Biological Control of Pseudomonas syringae pv. glycinea by Epiphytic Bacteria under Field Conditions

The efficacy of a bacterial strain as a biocontrol agent in the field may be related to the ecological similarity between the biocontrol agent and the target pathogen. Therefore, a number of different Pseudomonas syringae strains were evaluated for their antagonistic activities in vitro (agar-diffusion assay) and in planta (greenhouse assay) against the target pathogen, Pseudomonas syringae pv. glycinea. Six strains of five different pathovars were found to be antagonistic in vitro as well as in planta. The epiphytic fitness of the antagonistic Pseudomonas syringae strain 22d/93 and its two antibiotic-resistant mutants were examined on soybean plants in the fields. After adaptation the parental strain and its mutants had the ability to establish and maintain large epiphytic populations (about 10⁶ cfu/g FW) over the whole growing season after a single spray inoculation. The epiphytic behaviors of the mutants and the parent were not significantly different. The introduced bacteria did not influence the total bacterial population size. When the antagonist was coinoculated with the pathogen, the development of the pathogen was significantly reduced during the whole growing season. When the antagonistic strain was inoculated 4 weeks in advance of the pathogen, this antagonistic effect could be markedly enhanced. The final population size of the pathogen reached just 10⁴ cfu/g FW and was significantly reduced to 0.12% compared to the pathogen alone. This study demonstrates that biological control of foliar pathogens through colonization of the host plants with near isogenic or ecologically similar antagonistical strains seems to be a realistic goal.     

Dispersal of an introduced shrub (Crataegus monogyna) by the Brush-tailed Possum (Trichosurus vulpecula)

The Brush-tailed possum Trichosurus vulpecula eats the fleshy fruits of the introduced shrub Crataegus monogyna and defaecates intact seeds at least 0–50 m away. Viability of seeds recovered from faeces was >73%. comparable to the viability of fresh seeds (87.6%). Seed dispersal by possums has been largely neglected and may have important implications for invasion by woody weeds in Australasia.     

Biological suppression of rice diseases by Pseudomonas spp. under saline soil conditions

The aim of this study was to develop antagonistic strains specific for the coastal agricultural niche in Southern India. Indigenous Pseudomonas strains isolated from rhizosphere of rice cultivated in the coastal agri-ecosystem were screened for in vitro antibiosis against Xanthomonas oryzae pv. oryzaeand Rhizoctonia solani– the bacterial leaf blight (BB) and sheath blight (ShB) pathogens of rice (Oryza sativa) respectively. The strains exhibiting antibiosis were tested in the greenhouse under normal and saline soil conditions. The antagonists suppressed BB by 15 to 74% in an unamended soil. The efficient strains were tested under saline soil conditions and found to suppress disease by 46 to 82%. Similarly, incidence of ShB was also suppressed by 30 to 57% in the unamended soil by the efficient strains which, under saline soil conditions, were found to suppress ShB by 19 to 51%. Four strains of Pseudomonas tested suppressed both BB and ShB diseases in rice, of which three were efficient under both natural and saline soil conditions.     

Characterization of Pseudomonas spp. Associated with Spoilage of Gilt-Head Sea Bream Stored under Various Conditions

The population dynamics of pseudomonads in gilt-head sea bream Mediterranean fish (Sparus aurata) stored under different conditions were studied. Phenotypic analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins were performed to identify a total of 106 Pseudomonas strains isolated from S. aurata stored under different temperatures (at 0, 10, and 20°C) and packaging conditions (air and a modified atmosphere of 40% CO₂-30% N₂-30% O₂). Pseudomonas lundensis was the predominant species, followed by Pseudomonas fluorescens, while Pseudomonas fragi and Pseudomonas putida were detected less frequently. Fluorescent Pseudomonas strains dominated under air conditions, while proteolytic and less lipolytic strains dominated under modified-atmosphere packaging. Different storage conditions appear to govern the selection of pseudomonads in gilt-head sea bream fish.     

