A note on Pseudomonas syringae pv. berberidis infections of Berberis: aetiology of a leaf spot and leaf fall disease in England

R oberts , S.J. & P reece , T.F. 1984. A note on Pseudomonas syringae pv. berberidis infections of Berberis : aetiology of a leaf spot and leaf fall disease in England. Journal of Applied Bacteriology 56 , 507–513.
A leaf spot and leaf fall disease is currently causing problems among certain Berberis spp. in England. Evergreens affected include B. julianae, B. gagnepainii, B. candidula, B. hookeri , and deciduous spp. include B. x ottawensis and B. thunbergii . The pathogen, Pseudomonas syringae pv. berberidis , which was confirmed as a distinct pathover by biochemical and host tests, was isolated from less than half of leaves showing typical symptoms. Isolates of Ps. syringae pv. berberidis from England, New Zealand and USA differed in cultural characteristics and in patho-genicity to certain Berberis spp.     

The relation between dosage, bacterial growth and time for disease response during infection of bean leaves by Pseudomonas syringae pv. phaseolicola

Pseudomonas syringae pv. phaseolicola 1L3 was infiltrated at a dosage of 0mD5 to 512 times the median effective dose (ED₅₀) into the leaves of two bean cultivars, Borlotto di Vigevano (ED₅₀ 15 bacteria) and Saluggia (ED₅₀ 34 bacteria). The distributions of time for production of disease symptoms after inoculation with up to 64 ED₅₀ were in agreement with those predicted by the simple birth-death model for microbial infection. Individual times of response to higher doses were longer and more widely distributed than expected from the model. Growth curves of bacteria in leaves inoculated with 16, 64 or 512 ED₅₀ could be viewed conventionally as a sequence of an exponential phase, a phase of decelerating growth and a stationary phase. Viable counts, however, were also compatible with parabolic population trends during the period preceding, accompanying and immediately following the appearance of disease symptoms. Bacterial growth parameters estimated from response times and infectivity titration data were consistent with those calculated from viable counts in vivo.     

Hydrogen peroxide does not function downstream of salicylic acid in the induction of PR protein expression

The roles of salicylic acid (SA) and H₂O₂ in the induction of PR proteins in tobacco have been examined. Studies were conducted on wild-type tobacco and plants engineered to express a bacterial salicylate hydroxylase capable of metabolizing SA to catechol (SH-L plants). Wild-type and PR-1a—GUS-transformed plants express PR-1a following challenge with Pseudomonas syringae pathovar syringae , SA or 2,6-dichloro-isonicotinic acid (INA). In contrast, SH-L plants failed to respond to SA but did express PR-1a following INA treatment. H₂O₂ and the irreversible catalase inhibitor 3-amino-1,2,4-triazole (3-AT) were found to be weak inducers of PR-1a expression (relative to SA) in wild-type tobacco but were unable to induce PR-1a in SH-L plants, suggesting that the action of these compounds depends upon the accumulation of SA. A model has been proposed suggesting that SA binds to and inhibits a catalase inducing an increase in H₂O₂ leading to PR protein expression. Catalase activity has been measured in tobacco and no significant changes in activity following infection with P. syringae pv. syringae were detected. Furthermore, inhibition of catalase activity in vitro in plant extracts requires pre-incubation and only occurs at SA concentrations above 250 µM. Leaf disks pre-incubated with 1 mM SA do accumulate SA to these levels and PR-1a is efficiently induced but there is no apparent inhibition of catalase activity. It is also shown that a SA-responsive gene, PR-1a, and a H₂O₂-sensitive gene, AoPR-1, are both relatively insensitive to 3-AT suggesting that induction of these genes is unlikely to be due entirely to inhibition of an endogenous catalase.     

