Protection of tomato seedlings against infection by Pseudomonas syringae pv. tomato by using the plant growth-promoting bacterium Azospirillum brasilense

Pseudomonas syringae pv. tomato, the causal agent of bacterial speck of tomato, and the plant growth-promoting bacterium Azospirillum brasilense were inoculated onto tomato plants, either alone, as a mixed culture, or consecutively. The population dynamics in the rhizosphere and foliage, the development of bacterial speck disease, and their effects on plant growth were monitored. When inoculated onto separate plants, the A. brasilense population in the rhizosphere of tomato plants was 2 orders of magnitude greater than the population of P. syringae pv. tomato (10(7) versus 10(5) CFU/g [dry weight] of root). Under mist chamber conditions, the leaf population of P. syringae pv. tomato was 1 order of magnitude greater than that of A. brasilense (10(7) versus 10(6) CFU/g [dry weight] of leaf). Inoculation of seeds with a mixed culture of the two bacterial strains resulted in a reduction of the pathogen population in the rhizosphere, an increase in the A. brasilense population, the prevention of bacterial speck disease development, and improved plant growth. Inoculation of leaves with the mixed bacterial culture under mist conditions significantly reduced the P. syringae pv. tomato population and significantly decreased disease severity. Challenge with P. syringae pv. tomato after A. brasilense was established in the leaves further reduced both the population of P. syringae pv. tomato and disease severity and significantly enhanced plant development. Both bacteria maintained a large population in the rhizosphere for 45 days when each was inoculated separately onto tomato seeds (10(5) to 10(6) CFU/g [dry weight] of root). However, P. syringae pv. tomato did not survive in the rhizosphere in the presence of A. brasilense. Foliar inoculation of A. brasilense after P. syringae pv. tomato was established on the leaves did not alleviate bacterial speck disease, and A. brasilense did not survive well in the phyllosphere under these conditions, even in a mist chamber. Several applications of a low concentration of buffered malic acid significantly enhanced the leaf population of A. brasilense (>10(8) CFU/g [dry weight] of leaf), decreased the population of P. syringae pv. tomato to almost undetectable levels, almost eliminated disease development, and improved plant growth to the level of uninoculated healthy control plants. Based on our results, we propose that A. brasilense be used in prevention programs to combat the foliar bacterial speck disease caused by P. syringae pv. tomato.     

Construction and Use of a Nonradioactive DNA Hybridization Probe for Detection of Pseudomonas syringae pv. Tomato on Tomato Plants

Pseudomonas syringae pv. tomato, the causal agent for bacterial speck of tomato, produces the phytotoxin coronatine. A 5.3-kilobase XhoI fragment from the chromosomal region controlling toxin production was cloned into the plasmid pGB2, and the resulting recombinant plasmid, pTPR1, was tested for its ability to serve as a diagnostic probe for P. syringae pv. tomato. In a survey of 75 plant-associated bacteria, pTPR1 hybridized exclusively to those strains that produced coronatine. The detection limit for this probe, which was labeled with the Chemiprobe nonradioactive reporter system, was approximately 4 × 10³ CFU of lesion bacteria. During the 1989 growing season, a total of 258 leaf and fruit lesions from nine tomato fields were screened for P. syringae pv. tomato by using pTPR1 and the culture method of detection. The best agreement between the two methods, 90%, occurred early in the season with samples taken from relatively young (5-week-old) plants. Young plants also had a higher percentage of P. syringae pv. tomato-positive lesions. P. syringae pv. tomato was the only coronatine producer recovered from the nine tomato fields. All 244 P. syringae pv. tomato strains isolated during this study reacted strongly with the probe. The P. syringae pv. tomato population of healthy field tomato leaves was determined by a pTPR1 colony hybridization procedure. Every probe-positive colony that was isolated and characterized was identified as P. syringae pv. tomato. The pTPR1 probe should expedite disease diagnosis and facilitate epidemiological studies of this pathogen. It also should aid in screening transplant seedlings for bacterial speck infestation.     