Phenotypic plasticity in leaf morphology of Crataegus monogyna (Rosaceae): an experimental study with taxonomic implications

Many leaf characters are considered in the taxonomy of Crataegus in Europe, and several have been used in studies of the extent of hybridization in populations of northwest Europe. In such analyses it is assumed that the environmental component of phenotypic variation in such characters is insignificant. We tested this assumption by analysing the variation in the size and shape of leaves borne on clone cuttings of Crataegus monogyna maintained under identical conditions apart from the availability of soil nutrients. The resulting variation among leaves from this single genotype was as great as that observed previously across populations. Furthermore, although most of this variation could not be explained, a part could be attributed to differences in nutrient availability; of nine leaf characters investigated, eight showed significant variation due to this source, and in seven, the variation covaried significantly with nutrient level. The systematic implications of this are briefly explored.     

Variation in Sensitivity of Gaeumannomyces graminis to Antibiotics Produced by Fluorescent Pseudomonas spp. and Effect on Biological Control of Take-All of Wheat

Isolates of Gaeumannomyces graminis var. tritici, the causal agent of take-all of wheat, varied in sensitivity in vitro to the antibiotics phenazine-1-carboxylic acid (PCA) and 2,4-diacetylphloroglucinol (Phl) produced by fluorescent Pseudomonas spp. shown previously to have potential for biological control of this pathogen. None of the four isolates of G. graminis var. avenae examined were sensitive to either of the antibiotics in vitro at the concentrations tested. The single isolate of G. graminis var. graminis tested was insensitive to PCA at 1.0 (mu)g/ml. Pseudomonas fluorescens 2-79 and Pseudomonas chlororaphis 30-84, both of which produce PCA, effectively suppressed take-all caused by each of two PCA-sensitive isolates of G. graminis var. tritici. PCA-producing strains exhibited a reduced ability or complete inability to suppress take-all caused by two of three isolates of G. graminis var. tritici that were insensitive to PCA at 1.0 (mu)g/ml. P. fluorescens Q2-87, which produces Phl, suppressed take-all caused by three Phl-sensitive isolates but failed to provide significant suppression of take-all caused by two isolates of G. graminis var. tritici that were insensitive to Phl at 3.0 (mu)g/ml. These findings affirm the role of the antibiotics PCA and Phl in the biocontrol activity of these fluorescent Pseudomonas spp. and support earlier evidence that mechanisms in addition to PCA are responsible for suppression of take-all by strain 2-79. The results show further that isolates of G. graminis var. tritici insensitive to PCA and Phl are present in the pathogen population and provide additional justification for the use of mixtures of Pseudomonas spp. that employ different mechanisms of pathogen suppression to manage this disease.     

Survival of Campylobacter jejuni under Conditions of Atmospheric Oxygen Tension with the Support of Pseudomonas spp.

