HydroxyproUne-rich Glycoprotein Accumulation in TMV-infected Tobacco Showing Systemic Acquired Resistance to Powdery Mildew

Tobacco cv. Havana 425, with systemic-acquired resistance (SAR) to an otherwise compatible Erysiphe cicho-racearum DC. race after TMV infection, was infected with TMV basaily (4^th and 5^th leaves) and the hydroxyproline-rich glycoproteins (HRGPs) were determined in two experiments (experiment 1 and experiment 2) by analysing cell wall hydroxyproline (Hyp) in the 6^th leaf. In basal TMV infected plants Hyp content (μmol^-1 FW) was greater than in controls in both experiments: the increase was significant at all times except 16 days after TMV inoculation in experiment 1; the pool of data of experiment 2 was also significantly increased. In TMV protected + E. cichoracearum challenged leaves compared to untreated controls significant increases in Hyp were also noted between the pools of data in both experiments. No differences were found between Hyp content in protected compared to protected + challenged leaves in both experiments. These results show accumulation of HRGPs in Havana tobacco associated with TMV infection and SAR activation against E, cichoracearum. The accumulation appears to be due to the inducer, since no further increase was detected in protected leaves after challenging.     

Effects of ethylene biosynthesis in carrot root slices on 6-methoxymellein accumulation and resistance to Botrytis cinerea

The role of ethylene biosynthesis in the resistance response of carrot ( Daucus carota L., cv. Chantenay red-cored) slices to infection by Botrytis cinerea Pers. ex Pers. was investigated using aminoethoxyvinylglycine (AVG) and inhibitor of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (EC 4.4.1.-), and norbornadiene, an inhibitor of ethylene binding. Carrot slices became susceptible to a normally non-invasive level (10⁵ ml^-1) of spores of B. cinerea after treatment with AVG. ACC partially reversed the susceptibility induced by AVG. The ability of a crude pectic enzyme preparation from B. cinerea to induce resistance to a normally invasive level of B. cinerea spores (10⁶ ml^-1) was prevented by AVG. Accumulation of the carrot phyto-alexin 6-methoxymellein (6-MM) was prevented by norbornadiene, but it had no effect on the resistance response. An event associated with ethylene biosynthesis other than 6-MM accumulation appears to be responsible for the resistance of carrot slices to infection by B. cinerea.     

Multiplication of Pseudomonas syringae pv. phaseolicola "in planta"

Multiplication of Pseudomonas phaseolicola was determined in 17 different bean cultivars ( Phaseolus vulgaris ) and 9 other plant species, and the effect of different inoculation methods and conditions was also studied.
In susceptible leaves, a generation time of 2.1 h was determined in the early phase (2 days after inoculation). Different multiplication rates in susceptible and resistant leaves were clearly observed 4 days after inoculation. At this time the first small water-soaked spots were visible in the susceptible cultivars. Bacteria multiplied up to the 7th day after inoculation with a maximum of 10⁹ cells per cm² leaf (equal to ca. 4 × 10¹⁰ bacterial cells/cm³). At the same time, the water-soaked spots had reached their maximum size in most cases. Thus, bacterial multiplication and development of water-soaked spots paralleled each other.
In resistant leaves, no water-soaked spots appeared, and the final bacterial concentration was 1/1000–1/100 of that in susceptible leaves. Gomparison of races 1 and 2 in several bean cultivars indicated the non-existence of a gene-for-gene relationship with this disease. Old leaves were less susceptible to infection. Some bacterial multiplication was also observed in non-host plants. There was a general correlation between bacterial multiplication in the non-host plants and their botanical relation to Phaseolus vulgaris .     

