynthesis were as susceptible to Pst DC3000 as wild-type (WT) plants. Treatment of WT plants with exogenous methyl-JA (MeJA) complemented the virulence defect of a bacterial mutant deficient in COR production, but not a mutant defective in the type III secretion system. Analysis of host gene expression using cDNA microarrays revealed that COR works through Jai1 to induce the massive expression of JA and wound response genes that have been implicated in defense against herbivores. Concomitant with the induction of JA and wound response genes, the type III secretion system and COR repressed the expression of pathogenesis-related (PR) genes in Pst DC3000-infected WT plants. Resistance of jai1 plants to Pst DC3000 was correlated with a high level of PR gene expression and reduced expression of JA/wound response genes. These results indicate that COR promotes bacterial virulence by activating the host's JA signaling pathway, and further suggest that the type III secretion system might also modify host defense by targeting the JA signaling pathway in susceptible tomato plants.     

The phytotoxin coronatine from Pseudomonas syringae pv. tomato DC3000 functions as a virulence factor and influences defence pathways in edible brassicas

The phytotoxin coronatine (COR) contributes to the virulence of Pseudomonas syringae pv. tomato ( Pst ) strain DC3000 on Arabidopsis thaliana and tomato. However, little is known regarding the role of COR in the virulence of DC3000 on cultivated Brassica spp. In this study, the role of COR and its precursors, coronafacic acid (CFA) and coronamic acid (CMA), were examined in the virulence of Pst DC3000 on collard and turnip, two important edible brassicas. Pst DC3000 and three well-defined COR⁻ biosynthetic mutants of DC3000 exhibited substantial differences in the timing and phenotype of disease lesions on collard and turnip. When examined 3 days post-inoculation (dpi), collard inoculated with DC3000 exhibited visible anthocyanin production and lesions were chlorotic and water-soaked. On turnip, chlorotic and necrotic lesions were evident on DC3000-inoculated leaves 5 dpi. The bacterial population dynamics on plants inoculated with DC3000 and the COR⁻ mutants indicated that COR was essential for DC3000 to maintain high populations in turnip, but not collard. Real-time quantitative PCR revealed that the jasmonic acid pathway responsive genes, LOX2 and CORI1 , were expressed in both hosts inoculated with Pst DC3000. PR1 , a marker associated with the salicylic acid pathway, was expressed in collard and turnip inoculated with the CFA⁻ CMA⁻ mutant DB29, but not DC3000. Further comparison of PR1 and LOX2 expression indicated that CFA plays a subtle role in modulating defence in turnip. This is the first study to investigate the role of COR in the interaction of Pst DC3000 and cultivated brassicas using genetically and biochemically defined COR⁻ mutants.     

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.     

Transformation of Methanococcus maripaludis and identification of a Pst I-like restriction system

Abstract An optimized polyethylene glycol (PEG) method of transformation was developed for Methanococcus maripaludis using the pKAS102 integration vector. The frequency of transformation with 0.8 μg of plasmid and 3×10⁹ cells was 4.8×10⁻⁵ transformants cfu⁻¹, or 1.8×10⁵ transformants μg⁻¹, which was four orders of magnitude greater than with the natural transformation method. A Pst I restriction activity in M. maripaludis was also identified. Methylation of the plasmid with Pst I methylase increased the methanococcal transformation frequency at least four-fold. Also, chromosomal DNA from M. maripaludis was resistant to digestion by the Pst I endonuclease.     

Detached leaf enrichment: a method for detecting small numbers of Pseudomonas syringae pv. tomato and Xanthomonas campestris pv. vesicatoria in seed and symptomless leaves of tomato and pepper

A method for detecting 10¹-10² cells of phytopathogenic bacteria ( Pseudomonas syringae pv. tomato and Xanthomonas campestris pv. vesicatoria ) in either tomato or pepper seed was developed. The method is based on the enrichment of the compatible pathogen inside a detached leaf of its host when placed on a water agar medium. It was found to be superior to the diagnostic growth media method commonly used and to permit the detection of the pathogens in symptomless plants.     