Campylobacter jejuni is a major food-borne pathogen. Despite causing enteritis in humans, it is a well-adapted intestinal microorganism in animals, hardly ever generating disease symptoms. Nevertheless, as a true microaerophilic microorganism it is still puzzling how Campylobacter cells can survive on chicken meat, the main source of human infection. In this study, we demonstrate that C. jejuni is able to withstand conditions of atmospheric oxygen tension when cocultured with Pseudomonas species, major food-spoiling bacteria that are frequently found on chicken meat in rather high numbers. Using an in vitro survival assay, interactions of 145 C. jejuni wild-type strains and field isolates from chicken meat, broiler feces, and human clinical samples with type strains and food isolates of Pseudomonas spp., Proteus mirabilis, Citrobacter freundii, Micrococcus luteus, and Enterococcus faecalis were studied. When inoculated alone or in coculture with Proteus mirabilis, Citrobacter freundii, Micrococcus luteus, or Enterococcus faecalis type strains, Campylobacter cells were able to survive ambient oxygen levels for no more than 18 h. In contrast, Campylobacter bacteria inoculated with type strains or wild-type isolates of Pseudomonas showed a prolonged aerobic survival of up to >48 h. This microbial commensalism was diverse in C. jejuni isolates from different sources; isolates from chicken meat and humans in coculture with Pseudomonas putida were able to use this survival support better than fecal isolates from broilers. Scanning electron microscopy revealed the development of fiberlike structures braiding P. putida and C. jejuni cells. Hence, it seems that microaerophilic C. jejuni is able to survive ambient atmospheric oxygen tension by metabolic commensalism with Pseudomonas spp. This bacterium-bacterium interaction might set the basis for survival of C. jejuni on chicken meat and thus be the prerequisite step in the pathway toward human infection.Campylobacter food-borne infections are the most prevalent bacterial enteric infections in humans in industrialized and developing countries (1). It has been shown that most human infections are related to poultry meat and food produced from cattle or sheep (34, 41). Campylobacter jejuni, the species most frequently causing human disease, can be isolated from the animal intestinal tract at levels of up to 10⁹ CFU per gram of feces and can thus be called a well-adapted intestinal microorganism (30, 37). Nevertheless, because it causes human disease as a food-borne pathogen, it has to survive outside the gut. By cross-contamination at the level of the abattoir, Campylobacter bacteria hit the meat surface and have to adapt to different environmental challenges. C. jejuni is a true microaerophilic bacterium; thus, on the one hand it requires oxygen, but on the other hand it cannot grow under normal atmospheric oxygen tension conditions (15). Despite its sensitivity to high oxygen tension in vitro, viable and culturable Campylobacter bacteria can be isolated from nonskinned chicken meat at frequencies of 10⁴ CFU/g (9, 19). Assumptions on the mechanisms by which Campylobacter cells survive on meat surfaces are diverse, for example, by growing in biofilms, entering a “viable but nonculturable state,” or interacting with other microorganisms.For instance, C. jejuni is able to resist protozoa digestion and can parasitize inside protozoa, e.g., Tetrahymena pyriformis (35). This mechanism provides survival in harsh environments and resistance to antimicrobial substances and thus enhances the potential for transmission. But bacterium-bacterium interaction has also been demonstrated to be of a high level of importance for intestinal survival and uptake (20). Accordingly, members of Campylobacter have been identified to initiate cellular uptake of commensal bacteria into enterocytes (14). However, a bacterial community can also mean competition, e.g., bacteriocin production by Lactobacillus salivarius that is effective against Campylobacter colonization (36).Meat surfaces harbor numerous bacterial species (24). Some of these bacteria have adapted to this specific environmental niche and are well-known spoilage bacteria. Most relevant species belong to the family Pseudomonadaceae. But also different members of the Enterobacteriaceae can be found on meat. To date, information regarding the interaction between spoilage bacteria and pathogens is of increasing importance for public health safety measures.Hence, experimental data on the survival of C. jejuni isolates in the presence of selected meat-spoiling bacteria were analyzed and clearly demonstrated a specific interaction with type strains and isolates of Pseudomonas putida, Pseudomonas fragi, and Pseudomonas fluorescens from chicken meat surfaces.     

Symptoms of Fern Distortion Syndrome Resulting from Inoculation with Opportunistic Endophytic Fluorescent Pseudomonas spp.

Background

Fern Distortion Syndrome (FDS) is a serious disease of Leatherleaf fern (Rumohra adiantiformis). The main symptom of FDS is distortion of fronds, making them unmarketable. Additional symptoms include stunting, irregular sporulation, decreased rhizome diameter, and internal discoloration of rhizomes. We previously reported an association of symptoms with increased endophytic rhizome populations of fluorescent pseudomonads (FPs). The aim of the current study was to determine if FPs from ferns in Costa Rica with typical FDS symptoms would recreate symptoms of FDS.