Purification of an α-L-arabinofuranosidase from carrot cell cultures and its involvement in arabinose-rich polymer degradation

Konno, H., Yamasaki, Y. and Katoh, K. 1987. Purification of an α-L-arabinofurano-sidase from carrot cell cultures and its involvement in arabinose-rich polymer degradation.
An α-L-arabinofuranosidase (α-L-arabinofuranoside arabinofuranohydrolase, EC 3.2.1.55) was isolated from a homogenate of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The buffer-soluble enzyme was purified to homogeneity by a procedure involving ammonium sulfate fractionation, chromatography on DEAE-Sephadex A-50, Sephadex G-150, Con A-Sepharose 4B and CM-Sephadex C-50, and preparative polyacrylamide gel electrophoresis. The size of this enzyme as determined by polyacrylamide gel electrophoresis in the presence of sodium laurylsulfate and by Sephadex G-200 gel filtration was 94 and 110 kDa, respectively. The isoelectric point was at pH 4.7. The K_m and V_max values for p-nitrophenyl α-L-arabinofuranoside were 1.33 mM and 20.2 μimol (mg protein)^-1 h^-1, respectively. The optimal activity occurred at pH 4.2 with Mcllvaine buffer. The enzyme was stimulated by Ca²⁺ and Zn²⁺, whereas it was strongly inhibited by Cu²⁺, Ag²⁺, Hg²⁺, p-chloromercuri-benzoate and L-arabono-l,4-lactone. The enzyme acted on beet arabinan in an exo-fashion. Furthermore, the enzyme was partially involved in the hydrolysis of the ara-binogalactan and pectic polymer purified from carrot cell walls.     

Vegetable juice aids the recovery of heated spores of non-proteolytic Clostridium botulinum

Heating spores of non-proteolytic Clostridium botulinum at 85C for 2 min followed by plating on a standard laboratory medium reduced the count of viable spores by a factor of greater than 10⁴. A similar result was obtained when the plating medium was supplemented with juice from courgette, carrot or mung bean sprout. When plating was on media supplemented with hen egg white lysozyme or juice from turnip, swede, flat bean, cabbage or potato, heating at 85C for 10 min did not reduce the viable count by a factor of 10⁴. Thus these vegetable juices increased the measured heat resistance of spores of non-proteolytic Cl. botulinum . These findings are relevant to the safety of minimally processed (e.g. sous-vide ) foods.     

Extracellular exo-polygalacturonase secreted from carrot cell cultures. Its purification and involvement in pectic polymer degradation

Exo-polygalacturonase (exo-PGase, EC 3.2.1.67) activity has been detected in a culture filtrate of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The extracellular exo-PGase was purified to electrophoretic homogeneity using DEAE-Sephadex A-50 ion-exchange chromatography, Sephadex G-150 gel filtration, and preparative polyacrylamide gel electrophoresis (PAGE). The molecular mass of the purified enzyme was calculated to be 48 kDa from Sephadex G-200 gel filtration, and 50 kDa from sodium dodecyl sulfate (SDS)-PAGE after treatment with SDS and 2-mercaptoethanol. The isoelectric point was at pH 6.2. The K_m and V_max values for polygalacturonate (degree of polymerization: 52) were 14.4 μ M and 25.6 μmol (mg protein)⁻¹ h⁻¹, respectively. The optimal activity in McIlvaine's buffer occurred at pH 4.6. The enzyme activity was inhibited by Ba²⁺, Cu²⁺, Mn²⁺ and Hg²⁺. The enzyme was involved in ca 15% hydrolysis of the acidic polymer purified from carrot pectic polysaccharides, and connected with the release of galacturonic acid. Even after an exhaustive reaction the enzyme had, however, little or no effect on cell walls from carrot cell cultures.     

The immune response against Francisella tularensis live vaccine strain in Lpsn and Lpsd mice

Abstract The impact of Lps gene on the course of immune response against subcutaneous infection of mice with Francisella tularensis live vaccine strain was studied. Production and specificity of antibodies, cytotoxic responses of macrophages and NK-cells, spontaneous production ex vivo of cytokines IL-1α, IL-2, IL-4, IL-6, IL-10, IFN-γ, and TNF-α in spleen cell cultures in C3H/HeJ ( Lps ^d) mice in comparison with C3H/HeN ( Lps ^r) mice were tested. The value of LD₅₀ was significantly different in the two strains of mice (8.0 × 10³ cfu for C3H/HeJ versus 4.61 × 10⁵ cfu for C3H/HeN mice after subcutaneous inoculation). The production of NO₂ is also impaired in C3H/HeJ mice in the early intervals after infection. Thus, the defective Lps gene of C3H/HeJ mice influences both the level of innate resistance of mice to F. tularensis live vaccine strain infection and the process of induction and regulation of immune response against this intracellular bacterial pathogen.     