Relationship between polar basipetal auxin transport and acropetal Ca2+ transport into tomato fruits

The dependence of acropetal Ca²⁺ transport on polar basipetal indoleacetic acid (IAA) transport was investigated in excised tomato fruits ( Lycopersicon esculentum L. Mill.) using an in vitro fruit system. Auxin transport inhibitors like triiodobenzoic acid (TIBA), chlorofluorenolmethyl ester (CME) and naphthylphthalamic acid (NPA) were used in order to investigate the effect of restricted polar basipetal auxin transport on the acropetal transport of ⁴⁵Ca²⁺, ⁸⁶Rb⁺ and ⁹⁸Sr²⁺ into the same fruits. TIBA and CME inhibited basipetal transport of IAA. particularly in 10- to 12-day-old tomato fruits, and simultaneously restricted the acropetal transport of ⁴⁵Ca²⁺. The auxin transport inhibitors failed to significantly reduce the upward transport of ⁸⁶Rb⁺ and the transport of ⁹⁶Sr²⁺ was less inhibited than that of ⁴⁵Ca²⁺. TIBA and CME did not significantly affect the acropetal transport of labelled water into the fruit, nor the cation-exchange capacity or K⁺ and Mg²⁺ concentrations in the tomato fruit. These results support the view that a part of the Ca²⁺-specific acropetal transport into tomato fruits is associated with the polar basipetal IAA transport. This Ca²⁺ transport is independent of the transpiration stream into the fruit and the cation exchange capacity of the fruit tissue.     

In Human Fetal Astrocytes Exposure to Interleukin-1β Stimulates Acquisition of the GD3+ Phenotype and Inhibits Cell Division

Abstract: In human astrocyte cultures established from second-trimester fetal brain tissue, ∼5–10% of total astrocyte population in unstimulated cultures were GD3⁺/glial fibrillary acidic protein (GFAP)⁺. The GD3⁺ cells were always GFAP⁺ and grew as flat, highly spread cells but changed to process-bearing cells after interleukin-1β (IL-1β) stimulation. It is interesting that IL-1β, a known mitogen for rat astrocytes, suppressed human fetal astrocyte proliferation as determined by [³H]thymidine incorporation, bromodeoxyuridine (BrdU) labeling, and cell counting. The GD3⁺ population, however, consistently increased in absolute number after IL-1β stimulation, in a dose- and time-dependent manner. The IL-1β-mediated increase in number of GD3⁺ astrocytes was independent of initial cell density or serum concentration. By flow cytometry, IL-1β enhanced both the mean fluorescence intensity and the percentage of GD3⁺ cells. To investigate whether the increase in GD3⁺ astrocyte cell number was due to proliferation of preexisting GD3⁺ astrocytes or due to conversion of GD3⁻ to GD3⁺ cells, we performed BrdU/GD3 double immunocytochemistry. BrdU/GD3 double-positive cells were extremely rare in both control and IL-1β-stimulated cultures. Moreover, an increase in number of GD3⁺ astrocytes was still observed in control and IL-1β-stimulated cultures where GD3⁺ cells had been initially eliminated by cell sorting. These results indicate that GD3⁺ astrocytes in human fetal culture may represent a postmitotic, differentiated, distinct phenotype.     

Behaviour of Staphylococcus aureus strains, producers of enterotoxins C1 or C2, during the manufacture and storage of Burgos cheese

Burgos cheese was manufactured from pasteurized ewes milk inoculated with Staphylococcus aureus strains FRI 137 and FRI 361, at levels of ca 10³ and 10⁵ cfu/ml and stored at 4°, 10° and 15°C and at room temperature (10°-15°C). Populations of Staph. aureus and mesophilic aerobes, pH, and production of thermonuclease and enterotoxins C₁ and C₂ were investigated. Aerobic counts increased during cheese-making and storage. With both test strains, important growth was observed only during the storage period, the larger levels corresponding to the higher temperatures. Although Staph. aureus strains attained populations of over 10⁸ cfu/g, no enterotoxin was detected. Strain FRI 361 reached 10⁷ cfu/g without production of a detectable amount of thermonuclease. With strain FRI 137, the minimal population associated with enzyme activity was influenced by the inoculum size. Staphylococcus aureus counts are better indicators of staphylococcal growth in Burgos cheese than the thermonuclease test.     