Methodology and Findings

Greenhouse tests were conducted over a 29-month period. Micro-propagated ferns derived from tissue culture were first grown one year to produce rhizomes. Then, using an 8×9 randomized complete block experimental design, 8 replicate rhizomes were inoculated by dipping into 9 different treatments before planting. Treatments included water without bacteria (control), and four different groups of FPs, each at a two concentrations. The four groups of FPs included one group from healthy ferns without symptoms (another control treatment), two groups isolated from inside rhizomes of symptomatic ferns, and one group isolated from inside roots of symptomatic ferns. Symptoms were assessed 12 and 17 months later, and populations of FPs inside newly formed rhizomes were determined after 17 months. Results showed that inoculation with mixtures of FPs from ferns with FDS symptoms, but not from healthy ferns, recreated the primary symptom of frond deformities and also the secondary symptoms of irregular sporulation, decreased rhizome diameter, and internal discoloration of rhizomes.

Conclusions

These results suggest a model of causation of FDS in which symptoms result from latent infections by multiple species of opportunistic endophytic bacteria containing virulence genes that are expressed when populations inside the plant reach a minimum level.     

Latitudinal variation in the fruiting phenology of a bird-dispersed plant (shape Crataegus monogyna) in Western Europe

A number of fleshy-fruit-bearing plants of temperate regions are dispersed by migratory frugivorous birds. It has been suggested that the more southerly populations of such species should produce ripe fruits later than more northerly populations, to ensure that fruit is available when the birds arrive. I will call this the adaptive delay hypothesis. To test this hypothesis, I monitored fruiting phenology of Crataegus monogyna Jacq. at ten sites (in all of which fruit consumption is very largely by redwings, Turdus iliacus, and fieldfares, Turdus pilaris) between northern Spain (42° N) and northern Scotland (59° N). There was no negative correlation between latitude and date of fruit ripening (earliest recorded date on which ripe fruits had appeared, or earliest recorded date by which all fruits had ripened). My results thus argue against the adaptive delay hypothesis.     

4'-Acetylvitexin-2'-O-rhamnoside, isoorientin, orientin, and 8-methoxykaempferol-3-O-glucoside as markers for the differentiation of Crataegus monogyna and Crataegus pentagyna from Crataegus laevigata (Rosaceae)

In our chemotaxonomic investigation of pharmaceutically relevant Crataegus species, the qualitative and quantitative flavonoid fingerprint of Crataegus monogyna and C. pentagyna is presented. Six flavonoids were identified as vitexin-2'-O-rhamnoside (1), vitexin (2), isovitexin (3), rutin (4), hyperoside (5), and isoquercitrin (6). Besides the verification of the main compounds isoorientin (7) and orientin (8) in C. pentagyna, further four flavonoids were isolated and identified as isoorientin-2'-O-rhamnoside (9), orientin-2'-O-rhamnoside (10), isovitexin-2'-O-rhamnoside (11), and 8-methoxykaempferol-3-O-glucoside (12) by means of 1D- and 2D-NMR, MS, and UV analyses. Compound 12 was isolated for the first time from C. pentagyna. In contrast to C. pentagyna, C. monogyna samples were predominated by 4'-acetylvitexin-2'-O-rhamnoside (13), which was missing in C. pentagyna. Hence, 13 represents an interesting compound for chemotaxonomy of C. monogyna, whereas the main flavonoids 7, 8, and 12 could be proposed as markers for C. pentagyna. The absence of 7, 8, 12, and 13 in C. laevigata offers an appropriate tool for additional differentiation from C. monogyna and C. pentagyna, and for sample identification and quality control of the three main Crataegus species used in European phytotherapy.     

Fruit fate,frugivory, and fruit characteristics: a study of the hawthorn,Crataegus monogyna (Rosaceae)

Summary The fate of fruits from a population of European hawthorn (Crataegus monogyna) in western Oregon, USA, was examined over a two-year period. Only one frugivore, the American robin (Turdus migratorius) foraged on the C. monogyna fruits, making this an unusually straightforward fruit-frugivore system. Dispersal efficiency was low, with an average 21% of seeds being dispersed (carried away from parent plants) each year; the most common fate of fruits was to simply fall. Robins dropped 20% of the fruits that they picked, and defecated/regurgitated 40% of the fruits (seeds) that they swallowed, beneath parent plants. One trait, gruit abundance, strongly affected the probability of bush visitation by robins, bushes with larger fruit displays being preferred. Both absolute dispersal success (number of seeds) and dispersal efficiency (proportion of seeds; success per propagule) were also found to be correlated most strongly with initial fruit abundance. Individual plant fecundity and fruit quality were found to vary little between years; as a result, dispersal efficiencies for individual plants were also annully consistent. Larger (older) plants produced more fruits and therefore had higher fitness. These results suggest that the optimal fruiting strategy for C. monogyna is therefore to get as big as possible as quickly as possible by delaying fruiting until later in life.     