The Ability of Barley Powdery Mildew to Grow in vitro

A technique was developed for the in vitro culture of Blumeria graminis f. sp. hordei , an obligate biotrophic pathogen of barley. Optimal growth occurred at pH 5.6 on a medium containing 39 gl^–1 potato dextrose agar, 40 gl^–1 shredded fresh barley leaves, 20 gl^–1 sucrose, 13 mgl^–1 kanamycin and 80 mgl^–1 benzimidazole. At 20°C (90% relative humidity), conidia germinated 48 h after inoculation, producing an average colony diameter of 1 cm after 10 days. However, numerous colonies were present on the medium after 15 days. Light microscopy showed that there was a positive relationship between the amount of leaf in the medium and fungus growth. The fungus retained its virulence during 60 days of storage in vitro , and was able to infect barley. This is a useful and novel technique that could be beneficial in barley pathology breeding programs.     

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.     

Yam mosaic, a new potyvirus infecting Dioscorea cayenensis in the Ivory Coast

The major disease affecting Dioscorea cayenensis in the Ivory Coast is caused by a virus which was transmitted by mechanical inoculation to some Dioscorea spp. and Nicotiana benthamiana . In extracts of D. cayenensis leaves infectivity was lost after 10 min at 60 C but not 55 C and after dilution to 10^-3 but not 10^-2. A purification procedure is described. The virus particles are flexuous filaments c . 785 nm long. The virus was transmitted by four aphid species in the non-persistent manner, and is serologically related to four African potyviruses. The name yam mosaic virus is proposed; the present cryptogram is: */*:*/6:E/E:S/Ve/Ap, potyvirus group.     

An extracellular α-L-arabinofuranosidase secreted from cell suspension cultures of carrot

Carrot ( Daucus carota L. cv. Kintoki) cell cultures secrete an α-L-arabinofuranosidase (α-L-AFase, EC 3.2.1.55) into their culture medium during growth. The extracellular α-L-AFase (α-L-AFase-II) was purified to electrophoretic homogeneity from the concentrated medium using ammonium sulfate precipitation, chromatography on DEAE-Sepharose CL-6B, CM-Sepharose CL-6B, Sephacryl S-200HR and Concanavalin A-Sepharose, and preparative PAGE. The molecular mass of the purified enzyme was estimated to be 84 kDa by Sephacryl S-200HR gel-permeation, and 80 kDa by SDS-PAGE under denaturing conditions. The enzyme contained carbohydrate and protein in a ratio of 1:5 (w/w), and was analyzed for amino acid composition and the sequence of the first 21 amino acids of the N-terminus. The isoelectric point was pH 5.6, the pH optimum 3.8, and the temperature optimum 55°C. The activity was inhibited by Zn²⁺, Ag²⁺, Cu²⁺, Hg²⁺ and p -chloromercuribenzoate. The K_m and V_max values for p -nitrophenyl-α-L-arabinofuranoside were 0.22 m M and 0.11 mmol (mg protein)⁻¹ h⁻¹, respectively. The enzyme acted on beet arabinan in an exo-fashion, and was capable of hydrolysing arabinose-rich polymers purified from pectic polysaccha-rides of carrot cell cultures. However, even after an exhaustive reaction, the enzyme had little or no effect on cell walls from carrot cell cultures.     

