In vitro Production of Halo Blight Inducing Toxin by Pseudomonas phaseolicola (Burkh.) Dowson

Three pathogenic strains of Pseudomonas phaseolicola (strain 1 and 3 virulent and strain 5 weakly virulent) were tested for their toxic activity. All three strains produced detectable amounts of toxin in vitro. Cultural conditions and length of incubation greatly influenced toxin production. Maximum amount of toxin was produced at 20°C and pH 6.5. Glycerol served as the best carbon source and 1-cysteine as the best amino acid for toxin production.     

The quantitative estimation of the infection of bean seed with Pseudomonas phaseolicola (Burkh.) Dowson

A routine laboratory method for the detection of Pseudomonas phaseolicola in bean seed is described. The method will detect low levels of seed-borne infection and has been used in a statistical procedure (the most probable number method) to give an estimate of percentage infection. Infection in seeds harvested from heavily infected crops varied from 10 to 1%, compared with from 1% to less than 0.1% in commercial seed stocks. A high proportion of infected seeds failed to produce infected plants and this may account for the very low levels of primary infection reported in the field. Removal of seeds showing possible ‘symptoms’ of disease reduced, but did not eliminate, infection from seed stocks.     

Detection of infection by Pseudomonas phaseolicola (Burkh.) Dowson in white-seeded dwarf bean seed stocks

Seeds of white-seeded varieties of dwarf beans infected with Pseudomonas phaseolicola may show fluorescent areas in the testa under ultra-violet light. Fluorescence is not visible in seed with dark-coloured testas and is masked by coloured seed dressings. Bacterial infection is not the only cause of fluorescence in testas and the type and colour of the fluorescence cannot be used to diagnose infection. Of 542 fluorescing seeds examined 112 were infected by the pathogen, and only six infected seeds were found in 495 non-fluorescing seeds from the same samples. It is estimated that in two seed stocks used for these experiments 64 and 68 % of infected seeds were of the fluorescent type. A 34 lb sample of seedstock has to be taken for examination to detect, with 95 % confidence, ten infected seeds in 1 cwt. The selection of all fluorescing seeds from such large samples effectively concentrates the infected seed into a sample small enough to be tested by bacteriological methods. Test seeds are examined bacteriologically by enrichment-culture in nutrient broth. Ps. phaseolicola is identified in these cultures from the diagnostic symptoms of halo-blight disease induced in bean seedlings inoculated with aliquots of the cultures.     

Production of the chlorosis inducing toxin in liquid cultures ofPseudomonas phaseolicola (Burkh.) Dowson

Amino acids affected amount and time course of the toxin production on complex as well as on chemically defined media. With glutamic acid the toxin content was high in the early growth stage but decreased later on, while the reverse was true with cysteine as single amino acid.Toxin production was highest at temperatures below 20°C. Generally, the toxin production started during the rapid growth phase and reached its maximum shortly after growth ceased. Chloramphenicolinhibited bacteria did not produce much toxin. Bacterial cells contained relatively low amounts of the toxin. Conditions for toxin production in large quantities are described. The feasibility to produce tritiummarked toxin was studied.Comparison of different strains showed that avirulent or weakly virulent halo-less strains do not produce detectable amounts of toxin. However, there was no correlation of the toxin production to the grouping of the bacterial strains in race 1 or the more virulent race 2.     

Multiple Hydrolases in Bean Leaves (Phaseolus vulgaris L.) and the Effect of the Halo Blight Disease Caused by Pseudomonas phaseolicola (Burkh.) Dowson

Multiple forms of hydrolytic enzymes were demonstrated in extracts of healthy bean leaves (Phascolus vulgaris L.) and bean leaves infected with the halo blight organism [Pseudomonas phaseolicola (Burkh.) Dowson] by polyacrylamide disc electrophoresis.Bean leaves contained up to 4 acid phosphatase bands, 9 esterase bands active towards alpha-naphthyl acetate, and 7 esterase bands towards alpha-naphthyl butyrate. Only low or no activity was found for alkaline phosphatase, lipase, and aminopeptidase.Two artifacts are described which were observed with the lead phosphate method for acid phosphatase and the Tween method for demonstration of lipase.After infection with the halo blight organism the major acid phosphatase of the host increased during early and decreased at later infection stages. An acid phosphatase of bacterial origin with a more neutral pH optimum could be demonstrated in infected leaves. It is suggested that the bacterial acid phosphatase plays a role in uptake of metabolites by the pathogen.Several esterase bands decreased after infection. One host band with activity towards alpha-naphthyl butyrate increased. Also the pathogen showed an esterase band with high activity towards alpha-naphthyl butyrate.     

Bacteriophage phi6: a Lipid-Containing Virus of Pseudomonas phaseolicola

The purification and properties of a lipid-containing bacteriophage, 6, are described. The phage contains a lipid envelope which is probably essential for infection. Infectivity of 6 was lost in the presence of organic solvents, sodium deoxycholate, and phospholipase A. The fatty acid composition of the phage lipid was similar to that of the Pseudomonas phaseolicola host cells. The phage was composed of about 25% lipid, 13% RNA, and 62% protein. The buoyant density of 6 was 1.27 g/ml in cesium chloride. The morphology of 6 was unusual; it had a polyhedral head of about 60 nm surrounded by a membranous, compressible envelope which appeared to assume an elongated configuration upon attachment to pili. The adsorption rate constant was 3.3 × 10⁻¹⁰ ml/min in a semi-synthetic medium and 3.8 × 10⁻¹⁰ ml/min in a nutrient broth-yeast extract medium. The latent period was shorter in the former medium (80-115 min compared with 120-160 min), and the average burst size was larger (250-400 compared with 125-150). The eclipse period coincided with the latent period.     