Role of Extracellular Polysaccharide (EPS) Slime of Plant Pathogenic Bacteria in Protecting Cells to Reactive Oxygen Species

Pseudomonas syringae pv. phaseolicola , a phytopathogenic bacterium, seemed very sensitive in planta to the adverse action of reactive oxygen species (ROS) produced by two chemical systems. The disease symptoms in host plants were also suppressed by ROS. Several other plant pathogenic bacteria ( P. syringae pv. pisi, Erwinia amylovora, Xanthomonas campestris pv. pelargonii ) as well as P. fluorescens were also sensitive in vitro to the inhibiting or killinig action of ROS. It was shown that O₂ and H₂O₂ were produced in our two chemical systems and were involved in the killing action. OH'however was not involved in the adverse action on bacteria of the ROS. Superoxide dismutase and catalase were able to reverse the killing action of ROS. When the EPS slime around bacteria was removed by washing and centrifuging the cells, bacteria were more sensitive to ROS. However, when the cells of EPS- mutants were washed and centrifuged, their sensitivity to the killing action of ROS did not change because the lack of slime around the mutant cells.
The EPS^- Tn5 mutants of P. syringae pv. phaseolicola and the natural EPS^- mutant of E. amylovora were more sensitive to ROS than the wild type strains. These results support the idea that the EPS slime protects bacteria from ROS (O^-₂ and H₂O₂).     

Inhibition of photosynthesis and export in geranium grown at two CO2 levels and infected with Xanthomonas campestris pv. Pelargonii

The effects of CO₂ enrichment on growth of Xanthomonas campestris pv. pelargonii and the impact of infection on the photosynthesis and export of attached, intact, 'source' leaves of geranium ( Pelargonium x domesticum, 'Scarlet Orbit Improved' ) are reported. Two experiments were performed, one with plants without flower buds, and another with plants which were flowering. Measurements were made on healthy and diseased leaves at the CO₂ levels (35 Pa or 90 Pa) at which the plants were grown. There were no losses of chlorophyll, or any signs of visible chlorosis or necrosis due to infection. Lower numbers of bacteria were found in leaves at high CO₂, suggesting growth at elevated CO₂ created a less favourable condition in the leaf for bacterial growth. Although high CO₂ lowered the bacterial number in infected leaves, reductions in photosynthesis and export were greater than at ambient CO₂. The capacity of infected source leaves to export photoassimilates at rates observed in the controls was reduced in both light and darkness. In summary, the severity of infection on source leaf function by the bacteria was increased, rather than reduced by CO₂ enrichment, underscoring the need for further assessment of plant diseases and bacterial virulence in plants growing under varying CO₂ levels.     

LEAF INFECTION OF COTTON BY XANTHOMONAS MALVACEARUM (E.F.SM.) DOWSON

Xanthomonas malvacearum spread more rapidly along vascular tissue than into mesophyll when inoculated to the main veins of susceptible cotton leaves. The extent of spread varied with the concentrations of inocula, tissue age and cotton variety.
Increasing concentrations of inocula accelerated the initial spread of disease.
Bacteria spread more rapidly in young leaves than in old—increasing age greatly decreased disease in the mesophyll. The initial invasion was quicker in young leaves of young plants than in young leaves of old plants.
Three types of behaviour, according to the host's reaction, distinguish Knight's resistance factors: ( a ) where X. malvacearum spread extensively along veins and into mesophyll of plants containing factors B₃ and B₅; ( b ) where it was restricted to the point of inoculation in plants containing B ₄, B₉ and combinations with B _6m; and ( c ) where it spread along veins but not appreciably into mesophyll in varieties containing B ₂ and B ₂ B ₃.
From this and four other different types of tests, factors B ₂ and B ₃ seem to increase mesophyll resistance but only B ₂ gives appreciable vascular resistance. Further, the vascular bundles in varieties with B ₂ seem to be surrounded by an additional 'barrier' which resists X. malvacearum.     