Effect of auxins on spermidine uptake into carrot protoplasts

The effect of an auxin, indole-3-acetic-acid (IAA), on spermidine uptake into protoplasts of carrot ( Daucus carota L. cv. Ingrid) was studied. In the presence of 1 m M Ca²⁺, IAA (10⁻⁷ to 10⁻⁴ M ) enchances [¹⁴C]-spermidine uptake into carrot protoplasts, while no stimulation occurs in the absence of Ca²⁺. The time course of the uptake with and without IAA is very rapid and reaches saturation within 1 to 2 min. Preincubation of protoplasts with IAA inhibits the spermidine uptake. La³⁺, known not to penetrate the plasmalemma, exerts the same effect as Ca²⁺, but gives lower uptake values than Ca²⁺. The application of vanadate, an ATPase inhibitor, strongly inhibits IAA-stimulated spermidine uptake, suggesting that an energy-dependent mechanism may be involved in this transport. Neither spermidine nor Ca²⁺ alone stimulate IAA uptake. The synthetic auxin 2,4-dichlorophenoxyacetic acid, yields the same results as IAA with regard to time course of spermidine uptake with and without preincubation while, unlike IAA, no significant effect was observed on the Ca²⁺ -induced increase of spermidine uptake.     

Physiological effects of long-term exposure to low and moderate concentrations of atmospheric NH3 on poplar leaves

Abstract. Poplar shoots ( Populus euramericana L.) obtained from cuttings were exposed for 6 or 8 weeks to NH₃ concentrations of 50 and 100 μgm⁻³ or filtered air in fumigation chambers. After this exposure the rates of NH₃ uptake, transpiration, CO₂ assimilation and respiration of leaves were measured using a leaf chamber. During the long-term exposure also modulated chlorophyll fluorescence measurements were carried out to obtain information about the photosynthetic performance of individual leaves. Both fluorescence and leaf chamber measurements showed a higher photosynthetic activity of leaves exposed to 100 μg NH₃ m⁻³. These leaves showed also a larger leaf conductance and a larger uptake rate of NH₃ than leaves exposed to 50 μg m⁻³ NH₃ or filtered air. The long-term NH₃ exposure did not induce an internal resistance against NH₃ transport in the leaf, nor did it affect the leaf cuticle. So, not only at a short time exposure, but also at a long-term exposure NH₃ uptake into leaves can be calculated from data on the boundary layer and stomatal resistance for H₂O and ambient NH₃-concentration. Furthermore, the NH₃ exposure had no effect on the relation between CO₂-assimilation and stomatal conductance, indicating that NH₃ in concentrations up to 100 μg m⁻³ has no direct effect on stomatal behaviour; for example, by affecting the guard or contiguous cells of the stomata.     

Carbon source utilization of soil extracted microorganisms as a tool to detect the effects of soil supplemented with genetically engineered and non-engineered Corynebacterium glutamicum and a recombinant peptide at the community level

Abstract: Substrate utilization of microbial cells extracted from soil with a 0.85% aqueous sodium chloride solution, was determined to estimate effects on soil microorganisms at the community level with microtiter plates (Biolog GN®) containing 95 different sources of organic carbon. A consistent pattern of utilized substrates was obtained after 24 h of microtiter plate incubation at 28°C. The absorbance values (OD₅₉₀) obtained from a microtiter plate reader after background correction were transformed by using the average absorbance values of oxidized substrates as a threshold to distinguish between well utilized and poorly or non-utilized substrates and thereby reduce variances between replicates. Doubling times of the extracted soil microorganisms in the microtiter plates were tested with 12 substrates and ranged from 1.96 h to 3.23 h, depending on the carbon source. The carbon source utilization assay was used to assess the effects of soil inoculation with Corynebacterium glutamicum with and without a genetically engineered plasmid (pUN1; 6.3 kb), which encoded for the synthesis of the mammalian protease inhibiting peptide, aprotinin. Additionally, aprotinin itself was added at two concentrations to soil samples. An identical decrease in the number of carbon sources utilized, especially carbohydrates, occurred upon soil inoculation with both C. glutamicum strains after inoculation with 10⁶ cells g⁻¹ soil. This effect was only detectable during the first three weeks of incubation, as long as cell numbers of C. glutamicum (pUN1) were above 10⁵ cfu g⁻¹. Soil amendment with aprotinin resulted in utilization of additional substrates, most of them carbohydrates. With 0.1 mg aprotinin g⁻¹ soil this stimulation lasted 2 days and with 10 mg g⁻¹ it lasted for 7 days.     