The Effectiveness of Crataegus orientalis M Bieber. (Hawthorn) Extract Administration in Preventing Alveolar Bone Loss in Rats with Experimental Periodontitis

The purpose of this animal study was to evaluate the effects of hawthorn (Crataeus orientalis M Bieber.) extract on serum oxidative status and alveolar bone loss in experimental periodontitis. Twenty-seven Wistar rats were assigned to one of the following groups: non- ligated+placebo (saline) (NL, n = 9), ligature only+placebo (saline) (LO, n = 9), and ligature and treated with hawthorn extract in saline (H, n = 9) (100 mg/kg orogastrically, once a day for 11 days). Periodontitis was induced by submerging a 4/0 silk ligature in the sulcus of the mandibular right first molars of rats, and the animals were sacrificed after 11 days. Micro-CT examinations were performed for linear and volumetric parameter assessment of alveolar bone. Periodontal tissues were histopathologically examined to assess the differences among the study groups. Levels of serum total antioxidant status (TAS)/total oxidant status (TOS), and oxidative stress index (OSI) were also analyzed. Alveolar bone loss was significantly reduced by hawthorn administration compared to LO group (p<0.05). The number of inflammatory cells and osteoclasts in the LO group was significantly higher than that of the NL and H groups (p< 0.05). The number of osteoblasts in the LO and H groups was significantly higher than that of the NL group (p<0.05). TOS and OSI levels were significantly reduced in H group compared to LO group (P <0.05) and TAS levels were similar in H and NL group (p< 0.05). Hawthorn extract showed inhibitory effect on periodontal inflammation and alveolar bone loss by regulating TAS, TOS and OSI levels in periodontal disease in rats when administered systemically.     

Biological Control of Crown Gall in Grapevine and Raspberry by Two Pseudomonas spp. with a Wide Spectrum of Antagonistic Activity

Pseudomonas aureofaciens B-4117 and P. fluorescens CR330D inhibited the growth of a wide range of plant pathogens, including Agrobacterium tumefaciens , when tested on agar media. In a series of nursery-based trials with natural pathogen inoculum, application of either B-4117 or CR330D significantly reduced the incidence and severity of crown gall caused by A. tumefaciens on grapevine and raspberry. The extent of disease control depended upon the variety tested. Both bacteria reduced disease during seedling root production and grafting. The disease incidence on root cuttings of three grapevine varieties was reduced by 56-80% and the disease severity index (DSI) was decreased by 75-86%. Depending on the scion variety, the number of healthy rooted grafts increased by 2-3.5-fold, while the DSI was reduced by 1.5-3-fold. The results suggest that there is potential in using these antagonists to diminish the influence of latent rootstock infection on graft sensitivity to crown gall. Pretreatment of rooted raspberry seedlings with P. aureofaciens B-4117 prevented the development of crown galls caused by A. tumefaciens strain K24 or by a mixture of A. tumefaciens pathogenic strains previously isolated from raspberry. Both Pseudomonas spp. persisted on the root surfaces of inoculated vine cuttings and in non-sterile soil. The advantages of using the antagonistic bacteria as biocontrol agents of crown gall are discussed.     