Use of lysophosphatidylethanolamine, a natural lipid, to retard tomato leaf and fruit senescence

We studied the influence of lysophosphatidylethanolamine (LPE) on the pattern and rate of ethylene production and respiration of tomato ( Lycopersicon esculentum cv. H7155) leaflets and fruit. Leaflets that had been senescing on the plant showed a climacteric-like rise in ethylene production but not in respiration rate which decreased continuously with leaf age. Detached leaflets had a climacteric-like pattern in respiration whether they were incubated in complete darkness or in light. Detached leaflets incubated in the dark had higher rates of ethylene production and CO₂ evolution than did light-incubated leaves. There was no change in the pattern of ethylene production or CO₂ evolution as a result of LPE treatment. However, LPE-treated attached and detached leaflets had consistently lower rates of CO₂ evolution. The reduction in CO₂ evolution by LPE was most pronounced at the climacteric-like peak of the detached leaves. LPE-treated leaflets had a higher chlorophyll content and fresh weight and lower electrolyte leakage than the control. LPE-treated fruits had lower rates of ethylene and CO₂ production than did the control. LPE-treated fruits also had higher pericarp firmness and lower electrolyte leakage than the control. The results of the present study provide evidence that LPE is able to retard senescence of attached leaves and detached leaves and tomato fruits. Several recent studies suggest that lysolipids can act in a specific manner as metabolic regulators. Our results suggest a specific role of lysolipid LPE in aging and senescence     

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.     

Regulation of syringomycin synthesis in Pseudomonas syringae pv. syringae and defined conditions for its production

Production of the phytotoxin, syringomycin (SR), by Pseudomonas syringae pv. syringae strain B301D was regulated by both iron and inorganic phosphate similar to that of many bacterial secondary metabolites. Iron concentrations of 2 µmol/1 or more in deferrated potato-dextrose broth (PDB) resulted in the production of 1024 SR units/ml, a yield comparable to that produced in non-deferrated PDB. Moreover, production of one SR unit required approximately 0†4 ng of available FeCl₃. No SR was produced by strain B301D in deferrated PDB despite growth nearly identical with that of B301D in deferrated PDB supplemented with 10 µmol/1 FeCl₃. Furthermore, a phosphate concentration of 1 mmol/1 or more was suppressive to SR production. Of the amino acids tested, L-histidine at a concentration of ca 20 mmol/1 was the most effective nitrogen source for SR synthesis under defined conditions. Based on these observations, a synthetic medium, SR minimal, was formulated for SR or syringotoxin production by representative strains of Ps. syringae pv. syringae. The regulation of phytotoxin production is discussed in relation to pathogen survival and virulence.     

The cloning and characterization of phage promoters, directing high expression of luciferase in Pseudomonas syringae pv. phaseolicola, allowing single cell and microcolony detection

Regions of DNA containing promoter sequences from a Pseudomonas syringae pv. phaseolicola -specific phage (φ11P) were identified by shotgun cloning into a broad-host-range promoter-probe vector (pQF70). When used in conjunction with the luciferase reporter genes, one of these DNA fragments, 19H, directed gene expression at a level which enabled the subsequent light output (bioluminescence) of single cells of P. syringae pv. phaseolicola to be detected and visualized using a charge-coupled device (CCD). The P. syringae pv. phaseolicola φ11P, 19H and P. aeruginosa φPLS27, HcM promoters gave a 50-fold increase in bioluminescence (maximum relative light output) compared to similar constructs containing other well-characterized promoters, for example, tetracycline. Similar bioluminescent characteristics of the transformed bacterium, were observed during growth with and without antibiotic-selection. When lux ⁺ bacteria were inoculated onto French bean leaf ( Phaseolus vulgaris L.), the resultant secondary halo blight lesions were bioluminescent and during phylloplane colonization by the lux ⁺ bacterium, bioluminescence on leaf surfaces was detected and imaged by the CCD. Use of these newly identified promoters, combined with the greatly increased sensitivity of bioluminescence detection by the CCD, thus provided a new dimension for the study of natural ecological populations during the bacterial colonization of plants.     