Effect of high external NaCl concentration on ion transport within the shoot of Lupinus albus. II. Ions in phloem sap

Abstract. Phloem sap was collected from petioles of growing and fully expanded leaves of lupins exposed to 0–150 mol m⁻³ [NaCl]_ext, for various periods of time. Sap bled from growing leaves only after the turgor of the shoot was raised by applying pneumatic pressure to the root. Increased pressure was also needed to obtain sap from fully expanded leaves of plants at high [NaCl]_ext. Exposure to NaCl caused a rapid rise in the Na⁺ concentration in phloem sap to high levels. The Na⁺ concentration reached 20 mol m⁻³ within a day of exposure and reached a plateau of about 60 mol m⁻³ in plants at 50–150 mol m⁻³ [NaCl]_ext, after a week. There was a slower, smaller increase in the Cl⁻ concentration. K⁺ concentrations in phloem sap were not affected by [NaCl]_ext. Cl⁻ concentrations in phloem sap collected from growing leaves were similar to those from old leaves while Na⁺ concentrations were somewhat increased, suggesting that there was no reduction in the salt content of the phloem sap while it flowed within the shoot to the apex. Calculations of ion fluxes in xylem and phloem sap indicated that Na⁺ and Cl⁻ fluxes in the phloem from leaves of plants at high NaCl could be equal to those in the xylem. This prediction was borne out by observations that Na⁺ and Cl⁻ concentrations in recently expanded leaves remained constant.     

Effect of high external NaCl concentrations on ion transport within the shoot of Lupinus albus. I. Ions in xylem sap

Abstract. Xylem sap was collected from individual leaves of intact transpiring lupin plants exposed to increasing concentrations of NaCl by applying pneumatic pressure to the roots. Concentrations of Na⁺ and Cl⁻ in the xylem sap increased linearly with increases in the external NaCl concentration, averaging about 10% of the external concentration. Concentrations of K⁺ and NO₃⁻, the other major inorganic ions in the sap, were constant at about 2.5 and 1.5 mol m⁻³, respectively. There was no preferential direction of Na ⁺ or Cl⁻ to either young or old leaves: leaves of all ages received xylem sap having similar concentrations of Na⁺ and Cl⁻, and transpiration rates (per unit leaf area) were also similar for all leaves. Plants exposed to 120–160 mol m⁻³ NaCl rapidly developed injury of oldest leaves; when this occurred, the Na⁺ concentration in the leaflet midrib sap had increased to about 40 mol m⁻³ and the total solute concentration to 130 osmol m⁻³. This suggests that uptake of salts from the transpiration stream had fallen behind the rate of delivery to the leaf and that salts were building up in the apoplast.     

Characteristics of β-galactosidase purified from cell suspension cultures of carrot

Five glycosidase activities from cell homogenate of carrot ( Daucus carota L. cv. Kintoki) cell cultures were assayed after extraction successively by phosphate buffer (pH 7.0) and the buffer plus 2 M NaCl. A β-galactosidase (EC 3.2.1.23) was isolated in a highly purified state from the buffer-soluble protein fraction by ammonium sulfate fractionation and chromatography on CM-Sephadex C-50, DEAE-Sephadex A-50 and Sephadex G-200. The molecular weight of this enzyme was ca 104 000 and the isoelectric point was pH 7.8. The optimal activity occurred at pH 4.4 with McIlvaine buffer. The K_m and V_max values were 1.67 m M and 201 units (mg protein)⁻¹, respectively, for p -nitrophenyl β- d -galactopyranoside. The enzyme activity was strongly inhibited by Zn²⁺, Cu²⁺, Hg²⁺ and d -galactono-1,4-lactone. The enzyme acted on the β-1,4-linked galactan prepared from citrus pectin in an exo-fashion. Furthermore, the enzyme was slightly involved in the hydrolysis of the pectic polymer and cell walls purified from carrot cell cultures.     