In vitro susceptibility of Erwinia amylovora (Burrill) Winslow et al. to geraniol and citronellol

The in vitro antimicrobial activity of geraniol and citronellol towards seven strains of Erwinia amylovora , the causal agent of 'fire blight'of Rosaceous plants, was assessed in tube cultures. All of the strains tested at 1 × 10⁵ cfu/ml were inhibited for 24 h by geraniol in the range 600–1500 mg/1, whereas its minimum bactericidal concentration was 800–1700 mg/1. Citronellol was less effective, being bactericidal for only two of seven strains. RIF-NY, isolated from apple orchards, was relatively resistant to geraniol; 1700 mg/1 of the chemical only reduced the growth of an inoculum of 1 × 10⁷ cfu/ml. In general, such terpenoids commenced exerting a bactericidal effect 6 h after addition to the suspensions, even if geraniol added at 1700 mg/1 to 1 × 10³ cfu/ml of five strains, commenced its bactericidal activity earlier than 6 h.     

An Ecological Study of Marked Rhizobium trifolii Strains on the Host Plant Trifolium repens var. Huai in an Acidic Peat and a Neutral Mineral Soil

An ecological study of the nodulation of Trifolium repens var. grassland Huai by genetically marked Rhizobium trifolii was carried out in two Irish soils, a neutral mineral and an acidic peat. An indigenous population of 2 x 10⁴ R. trifolii /g was found in the mineral soil. In the peat soil, 4 x 10¹ R. trifolii /g was found in the uninoculated peat. This number increased to 4.5 x 10⁵ R. trifolii /g, however, eight weeks after the peat soil was neutralized, supplemented with nutrients and sown with uninoculated clover seed. Indigenous R. trifolii strains from the mineral soil were effective whereas strains from the peat soil were ineffective on the host plant T. repens under plant room conditions. The introduced strains were inoculated on to clover seed at the rate of 1 x 10⁵ R. trifolii /seed. In the mineral soil, the introduced inoculum failed to establish at any period during the growing season. In the peat soil, the percentage establishment of the introduced inoculum varied from 40-50% of nodules selected eight weeks after sowing to 70-90% of nodules selected at the end of the growing season.     

Conjugative transfer of the transposon Tn919 to lactic acid bacteria

Abstract The streptococcal transposon Tn 919 was transferred from Streptococcus faecalis GF590 to selected Group N Streptococcus strains and to one strain each of Lactobacillus plantarum and Leuconostoc cremoris , using the filter mating method. An S. lactis MG1363 Rif^r Tc^r transconjugant also acted as a donor, but was less efficient than GF590. Frequencies of transfer varied between 4.0 × 10⁻⁸ and 5.29 × 10⁻⁵ per recipient. Further analysis of S. lactis MG1363 Sm^r Tc^r transconjugants showed that insertion of Tn 919 into the chromosome was site-specific.     

Survival of genetically modified Pseudomonas fluorescens introduced into subtropical soil microcosms

Abstract A genetically modified strain of Pseudomonas fluorescens and its parent showed grossly similar decline rates following introduction into subtropical clay and sandy soils. In unplanted clay soit at pH 6.9 and 25°C, population densities declined progressively from about 10⁸ to 10³ colony forming units (cfu) g⁻¹ dry soil over 75 days, but in unplanted sandy soil the introduced populations could not be detected after 25 days. In clay soil at pH 8.7 or 4.7, or at environmental temperature, decay rates were enhanced as compared to those at pH 6.9 and 25°C. Counts of introduced strains in clay bulk soil and in rhizosphere and rhizoplane of maize suggested that the introduced bacteria competed well with the native bacteria, and colonized the roots at about 10⁶ cfu g⁻¹ dry root at 25°C, over 20 days. However, rhizoplane colonization was lower at environmental temperature. The decay rate of both strains was slower in planted than in unplanted sandy soil. The population densities in the rhizosphere and rhizoplane in the sandy soil were significantly lower than those in the clay soil. Both introduced strains colonized the maize roots in both soils, using seeds coated with bacteria in 1% carboxymethyl cellulose. Introduced cells were localized at different sites along the roots of plants developing in clay soil, with higher densities in the original (near the seeds) and root hair zones as compared to the intermediate zones. No significant difference was observed between the extent of root colonization of the genetically modified strain and its parent.     