Role of ethylene in senescence of watercress leaves

Detached watercress leaves showed a rapid senescence rate as compared with other herbs. It was therefore of interest to investigate the role of ethylene in the rapid senescence of watercress leaves, and So estimate the efficacy of various inhibitors of elhylene synthesis (aminoethoxyvinylglycine, AVG) and action (CO₂, Ag⁺) in retarding senescence processes. The progress of senescence in watercress bunches (leaves attached to cut stems) and in detached leaves was estimated by measuring the rate of chlorophyll (Chl) loss, proteolysis and lipid oxidation. Evidence is presented showing that application of 11% CO₂ to watercress bunches in a flow-through system had a long-lasting effect on senescence, exhibited by highly efficient retardation of all the senescence processes tested. On the other hand, application of AVG (O.1 m M ) or Ag-(30 μ M ) to detached leaves affected Chl loss much more than prciteolysis. These results suggest that the senescence-retarding activity of CO₂ cannot be attributed solely to its action as an anli-ethylene agent and that not all senescence-associated processes are regulated by ethylene.     

On the mechanism of rejuvenation of ageing detached bean leaves by low-concentration stressors

The effect of low concentrations of some stress-inducing compounds of different toxicity and chemical nature, such as Cd and Pb salts or DCMU, was investigated on the senescence of chloroplasts in detached primary leaves of bean ( Phaseolus vulgaris L . ). After 1 week of senescence followed by root development from the petiole, these agents stimulated chlorophyll accumulation and photosynthetic activity (¹⁴CO₂ fixation) as compared to the control, thus inducing rejuvenation. Low-concentration stressors increased the level of active cytokinins in roots and leaves during the treatment, as monitored by the Amaranthus betacyanin bioassay and high-pressure liquid chromatography. The lithium ion, an inhibitor of the PIP₂-IP₃/DAG signal transduction pathway, abolished the stimulating effect of stressors, both in roots (retarding cytokinin synthesis) and consequently also in leaves (reducing cytokinin-dependent chlorophyll accumulation). This suggests the involvement of the PIP₂-IP₃/DAG signal transduction pathway in generation of these consecutive organ-specific responses.     

Contributions of the effector gene hopQ1-1 to differences in host range between Pseudomonas syringae pv. phaseolicola and P. syringae pv. tabaci

To study the role of type III-secreted effectors in the host adaptation of the tobacco ( Nicotiana sp.) pathogen Pseudomonas syringae pv. tabaci , a selection of seven strains was first characterized by multilocus sequence typing (MLST) to determine their phylogenetic affinity. MLST revealed that all strains represented a tight phylogenetic group and that the most closely related strain with a completely sequenced genome was the bean ( Phaseolus vulgaris ) pathogen P. syringae pv. phaseolicola 1448A. Using primers designed to 21 P. syringae pv. phaseolicola 1448A effector genes, it was determined that P. syringae pv. phaseolicola 1448A shared at least 10 effectors with all tested P. syringae pv. tabaci strains. Six of the 11 effectors that failed to amplify from P. syringae pv. tabaci strains were individually expressed in one P. syringae pv. tabaci strain. Although five effectors had no effect on phenotype, growth in planta and disease severity of the transgenic P. syringae pv. tabaci expressing hopQ1-1 _Pph1448A were significantly increased in bean, but reduced in tobacco. We conclude that hopQ1-1 has been retained in P. syringae pv. phaseolicola 1448A, as this effector suppresses immunity in bean, whereas hopQ1-1 is missing from P. syringae pv. tabaci strains because it triggers defences in Nicotiana spp. This provides evidence that fine-tuning effector repertoires during host adaptation lead to a concomitant reduction in virulence in non-host species.     