Influence of the extraction mode on the yield of hyperoside, vitexin and vitexin-2'-O-rhamnoside from Crataegus monogyna Jacq. (hawthorn)

Introduction –  The extract of Crataegus monogyna shows sedative, hypotensive, vasodilator and cardio‐tonic actions. Although several papers dealing with the extraction of metabolites from Crataegus have been published, the plant productivity in terms of bioactive compounds is not easily understandable as yet. Objective –  To investigate the influence of the extraction mode on the yield of bioactive compounds from Crataegus monogyna Jacq. in order to evaluate plant productivity. Methodology –  Samples were prepared by extraction of powdered material obtained from top branches, flowers and leaves. Soxhlet extraction, maceration and ultrasound‐ and microwave‐assisted extraction at different experimental conditions were investigated for the exhaustive extraction of hyperoside, vitexin and vitexin‐2′′‐O‐rhamnoside. The phytocomponents were identified and quantified by HPLC‐UV/PAD, comparing HPLC retention times and UV spectra of individual peaks with those of the standards analysed under the same conditions. Results –  An easy‐to‐use HPLC isocratic method suitable for the quantification of hyperoside, vitexin and vitexin‐2′′‐O‐rhamnoside in raw plant extracts was developed. The optimised HPLC methodology was applied to evaluate different extraction procedures. The ultrasound and microwave‐assisted extraction protocols showed higher extraction efficiency than the others. In particular, the optimised microwave protocol gave rise to the highest extraction efficiency with high reproducibility. Conclusions –  A microwave protocol combined with isocratic HPLC analysis is proposed for the rapid screening of plant materials collected in different environmental conditions in order to evaluate the productivity of Crataegus monogyna Jacq. and to find out the best ecological conditions to cultivate hawthorn in Northern Italy. Copyright © 2008 John Wiley & Sons, Ltd.     

Biological Activity and Food Analysis ofCyttaria spp. (Discomycetes)

The biological activity and nutritional composition of Chilean collections ofCyttaria berteroi C. darwinii, C. espinosae, C. harioti andC. johowii have been determined. The crude protein, lipid, ash, and carbohydrate content of the samples examined were similar to that of other edible fungi. Amino acid analysis of ChileanCyttaria showed that proteins of all species are deficient in methionine and cysteine and excepting oneC. espinosae collection, all samples proved to be below the WHO values for the essential amino acids valine, isoleucine, leucine, and lysine. The acute oral toxicity test ofC. espinosae in rats showed that doses up to 2.5 g extract/kg body weight (corresponding to 25.7–38.7 g fresh weight/kg) did not produce mortality or macroscopic damage in the organs examined of the test animals. Cyttaria extracts assayed at 50 fig/ml were inactive or moderately active as inhibitors towards the enzymes xanthine oxidase (0-31%) and β-glucuronidase (0-65%), and lacked antifungal and antibacterial effects in a battery of antimicrobial assays. When administered intravenously at 2.5 mg/kg, the water-soluble extract ofCyttaria produced a hypotensive response in rats (-16 to -21%). Furthermore, most of the aqueous extracts ofC. espinosae andC. harioti showed DNA binding activity. The main sterols fromCyttaria espinosae andC. berteroi were identified as dihydrobrassicasterol derivatives. Our study suggests that edibleCyttaria species do not represent an acute toxicity risk for consumers and that their nutritional value is similar to that of other edible, cultivated mushrooms. The preservation ofCyttaria spp. as a food resource is linked to the protection of the temperateNothofagus forests.     

Biological Control Activities of Rice-Associated Bacillus sp. Strains against Sheath Blight and Bacterial Panicle Blight of Rice