Nutritional Similarity between Leaf-Associated Nonpathogenic Bacteria and the Pathogen Is Not Predictive of Efficacy in Biological Control of Bacterial Spot of Tomato

It has been demonstrated that for a nonpathogenic, leaf-associated bacterium, effectiveness in the control of bacterial speck of tomato is correlated with the similarity in the nutritional needs of the nonpathogenic bacterium and the pathogen Pseudomonas syringae pv. tomato. This relationship was investigated further in this study by using the pathogen Xanthomonas campestris pv. vesicatoria, the causal agent of bacterial spot of tomato, and a collection of nonpathogenic bacteria isolated from tomato foliage. The effects of inoculation of tomato plants with one of 34 nonpathogenic bacteria prior to inoculation with the pathogen X. campestris pv. vesicatoria were quantified by determining (i) the reduction in disease severity (number of lesions per square centimeter) in greenhouse assays and (ii) the reduction in leaf surface pathogen population size (log₁₀ of the number of CFU per leaflet) in growth chamber assays. Nutritional similarity between the nonpathogenic bacteria and X. campestris pv. vesicatoria was quantified by using either niche overlap indices (NOI) or relatedness in cluster analyses based upon in vitro utilization of carbon or nitrogen sources reported to be present in tomato tissues or in Biolog GN plates. In contrast to studies with P. syringae pv. tomato, nutritional similarity between the nonpathogenic bacteria and the pathogen X. campestris pv. vesicatoria was not correlated with reductions in disease severity. Nutritional similarity was also not correlated with reductions in pathogen population size. Further, the percentage of reduction in leaf surface pathogen population size was not correlated with the percentage of reduction in disease severity, suggesting that the epiphytic population size of X. campestris pv. vesicatoria is not related to disease severity and that X. campestris pv. vesicatoria exhibits behavior in the phyllosphere prior to lesion formation that is different from that of P. syringae pv. tomato.     

The plant growth-promoting rhizobacterium Bacillus cereus AR156 induces resistance in tomato with induction and priming of defence response

In a previous study, we demonstrated the ability of the rhizobacterium Bacillus cereus AR156 (AR156) to protect tomato against bacterial wilt caused by Ralstonia solanacearum and root-knot disease caused by Meloidogyne incognita. Here, we investigate the ability of AR156 to promote plant growth and its role in the systemic protection of tomatoes cultivated in greenhouses against bacterial speck disease caused by Pseudomonas syringae pv. tomato DC3000 (DC3000). In our experiments, the AR156 population reached 10⁵–10⁶ CFU/g rhizosphere soil, and remained at that level in the rhizosphere of tomato plants for more than 2 months. In terms of its ability to promote plant growth, AR156 increased the average biomass of the tomato by 47.7%. AR156 also elicited induced systemic resistance against DC3000, significantly reduced bacterial speck disease severity 1.6-fold, and inhibited proliferation of the pathogen by approximately 15-fold. This strain triggered the accumulation of defence-related genes (PR1 and PIN2) in tomato leaves and primed the leaves for accelerated defence-related gene expression upon challenge with DC3000. That suggested simultaneous activation of the salicylic acid and the jasmonic acid dependent signalling pathways by AR156 against DC3000. In conclusion, B. cereus AR156 was found to form robust colonies in the roots of tomato and had some beneficial effects, including biological control of bacterial speck disease via ISR and promotion of plant growth.     

Chemotaxis by Pseudomonas syringae pv. tomato

Optimal laboratory conditions for studying chemotaxis by Pseudomonas syringae pv. tomato were determined by using the Adler capillary tube assay. Although they are not an absolute requirement for chemotaxis, the presence of 0.1 mM EDTA and 1 mM MgCl₂ in the chemotaxis buffer (10 mM potassium phosphate [pH 7.2]) significantly enhanced the response to attractant. The addition of mannitol as an energy source had little effect. The optimal temperature for chemotaxis was 23°C, which is 5°C below the optimal growth temperature for this pathogen. The best response occurred when the bacteria were exposed to attractant for 60 min at a concentration of approximately 5 × 10⁶ CFU/ml. P. syringae pv. tomato was strongly attracted to citric and malic acids, which are the predominant organic acids in tomato fruit. With the exception of asparagine, the major amino acids of tomatoes were weak to moderate attractants. Glucose and fructose, which account for approximately 47% of tomato dry matter, also elicited poor responses. In assays with tomato intercellular fluid and leaf surface water, the bacterial speck pathogen could not chemotactically distinguish between a resistant and a susceptible cultivar of tomato.     