Effects of tabtoxin on nitrogen metabolism

Tabtoxin is a chlorosis-inducing toxin produced by the plant pathogenic bacterium Pseudomonas syringae pv. tabaci. Previous studies have indicated that tabtoxin inhibits glutamine synthetase (EC 6.3.1.2) in vitro. We report here that tabtoxin also inhibits glutamine synthetase in vivo. The main evidence was that assimilation of exogenous ¹⁵NH₃ into Asparagus sprengeri protein was rapidly inhibited in isolated cells exposed to tabtoxin. This was associated with an equivalent decline in glutamine synthetase activity in extracts of these cells and the accumulation of extracellular ammonia. Glutamine synthetase was also inhibited in leaves of Nicotiana tabacum L. cv. White Burley treated with tabtoxin and the affected tissue accumulated ammonia and became chlorotic. However, the development of symptoms and accumulation of ammonia was suppressed when the leaves were held in air containing 1% CO₂ to reduce photorespiration. This indicates that the chlorotic symptom did not result from the inhibition of nitrogen assimilation but was a consequence of the interruption of the photorespiratory nitrogen cycle.     

Stimulation of respiration by Pb2+ in detached leaves and mitochondria of C3 and C4 plants

The exposure of detached leaves of C₃ plants (pea, barley) and C₄ plant (maize) to 5 m M Pb (NO₃)₂ for 24 h caused a reduction of their photosynthetic activity by 40–60%, whereas the respiratory rate was stimulated by 20–50%. Mitochondria isolated from Pb²⁺-treated pea leaves oxidized substrates (glycine, succinate, malate) at higher rates than mitochondria from control leaves. The respiratory control (RCR) and the ADP/O ratio were not affected. Pb²⁺ caused an increase in ATP content and the ATP/ADP ratio in pea and maize leaves. Rapid fractionation of barley protoplasts incubated at low and high CO₂ conditions, indicated that the increased ATP/ADP ratio in Pb²⁺-treated leaves resulted mainly from the production of mitochondrial ATP. The measurements of membrane potential of mitochondria with a TPP⁺-sensitive electrode further showed that mitochondria isolated from Pb²⁺-treated leaves had at least as high membrane potential as mitochondria from control leaves. The activity of NAD-malate dehydrogenase in the protoplasts from barley leaves treated with Pb²⁺ was 3-fold higher than in protoplasts from control leaves. The activities of photorespiratory enzymes NADH-hydroxypyruvate reductase and glycolate oxidase as well as of NAD-malic enzyme were not affected. The presented data indicate that stimulation of respiration in leaves treated by lead is in a close relationship with activation of malate dehydrogenase and stimulation of the mitochondrial ATP production. Thus, respiration might fulfil a protective role during heavy metal exposure.     

Direct and indirect effects of Cd2+ on photosynthesis in sugar beet (Beta vulgaris)

Sugar-beet plants ( Beta vulgaris L. cv. Monohill) were cultivated for 4 weeks in a complete nutrient solution. Indirect effects of cadmium were studied by adding 5, 10 or 20 μ M CdCl₂ to the culture medium while direct effects were determined by adding 1, 5, 20, 50 or 2 000 μ M CdCl₂ to the assay media. The photosynthetic properties were characterized by measurement of CO₂ fixation in intact plants, fluorescence emission by intact leaves and isolated chloroplasts, photosystem (PS) I and PSII mediated electron transport of isolated chloroplasts, and CO₂-dependent O₂ evolution by protoplasts. When directly applied to isolated leaves, protoplasts and chloroplasts. Cd²⁺ impeded CO₂ fixation without affecting the rates of electron transport of PSI or PSII or the rate of dark respiration. When Cd²⁺ was applied through the culture medium the capacity for, and the maximal quantum yield of CO₂ assimilation by intact plants both decreased. This was associated with: (1) decreased total as well as effective chlorophyll content (PSII antennae size), (2) decreased coupling of electron transport in isolated chloroplasts, (3) perturbed carbon reduction cycle as indicated by fluorescence measurements. Also, protoplasts isolated from leaves of Cd²⁺-cultivated plants showed an increased rate of dark respiration.     