Potential biological control agents for two major rice diseases, sheath blight and bacterial panicle blight, were isolated from rice plants in this study. Rice-associated bacteria (RABs) isolated from rice plants grown in the field were tested for their antagonistic activities against the rice pathogens, Rhizoctonia solani and Burkholderia glumae, which cause sheath blight and bacterial panicle blight, respectively. Twenty-nine RABs were initially screened based on their antagonistic activities against both R. solani and B. glumae. In follow-up retests, 26 RABs of the 29 RABs were confirmed to have antimicrobial activities, but the rest three RABs did not reproduce any observable antagonistic activity against R. solani or B. glumae. According to16S rDNA sequence identity, 12 of the 26 antagonistic RABs were closest to Bacillus amyloliquefaciens, while seven RABs were to B. methylotrophicus and B, subtilis, respectively. The 16S rDNA sequences of the three non-antagonistic RABs were closest to Lysinibacillus sphaericus (RAB1 and RAB12) and Lysinibacillus macroides (RAB5). The five selected RABs showing highest antimicrobial activities (RAB6, RAB9, RAB16, RAB17S, and RAB18) were closest to B. amyloliquefaciens in DNA sequence of 16S rDNA and gyrB, but to B. subtilis in that of recA. These RABs were observed to inhibit the sclerotial germination of R. solani on potato dextrose agar and the lesion development on detached rice leaves by artificial inoculation of R. solani. These antagonistic RABs also significantly suppressed the disease development of sheath blight and bacterial panicle blight in a field condition, suggesting that they can be potential biological control agents for these rice diseases. However, these antagonistic RABs showed diminished disease suppression activities in the repeated field trial conducted in the following year probably due to their reduced antagonistic activities to the pathogens during the long-term storage in -70C, suggesting that development of proper storage methods to maintain antagonistic activity is as crucial as identification of new biological control agents.     

Assessment of the Environmental Fate of the Biological Control Agent of Fire Blight, Pseudomonas fluorescens EPS62e, on Apple by Culture and Real-Time PCR Methods

The colonization of apple blossoms and leaves by Pseudomonas fluorescens EPS62e was monitored in greenhouse and field trials using cultivable cell counting and real-time PCR. The real-time PCR provided a specific quantitative method for the detection of strain EPS62e. The detection level was around 10² cells g (fresh weight)⁻¹ and the standard curve was linear within a 5-log range. EPS62e actively colonized flowers reaching values from 10⁷ to 10⁸ cells per blossom. In apple flowers, no significant differences were observed between population levels obtained by real-time PCR and plating, suggesting that viable but nonculturable (VBNC) cells and residual nondegraded DNA were not present. In contrast, on apple leaves, where cultivable populations of EPS62e decreased with time, significant differences were observed between real-time PCR and plating. These differences indicate the presence of VBNC cells or nondegraded DNA after cell death. Therefore, the EPS62e population was under optimal conditions during the colonization of flowers but it was stressed and poorly survived on leaves. It was concluded that for monitoring this biological control agent, the combined use of cultivable cell count and real-time PCR is necessary.     

Influence of Two Plant Species (Flax and Tomato) on the Distribution of Nitrogen Dissimilative Abilities within Fluorescent Pseudomonas spp

The distribution of nitrogen-dissimilative abilities among 317 isolates of fluorescent pseudomonads was studied. These strains were isolated from an uncultivated soil and from the rhizosphere, rhizoplane, and root tissue of two plant species (flax and tomato) cultivated on this same soil. The isolates were distributed into two species, Pseudomonas fluorescens (45.1%) and Pseudomonas putida (40.4%), plus an intermediate type (14.5%). P. fluorescens was the species with the greatest proportion of isolates in the root compartments and the greatest proportion of dissimilatory and denitrifying strains. According to their ability to dissimilate nitrogen, the isolates have been distributed into nondissimilatory and dissimilatory strains, nitrate reducers and true denitrifiers with or without N(inf2)O reductase. The proportion of dissimilatory isolates was significantly enhanced in the compartments affected by flax and tomato roots (55% in uncultivated soil and 90 and 82% in the root tissue of flax and tomato, respectively). Among these strains, the proportion of denitrifiers gradually and significantly increased in the root vicinity of tomato (44, 68, 75, and 94% in uncultivated soil, rhizosphere, rhizoplane, and root tissue, respectively) and was higher in the flax rhizoplane (66%) than in the uncultivated soil. A higher proportion of N(inf2)O reducers was also found in the root compartments. This result was particularly clear for tomato. It is hypothesized that denitrification could be a selective advantage for the denitrifiers in the root environment and that this process could contribute to modify the specific composition of the bacterial communities in the rhizosphere.