Sodium partitioning within the shoot of soybean

Uptake and partitioning of Na⁺ and Cl⁻ in plants of soybean ( Glycine max L. Merr. cv. Hodgson) exposed to moderate NaCl concentrations were studied over an 8-day period. Plants showed marked retention of Na⁺ in the stems and low transport to laminae of young leaves. The xylem sap ascending the main axis was progressively depleted in Na⁺. The oldest leaf greatly contributed to Na⁺ depletion of the sap flowing to younger leaves. These results in combination with estimates of phloem recirculation indicated that Na⁺ accumulation in the young leaf was prevented both by depletion of Na⁺ from the xylem stream, and by a high recirculation of Na⁺ via the phloem. However, this protection of young leaves was effective only for very mild salinity treatment.     

Effect of Saccharomyces boulardii against experimental oral infection with Salmonella typhimurium and Shigella flexneri in conventional and gnotobiotic mice

A.C.P. RODRIGUES, R.M. NARDI, E.A. BAMBIRRA, E.C. VIEIRA AND J.R. NICOLI. 1996. Saccharomyces boulardii was shown to be capable of inhibiting multiplication of enteropathogenic bacteria in vitro and is currently used for its anti-diarrhoea properties. We studied the capacity of this yeast to antagonize Salmonella typhimurium and Shigella flexneri in the intestinal tract of conventional or gnotobiotic NMRI mice. Conventional animals were given daily 10 mg doses of S. boulardii , whereas germ-free animals were given a single 10 mg dose. Both groups were challenged orally 5 d later with the pathogenic bacteria (10⁸ or 10² viable cells, respectively). Control groups were treated with saline instead of S. boulardii. Mortality and/or histopathological data showed a protective effect against the pathogenic bacteria in yeast-treated mice. Saccharomyces boulardii colonized the digestive tract of gnotobiotic mice and the number of viable cells ranged around 10¹⁰ g^-1 of faeces. In experimental and control gnotobiotic animals, Salm. typhimurium and Sh. flexneri became rapidly established at a level of about 10¹⁰ viable cells g^-1 of faeces and remained at high levels until the animals died or were sacrificed. The protection against Salm. typhimurium and Sh. flexneri obtained in conventional and/or gnotobiotic mice previously associated with S. boulardii is not due to the reduction of the bacterial populations in the intestines.     

Uptake and distribution of calcium, magnesium and potassium in cucumber of different age

Uptake and distribution of Ca⁺, Mg²⁺ and K²⁺ were investigated in plants of cucumber ( Cucumis sativus L. var. Cila) which had been cultivated for 12, 19, 32, or 53 days in complete nutrient solution with 1.0 m M Ca²⁺, 2.0 m M Mg²⁺ and 2.0 m M K⁺. The ⁺ concentration was about the same in roots and shoots, while the Ca²⁺ and Mg²⁺ concentrations were low in roots compared to shoots. The K⁺ concentration decreased with increasing leaf age, while the Ca²⁺ and Mg²⁺ concentrations increased, except in older plants with flowers and fruits, where an increased concentration was found in the youngest leaves. This is discussed in connection with increased indoleacetic acid (IAA) synthesis in the shoot. Excision of leaves at different levels from 21-day-old plants, followed by uptake for 24 h from the nutrient solution on days 22 and 23, resulted in no immediate reduction in Ca²⁺ (⁴⁵Ca) uptake. Transport of Ca²⁺ increased to leaves above and below the excision point and total Ca²⁺ uptake remained at the same level as for the intact plant. It is suggested that regulation of Ca²⁺ uptake is primarily achieved in the root while the distribution in the shoot is regulated by the accessability of negative binding sites.