Changes in water relations and in some physiological functions of bean under very light osmotic shock induced by polyethylene glycol

Bean plantlets ( Phaseolus vulgaris L. cv. Topcrop) were stressed at the age of 16–18 days by gradual (2–8%) or abrupt addition of 6% (w/v) polyethylene glycol Mw 6000 (PEG 6000) to Hoagland solution. Leaf conductance, photosynthesis, internal CO₂ partial pressure (C_i), relative water content (RWC), water content/dry weight (H₂O/DW), apoplastic PEG concentrations and weight of leaves, stems and roots were determined. Leaf conductance, photosynthesis and C_i were determined on non-detached primary leaves, and leaf potentials (water, osmotic and turgor potentials) were investigated in freshly detached (non-rehydrated) primary leaves, both in treated and control plants; RWC and osmotic potential were also assessed at the null turgor point. Low PEG 6000 concentrations induced early and evident decrease in leaf conductance and photosynthesis, whereas C_i decreased only moderately and tended to recover during advanced stress. There were moderate though significant decreases in RWC and H₂O/DW, no change or increases in water potential, no significant changes in osmotic potential and a moderate but significant increase in turgor potential. Even when referred to null turgor point, RWC significantly decreased and osmotic potential was unchanged. It was concluded that apoplastic PEG 6000 accumulation at evaporating sites would account for the early decrease in conductance which would also justify the unchanged or the prevalent increase in water potential and turgor potential. The subsequent PEG diffusion and concentration in the leaf apoplastic water would have induced the RWC and H₂O/DW decrease and the final turgor flexion documented.     

Identification and Phytotoxicity of 3-methylthiopropanoic and Trans-3-methylthiopropenoic Acids Produced in Culture by Xanthomonas campestris pv. vitians

Two phytotoxic metabolites were isolated from culture filtrates of Xanthomonas campestris pv, vitians , the causal agent of lettuce leaf spots and headrot. The two compounds were identified as 3-methylthiopropanoic (1) and trans-3-methylthiopropenoic (2) acids by chemical and spectroscopic methods. Toxic effects of the two compounds on leaf tissues and protoplasts of lettuce and cabbage were investigated. Solutions of 1 and 2 induced chlorosis and necrosis on lettuce leaves at minimum concentrations of 300 and 50 μg/ml, respectively. Infiltration in cabbage leaves did not produce any symptoms. The LD₅₀ values for 1 and 2 against lettuce protoplasts were 15 and 16 μg/ml, respectively. Activity of the two metabolites against cabbage protoplasts was very low (LD₅₀ > 500 μg/ml).     

Water stress, carbon dioxide, and light effects on sucrosephosphate synthase activity in Phaseolus vulgaris

The characteristics of sucrose-phosphate synthase (SPS; EC 2.4.1.14) activity in leaves of Phaseolus vulgaris L. cv. Linden was studied in plants subjected to water stress and various CO₂ and light treatments. When water was withheld for 3 days causing mild water stress (–0.9 MPa), the activity of SPS measured in crude extracts was reduced ca 50%. The effect of water stress was most evident when the enzyme was assayed with saturating amounts of its substrates fructose 6-phosphate and UDP glucose. Placing a water-stressed plant in an atmosphere containing 1% CO₂ reversed the effect of water stress on SPS activity over 5 h even though the water stress was not relieved. Holding unstressed leaves in low CO₂ partial pressure reduced the extractable activity of SPS. After 1 h of low CO₂ treatment the effect of low CO₂ could be reversed by 20 min of 5% CO₂. However, after 24 h of low CO₂ treatment, less SPS activity was recovered by the 20 min treatment. The cytosolic protein synthesis inhibitor cycloheximide prevented the slow recovery of SPS activity, but did not affect the rapid recovery of SPS. We conclude that the effect of water stress on SPS activity was a consequence of the inhibition of photosynthesis caused by stomatal closure. Responses of Phaseolus vulgaris SPS to light were similar to the response to low CO₂ in that the effects were most pronounced under V_max assay conditions. This is the first report of this type of light response of SPS in a dicotyledonous species.