Morphology of Leaf Surfaces of Tomato Cultivars in Relation to Possible Invasion into the Leaf by Pseudomonas syringae pv. tomato

The role of morphological characteristics of tomato leaves inthe infection process of the pathogen Pseudomonas syringae pv.tomato was studied in 15 cultivars of varying susceptibility.Natural openings such as stomata, broken trichomes and cuticularcracks were counted. It was found that susceptibility increasedin proportion to the number of these potential penetration sites.However, their role is probably limited because even in thehighly resistant cultivars there are enough natural openingsto enable successful bacterial penetration. Bacterial speck of tomato, Lycopersicon esculentum, phytopathogenic bacteria, plant morphology, Pseudomonas syringae pv. tomato, tomato     

Response of tomato genotypes to bacterial speck (Pseudomonas syringae pv. tomato)

Two genotypes of tomato A 100 and Ontario 7710 which were inoculated separately with four strains of Pseudomonas syringae pv. tomato differed significantly in disease severity (susceptibility) to bacterial speck. At both concentrations of inoculum of each strain used (10⁷ and 10⁸ cfu/ml) A 100 appeared to be highly susceptible whereas Ontario 7710 showed very low or no susceptibility. The significant differences in virulence between strains and in response of tomato plants in three replicate experiments were found. Generally, concentration of inoculum 10⁷ cfu/ml was too low to induce consistent level of disease severity. The obtained results indicate the importance of consistent and favorable conditions for disease development in screening of tomato resistance to bacterial speck.     

Enhancement of Population Size of a Biological Control Agent and Efficacy in Control of Bacterial Speck of Tomato through Salicylate and Ammonium Sulfate Amendments

Sodium salicylate and ammonium sulfate were applied to leaf surfaces along with suspensions of the biological control agents Pseudomonas syringae Cit7(pNAH7), which catabolizes salicylate, and Cit7, which does not catabolize salicylate, to determine whether enhanced biological control of bacterial speck of tomato could be achieved. Foliar amendment with salicylate alone significantly enhanced the population size and the efficacy of Cit7(pNAH7), but not of Cit7, on tomato leaves. Application of ammonium sulfate alone did not result in enhanced population size or biological control efficacy of either Cit7(pNAH7) or Cit7; however, when foliar amendments with both sodium salicylate and ammonium sulfate were applied, a trend toward further increases in population size and biological control efficacy of Cit7(pNAH7) was observed. This study demonstrates the potential of using a selective carbon source to improve the efficacy of a bacterial biological control agent in the control of a bacterial plant disease and supports previous conclusions that the growth of P. syringae in the phyllosphere is primarily carbon limited and secondarily nitrogen limited.     

Assessment of the Importance of Similarity in Carbon Source Utilization Profiles between the Biological Control Agent and the Pathogen in Biological Control of Bacterial Speck of Tomato

Bacterial speck of tomato, caused by Pseudomonas syringae pv. tomato, was used to determine whether similarity in carbon source utilization between a preemptive biological control agent and the pathogen was significant in determining the ability of the bacterium to suppress disease. Similarity in carbon source utilization was quantified as the ratio of the number of tomato carbon sources utilized in vitro by the biological control agent to the number of tomato carbon sources utilized in vitro by the target pathogen (the niche overlap index [NOI]). Suppression of the disease was quantified as the percent reduction in disease severity compared to the pathogen-only control when nonpathogenic bacteria were applied to foliage 48 h prior to the pathogen. In the collection of 36 nonpathogenic bacterial strains, there was a significant (P < 0.01), but weak (r² = 0.25), correlation between reduction in disease severity and similarity in carbon source utilization, suggesting that similarity in carbon source use was significant in determining ability to suppress disease. The relationship was investigated further using catabolic mutants of P. syringae strain TLP2, an effective biological control agent of speck. Catabolic mutants exhibited lower levels of similarity (NOI = 0.07 to 0.90) than did wild-type TLP2 (NOI = 0.93). With these catabolic mutants there was a significant (P < 0.01), and stronger (r² = 0.42), correlation between reduction in disease severity and similarity in carbon source utilization. This suggests that similarity in carbon source utilization was a more important component of biological control ability for the catabolic mutants than for the nonpathogenic bacteria. Together, these studies indicate that suppression of bacterial speck of tomato was correlated with nutritional similarity between the pathogenic and nonpathogenic bacteria and suggest that preemptive utilization of carbon sources was probably involved in the biological control of the disease by both the naturally occurring nonpathogenic bacteria and the catabolic mutants.     