Growth analysis of wild-type and photomorphogenic-mutant tomato plants

A custom designed growth-measuring apparatus, controlled by a microcomputer has been used to study extension growth kinetics of wild-type and photomorphogenic-mutant tomato ( Lycopersicon esculentum Mill.) plants with and without end-of-day farred light (EODFR). The following photomorphogenic mutants were used. Far-red insensitive ( fri ^.1): deficient in phytochrome A (phyA); temporarily red light-insensitive ( tri ^.3): deficient in phytochrome Bl (phyB1), and their isogenic wild type (WT) cv. MoneyMaker. aurea (au) : deficient in phytochrome chromophore biosynthesis; high-pigment-1 ( hp-1 ): exhibiting exaggerated phytochrome responses, and their isogenic WT cv. Ailsa Craig. The stem elongation rate (SER) during a 24-h period of all the genotypes studied shows a similar pattern, having two dramatic transients, one shortly after the onset of the light period (a sharp decline in SER) and another shortly after the start of the dark period (a sharp increase in SER). These transients are probably associated with water relations as a consequence of opening and closure of the stomata. The fastest SER occurs during the dramatic oscillations early in the dark period. Between the genotypes there are large quantitative differences in SER. All the genotypes tested exhibited a strong EODFR response, resulting in a relative promotion of SER during the first period after the start of EODFR and in the subsequent light and dark periods. These results indicate that phyA, absent in the fri ^.1 mutant, does not play a major role in SER of light-grown tomato plants, whereas phyB 1, absent in the tri ^.3 mutant, is partly responsible for the compact stature of WT plants. An additional phytochrome other than phy A and phy B1 must therefore be capable of eliciting the EODFR response.     

Note: Comparison of media for the isolation of lactose-positive Salmonella

We studied the capacity of 10 selective media (Rambach agar, RB; salmonella-shigella agar, SS; SM-ID medium, SM; Hektoen enteric agar, HE; modified semisolid Rappaport-Vassiliadis agar, MSRV; bismuth sulphite agar, BS; MacConkey agar, MC; brilliant green agar, BG; novobiocin-brilliant green-glucose agar, NBG; and novobiocin-brilliant green-glycerol-lactose agar, NBGL), and the C₈-esterase test (MUCAP test, Biolife, Italy) to detect the growth of 14 strains of lactose-positive Salmonella (12 Salm. virchow and two Salm. montevideo ) and 16 Salm. arizonae. Suspensions of pure strain were plated on the aforementioned media and on Mueller-Hinton, used as a control, with inocula of 3 x 10² cfu ml^-1. The performance of BS was excellent, determining the 30 strains as typical Salmonella colonies (H₂S+). On NBG, 27 strains were detected. On MSRV, only some strains grew and only one produced swarming. On the other media, the two Salm. montevideo and the 12 Salm. virchow strains produced coliform colonies. Some of these latter were inhibited on BG and NBGL. The 16 Salm. arizonae strains produced typical colonies on all the media, except on RB, SM and MSRV. On NBGL, two strains did not produce H₂S. The C₈-esterase test was only successful with Salm. montevideo and Salm. virchow on NBG and RB (with a few exceptions on the latter). However, with Salm. arizonae the test was positive on SS, MC, HE, BG and NBG. In summary, BS was the best medium of those used (all the 30 strains were isolated), followed by NBG (27 isolates).     

Spatial organization and population density of the fish community of the litter banks within a central Amazonian blackwater stream

Results of a study of the fish community of the leaf litter banks in the lower reaches of the Taruma-Mirim, a small central Amazonian stream is presented. The Taruma-Mirim is an acidic, blackwater, forest stream which flows through igapo forest and enters the Rio Negro close to Manaus. The commonly held view that these blackwaters hold an impoverished ichthyofauna is challenged. The studied litter banks hold about 20 species of fish, all of which were found to live within distinct subregions of the habitat. Average fish density was about 100 individuals m⁻². Absolute population sizes were found to be remarkably small; within a 200 m² area the most abundant species had a population size of about 10⁴ and the least abundant <10² individuals. It is argued that high species richness linked to specialized habitat requirements and small population size indicates considerable population stability. The factors leading to the evolution of such species richness are discussed.