噬菌体受体及其鉴定方法

目的噬菌体又称细菌病毒,它可以侵入细菌使细菌裂解。噬菌体受体位于宿主细胞表面,能被噬菌体识别并特异性结合。噬菌体受体多种多样,包括细菌膜蛋白、LPS、磷壁酸,也可以是菌毛、鞭毛、荚膜多糖等。噬菌体在微生态平衡、微生物控制等方面具有重要意义,为了更好地研究噬菌体和挖掘噬菌体潜在价值,确定噬菌体受体是十分关键的一步。本研究就细菌噬菌体受体进行分类介绍,并对噬菌体受体鉴定方法进行总结。     

Seed treatment for the control of halo-blight of beans (Pseudomonas phaseolicola)

Streptomycin and kasugamycin were applied as soaks, slurries or dusts to dwarf bean seeds either naturally (internally) infected or externally contaminated with Pseudomonas phaseolicola. Slurries of streptomycin (2–5 g a. i./kg seed) or kasugamycin (0–25 g a.i./kg seed) were the most effective treatments against both types of infection and were generally non-phytotoxic at these rates. Combined analysis of eight field experiments made over a 3 yr period showed that on average both compounds applied in slurries reduced primary infection from infected seeds by 98 %. The method thus shows considerable promise as a commercial control treatment.     

Volatile Products of the Lipoxygenase Pathway Evolved from Phaseolus vulgaris (L.) Leaves Inoculated with Pseudomonas syringae pv phaseolicola

Activation of the "lipoxygenase pathway" in plants gives rise to a series of products derived from fatty acids. Analysis by gas chromatography-mass spectroscopy of volatile products produced by Phaseolus vulgaris (L.) cv Red Mexican leaves during a hypersensitive resistance response (HR) to the plant pathogenic bacterium Pseudomonas syringae pv phaseolicola showed evolution of several lipid-derived volatiles, including cis-3-hexenol and trans-2-hexenal, which arise from the 13-hydroperoxide of linolenic acid. These compounds were not produced in detectable amounts by buffer-inoculated leaves, nor did they evolve to such a high degree during comparable stages of the susceptible response. The absence of trans-2,cis-6-nonadienal, a product expected from 9-hydroperoxide of linolenic acid, suggests that lipid peroxidation during the HR proceeded primarily enzymically via bean lipoxygenase, which produces the 13-hydroperoxide, and not via autoxidative processes. The effects of trans-2-hexenal, cis-3-hexenol, and traumatic acid on P.s pv phaseolicola were investigaed. trans-2-Hexenal appeared to be highly bactericidal at low concentrations, whereas cis-3-hexenol was bactericidal only at much higher concentrations. Traumatic acid appeared to have no effect on P.s. pv. phaseolicola at the concentrations tested. These results demonstrate that during plant defense responses against microbial attack, several lipid-derived compounds are produced by the plant, some of which possess antimicrobial activity and conceivably are involved in plant disease resistance. The time of production of these substances, in amounts that would be expected to be antibacterial in vitro, correlated with a slowing down of the growth rate of bacteria in the leaves and was seen at a time before the accumulation of isoflavonoid phytoalexins in the host.     

Rapid identification of yeasts by serological methods: a combined serological and biological method.

A total of 387 yeasts from the contents of the digestive tracts of domestic animals and poultry were identified by slide agglutination tests using factor antisera and urease tests. The results of this serological test were very satisfactory with respect to accuracy and rapidity, particularly when performed in combination with concomitant physiological tests only for assimilation of inositol and potassium nitrate. It may be concluded that such a combination of serological and biological tests is very useful for identifying yeast strains from various sources.     

Effectiveness of late copper and streptomycin sprays for the control of halo-blight of beans (Pseudomonas phaseolicola)

Two sprays of copper oxychloride or streptomycin sulphate (0–1 % a.i., 675 1/ha) applied to dwarf beans at first flowering and at pod set reduced pod infection by c. 70%. When applied as single sprays at pod set, copper was more effective than streptomycin and reduced pod infection by 50%. Although a copper spray at pod set increased the proportion of pods suitable for processing, more effective control was obtained with a regular spray programme, commencing at the seedling stage.     

Studies on the Extracellular Polysaccharides (EPS) Produced in vitro by Pseudomonas phaseolicola

Native EPS produced by Pseudomonas syringae pv. phaseolicola in vitro was separated by ion exchange chromatography on DEAE fractogel into three different polysaccharide fractions. A neutral polysaccharide eluting with the void volume yielded only fructose upon hydrolysis and exhibited an IR spectrum similar to authentic levan. At about 300 mM KCl a mannuronan eluted. Comparison with authentic alginate by IR spectroscopy, elution behaviour during DEAE-fractogel column chromatography, and monomer composition (mannuronic acid and traces of guluronic acid) confirmed the identity of this fraction as a bacterial alginate. It contained about 56 mol% acetyl groups. A third polysaccharide eluted at about 160 mM KCl. Its monomeric composition (rhamnose, fucose, glucose, and amino sugars), elution behaviour upon DEAE-fractogel column chromatography, and TLC patterns, closely resembled the sugar moiety of lipopolysaccharides (LPS) from, Pseudomonas syringae pv. phaseolicola. The protein component of crude EPS represented a fourth macromolecular fraction. It was not covalently linked to any of the polysaccharides since it could be removed from the EPS by phenol extraction.