Reduction of Olive Knot Disease by a Bacteriocin from Pseudomonas syringae pv. ciccaronei

A bacteriocin produced by Pseudomonas syringae pv. ciccaronei, used at different purification levels and concentrations in culture and in planta, inhibited the multiplication of P. syringae subsp. savastanoi, the causal agent of olive knot disease, and affected the epiphytic survival of the pathogen on the leaves and twigs of treated olive plants. Treatments with bacteriocin from P. syringae pv. ciccaronei inhibited the formation of overgrowths on olive plants caused by P. syringae subsp. savastanoi strains PVBa229 and PVBa304 inoculated on V-shaped slits and on leaf scars at concentrations of 10⁵ and 10⁸ CFU ml⁻¹, respectively. In particular, the application of 6,000 arbitrary units (AU) of crude bacteriocin (dialyzed ammonium sulfate precipitate of culture supernatant) ml⁻¹ at the inoculated V-shaped slits and leaf scars resulted in the formation of knots with weight values reduced by 81 and 51%, respectively, compared to the control, depending on the strains and inoculation method used. Crude bacteriocin (6,000 AU ml⁻¹) was also effective in controlling the multiplication of epiphytic populations of the pathogen. In particular, the bacterial populations recovered after 30 days were at least 350 and 20 times lower than the control populations on twigs and on leaves, respectively. These results suggest that bacteriocin from P. syringae pv. ciccaronei can be used effectively to control the survival of the causal agent of olive knot disease and to prevent its multiplication at inoculation sites.     

Natural Variation for Responsiveness to flg22, flgII-28, and csp22 and Pseudomonas syringae pv. tomato in Heirloom Tomatoes

Tomato (Solanum lycopersicum L.) is susceptible to many diseases including bacterial speck caused by Pseudomonas syringae pv. tomato. Bacterial speck disease is a serious problem worldwide in tomato production areas where moist conditions and cool temperatures occur. To enhance breeding of speck resistant fresh-market tomato cultivars we identified a race 0 field isolate, NC-C3, of P. s. pv. tomato in North Carolina and used it to screen a collection of heirloom tomato lines for speck resistance in the field. We observed statistically significant variation among the heirloom tomatoes for their response to P. s. pv. tomato NC-C3 with two lines showing resistance approaching a cultivar that expresses the Pto resistance gene, although none of the heirloom lines have Pto. Using an assay that measures microbe-associated molecular pattern (MAMP)-induced production of reactive oxygen species (ROS), we investigated whether the heirloom lines showed differential responsiveness to three bacterial-derived peptide MAMPs: flg22 and flgII-28 (from flagellin) and csp22 (from cold shock protein). Significant differences were observed for MAMP responsiveness among the lines, although these differences did not correlate strongly with resistance or susceptibility to bacterial speck disease. The identification of natural variation for MAMP responsiveness opens up the possibility of using a genetic approach to identify the underlying loci and to facilitate breeding of cultivars with enhanced disease resistance. Towards this goal, we discovered that responsiveness to csp22 segregates as a single locus in an F₂ population of tomato.     

Integrated biological control of bacterial speck and spot of tomato under field conditions using foliar biological control agents and plant growth-promoting rhizobacteria

Integration of foliar bacterial biological control agents and plant growth promoting rhizobacteria (PGPR) was investigated to determine whether biological control of bacterial speck of tomato, caused by Pseudomonas syringae pv. tomato, and bacterial spot of tomato, caused by Xanthomonas campestris pv. vesicatoria and Xanthomonas vesicatoria, could be improved. Three foliar biological control agents and two selected PGPR strains were employed in pairwise combinations. The foliar biological control agents had previously demonstrated moderate control of bacterial speck or bacterial spot when applied as foliar sprays. The PGPR strains were selected in this study based on their capacity to induce resistance against bacterial speck when applied as seed and soil treatments in the greenhouse. Field trials were conducted in Alabama, Florida, and California for evaluation of the efficacy in control of bacterial speck and in Alabama and Florida for control of bacterial spot. The foliar biological control agent P. syringae strain Cit7 was the most effective of the three foliar biological control agents, providing significant suppression of bacterial speck in all field trials and bacterial spot in two out of three field trials. When applied as a seed treatment and soil drench, PGPR strain Pseudomonas fluorescens 89B-61 significantly reduced foliar severity of bacterial speck in the field trial in California and in three of six disease ratings in the field trials in Alabama. PGPR strains 89B-61 and Bacillus pumilus SE34 both provided significant suppression of bacterial spot in the two field trials conducted in Alabama. Combined use of foliar biological control agent Cit7 and PGPR strain 89B-61 provided significant control of bacterial speck and spot of tomato in each trial. In one field trial, control was enhanced significantly with combined biological control agents compared to single agent inoculations. These results suggest that some PGPR strains may induce plant resistance under field conditions, providing effective suppression of bacterial speck and spot of tomato, and that there may be some benefit to the integration of rhizosphere-applied PGPR and foliar-applied biological control agents.     

Polysaccharides of <Emphasis Type="Italic">Pseudomonas</Emphasis> Pathovar Strains That Infect Pea,Tomato, and Soya Bean

In order to understand the mode of action of taxonomically related Pseudomonas syringae pathovar strains that infect pea, tomato, and soya bean, we examined their extracellular polysaccharides (EPS). Maximum production of polysaccharide in shake culture of these pathogens was observed between 24 and 60 h. P. syringae pv. pisi 519, the bacterial blight pathogen of pea, produced a higher amount of polysaccharide (34.87 g/mL) at 60 h compared with 32.67 g/mL produced by P. syringae pv. glycinea NCPPB 1783, the bacterial blight pathogen of soya bean, and 30.03 g/mL produced by P. syringae pv. tomato NCPPB 269, the bacterial speck pathogen of tomato. EPS produced by P. syringae pv. pisi 519, P. syringae pv. tomato NCPPB 269, and P. syringae pv. glycinea NCPPB 1783 was characterized with infrared (FTIR), nuclear magnetic resonance (NMR), high performance thin layer chromatography, (HPTLC), and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. HPTLC profiles revealed the presence of glucose and glucuronic acid in all bacteria and mannose only in P. syringae pv. tomato. Molecular mass of EPS of P. syringae pv. pisi (m/z 933.8), P. syringae pv. tomato (m/z 950.4), and P. syringae pv. glycinea (m/z 933.5) was confirmed by MALDI-TOF mass spectrometry.     

FleQ Coordinates Flagellum-Dependent and -Independent Motilities in Pseudomonas syringae pv. tomato DC3000

Motility plays an essential role in bacterial fitness and colonization in the plant environment, since it favors nutrient acquisition and avoidance of toxic substances, successful competition with other microorganisms, the ability to locate the preferred hosts, access to optimal sites within them, and dispersal in the environment during the course of transmission. In this work, we have observed that the mutation of the flagellar master regulatory gene, fleQ, alters bacterial surface motility and biosurfactant production, uncovering a new type of motility for Pseudomonas syringae pv. tomato DC3000 on semisolid surfaces. We present evidence that P. syringae pv. tomato DC3000 moves over semisolid surfaces by using at least two different types of motility, namely, swarming, which depends on the presence of flagella and syringafactin, a biosurfactant produced by this strain, and a flagellum-independent surface spreading or sliding, which also requires syringafactin. We also show that FleQ activates flagellum synthesis and negatively regulates syringafactin production in P. syringae pv. tomato DC3000. Finally, it was surprising to observe that mutants lacking flagella or syringafactin were as virulent as the wild type, and only the simultaneous loss of both flagella and syringafactin impairs the ability of P. syringae pv. tomato DC3000 to colonize tomato host plants and cause disease.     

Nutritional similarity between leaf-associated nonpathogenic bacteria and the pathogen is not predictive of efficacy in biological control of bacterial spot of tomato

It has been demonstrated that for a nonpathogenic, leaf-associated bacterium, effectiveness in the control of bacterial speck of tomato is correlated with the similarity in the nutritional needs of the nonpathogenic bacterium and the pathogen Pseudomonas syringae pv. tomato. This relationship was investigated further in this study by using the pathogen Xanthomonas campestris pv. vesicatoria, the causal agent of bacterial spot of tomato, and a collection of nonpathogenic bacteria isolated from tomato foliage. The effects of inoculation of tomato plants with one of 34 nonpathogenic bacteria prior to inoculation with the pathogen X. campestris pv. vesicatoria were quantified by determining (i) the reduction in disease severity (number of lesions per square centimeter) in greenhouse assays and (ii) the reduction in leaf surface pathogen population size (log(10) of the number of CFU per leaflet) in growth chamber assays. Nutritional similarity between the nonpathogenic bacteria and X. campestris pv. vesicatoria was quantified by using either niche overlap indices (NOI) or relatedness in cluster analyses based upon in vitro utilization of carbon or nitrogen sources reported to be present in tomato tissues or in Biolog GN plates. In contrast to studies with P. syringae pv. tomato, nutritional similarity between the nonpathogenic bacteria and the pathogen X. campestris pv. vesicatoria was not correlated with reductions in disease severity. Nutritional similarity was also not correlated with reductions in pathogen population size. Further, the percentage of reduction in leaf surface pathogen population size was not correlated with the percentage of reduction in disease severity, suggesting that the epiphytic population size of X. campestris pv. vesicatoria is not related to disease severity and that X. campestris pv. vesicatoria exhibits behavior in the phyllosphere prior to lesion formation that is different from that of P. syringae pv. tomato.     

Differences between Pseudomonas syringae pv. syringae B728a and Pantoea agglomerans BRT98 in Epiphytic and Endophytic Colonization of Leaves

The leaf colonization strategies of two bacterial strains were investigated. The foliar pathogen Pseudomonas syringae pv. syringae strain B728a and the nonpathogen Pantoea agglomerans strain BRT98 were marked with a green fluorescent protein, and surface (epiphytic) and subsurface (endophytic) sites of bean and maize leaves in the laboratory and the field were monitored to see if populations of these strains developed. The populations were monitored using both fluorescence microscopy and counts of culturable cells recovered from nonsterilized and surface-sterilized leaves. The P. agglomerans strain exclusively colonized epiphytic sites on the two plant species. Under favorable conditions, the P. agglomerans strain formed aggregates that often extended over multiple epidermal cells. The P. syringae pv. syringae strain established epiphytic and endophytic populations on asymptomatic leaves of the two plant species in the field, with most of the P. syringae pv. syringae B728a cells remaining in epiphytic sites of the maize leaves and an increasing number occupying endophytic sites of the bean leaves in the 15-day monitoring period. The epiphytic P. syringae pv. syringae B728a populations appeared to originate primarily from multiplication in surface sites rather than from the movement of cells from subsurface to surface sites. The endophytic P. syringae pv. syringae B728a populations appeared to originate primarily from inward movement through the stomata, with higher levels of multiplication occurring in bean than in maize. A rainstorm involving a high raindrop momentum was associated with rapid growth of the P. agglomerans strain on both plant species and with rapid growth of both the epiphytic and endophytic populations of the P. syringae pv. syringae strain on bean but not with growth of the P. syringae pv. syringae strain on maize. These results demonstrate that the two bacterial strains employed distinct colonization strategies and that the epiphytic and endophytic population dynamics of the pathogenic P. syringae pv. syringae strain were dependent on the plant species, whereas those of the nonpathogenic P. agglomerans strain were not.     

A Lipoxygenase from Leaves of Tomato (Lycopersicon esculentum Mill.) Is Induced in Response to Plant Pathogenic Pseudomonads

Lipoxygenase (LOX) mRNA, enzyme protein, and enzyme activity were found to be induced in leaves of tomato (Lycopersicon esculentum Mill. cv Moneymaker) on inoculation with plant pathogenic bacteria. The rate of enzyme activity with linoleic or linolenic acid as substrate was approximately 10 times greater than that with arachidonic acid. Optimum activity was at pH 7.0. In the incompatible interaction, which was associated with a hypersensitive reaction (HR), a single band with relative molecular weight approximately 100,000 was revealed by probing western blots of enzyme extracts with antiserum raised against a pea lipoxygenase. Changes in the intensity of this band reflected the changes observed in LOX enzyme activity after bacterial inoculations. In the hypersensitive reaction, i.e. after inoculation with Pseudomonas syringae pv syringae, LOX mRNA was induced by 3 hours and enzyme activity began to increase between 6 and 12 hours and had reached maximum levels by 24 to 48 hours. In tomato leaves inoculated with P. syringae pv tomato (compatible interaction), LOX mRNA was induced later and enzyme activity changed only marginally in the first 24 hours, then increased steadily up to 72 hours, reaching the levels seen in the HR.     

Enzyme-Linked Immunosorbent Assay for Specific Identification and Enumeration of Azospirillum brasilense Cd. in Cereal Roots

The enzyme-linked immunosorbent assay is suggested as a reliable, sensitive, and highly specific method for the identification and enumeration of Azospirillum brasilense Cd. As few as 10⁵ CFU/ml can be practically identified by this method. At higher bacterial numbers, sensitivity increased linearly up to 5 × 10⁸ CFU/ml, yielding useful standard curves. No cross-reaction was found either with different closely related Azospirillum strains or with other rhizosphere bacteria. The method allows for a specific identification of A. brasilense Cd. both in pure cultures and in mixtures with other bacterial species, even when the colony morphology is variable. The method was successfully applied to assess the degree of root colonization on various cereals by A. brasilense Cd.     

Effects of Tabtoxinine-beta-Lactam on Nitrogen Metabolism in Avena sativa L. Roots

The effects of tabtoxinine-β-lactam (T-β-L) on nitrate uptake and glutamine synthetase (GS) and nitrate reductase (NR) activities in roots of Avena sativa seedlings were determined. Seven-day-old oat seedlings placed in a 10 mm KNO₃ and 0.5 mm T-β-L solution for 24 hours took up T-β-L and lost approximately 90% of their root GS activity. [³H]-T-β-L taken up by roots of seven-day-old oat seedlings was associated with GS immunoprecipitated from the extract of these roots. Total nitrate uptake and in vivo NR activity were decreased approximately 50% in the T-β-L treated roots. However, T-β-L uptake did not affect the induction phases of nitrate uptake or reduction, nor did it inhibit in vitro NR activity. Thus, the decrease in nitrate uptake and reduction is a secondary effect of T-β-L action. Roots of seven-day-old oat seedlings were inoculated with Pseudomonas syringae pv tabaci (Tox+) and the pathogen population in the rhizosphere was estimated by dilution plate count; 6 × 10¹³ bacteria were recovered after 3 days, as compared to the original inoculation with 7 × 10⁹ bacteria, indicating a significant growth of the pathogen in the rhizosphere. The bacteria recovered from the rhizosphere caused chlorosis in tobacco leaves and produced T-β-L in culture; 1 × 10¹⁴ bacteria were recovered from roots of seedlings inoculated with P. syringae pv tabaci (Tox−) using the same inoculation and assay procedure as for the pv tabaci (Tox+). Extracts of surface-sterilized roots previously inoculated with P. syringae pv tabaci (Tox+) did not produce viable bacterial cultures when plated out on a complete medium. Oat seedlings growing in sand culture and inoculated with P. syringae pv tabaci (Tox+) had developed chlorosis, and root GS activity had declined to less than 10% of controls after 3 days. Conversely, seedlings inoculated with P. syringae pv tabaci (Tox−) never developed chlorosis and maintained normal levels of GS activity. All oat plants inoculated with P. syringae pv tabaci (Tox+) died within 7 days after inoculation as compared to the plants inoculated with P. syringae pv tabaci (Tox−) which grew to maturity.     

Induced Resistance in Tomato Against Corynebacterium michiganense pv. michiganense by Prior Inoculation with Pseudomonas syringae Pathovars

Prior inoculation of wounded tomato petioles with a minimum concentration of 5 × 10⁴ cells per wound of various Pseudomonas syringae pathovars completely protected plants against subsequent infection with Corynebacterium michiganense pv. michiganense inoculated on the same site. Only living cells induced effective protection. In protected tissue, cells of Corynebacterium michiganense pv. michiganense remained localized at the inoculation site and their multiplication was restricted. Irrespective of the cell number introduced, initial population decreased slowly and then levelled off below the initial inoculum level. This level remained constant throughout the experimental period (15 days). Similarly, the, cell number of the inducer Pseudomonas syringae pv. phaseolicola levelled off at ca. 10⁶ cells per plant. The protection was not systemic and could be eliminated by removing the upper 5 mm of the inoculated wound tissues containing, the inducer.