Do genetic make-up and growth manipulation affect tomato fruit size by cell number, or cell size and DNA endoreduplication?

This work investigated the link between genetic and developmental controls of fruit size and composition. On two isogenic lines (CF12-C and CF14-L), differing by fruit weight and sugar content quantitative trait loci (QTLs) identified previously, basal and tip fruits were characterized at anthesis and at maturity through their growth, dry matter and sugar content, number and size of cells and nuclei DNA content. The influence of competition was assessed by removing either basal or tip ovaries at anthesis. On an intact inflorescence, CF12-C fruits grew less than CF14-L fruits, with 1.67 fewer cell layers and similar cell size, suggesting that genes controlling cell division may be responsible for this fruit size variation. Truss thinning masked the QTL effect on fruit size, mainly by reducing the difference in cell number between the two lines and by promoting cell expansion in tip fruits, so that fruit growth was similar at both positions and for both lines. Thus, in these lines, cell number exerts a control on final fruit size only when there is competition among fruits. Different responses of basal and tip fruits after flower removal suggested that this treatment induced changes in hormonal relationships within the truss. No fixed relationship between DNA endoreduplication and cell size was found, as while cell size and dry matter and sugar contents differed with tomato lines, fruit position and truss size, endoreduplication patterns were the same. CF12-C fruits had a higher dry matter (+0.3% of fresh weight) and carbohydrates (+8% of dry matter) content than CF14-L fruits. The percentage dry matter was independent of truss size but decreased slightly from basal to tip fruits.

Polyphasic classification of the genus Photorhabdus and proposal of new taxa: P. luminescens subsp. luminescens subsp. nov., P. luminescens subsp. akhurstii subsp. nov., P. luminescens subsp. laumondii subsp. nov., P. temperata sp. nov., P. temperata subsp. temperata subsp. nov. and P. asymbiotica sp. nov.

The taxonomic position of Photorhabdus strains was examined through the results of DNA relatedness (S1 nuclease method) studies associated with the determination of delta Tm, 16S rRNA phylogenetic inferences and phenotypic characterization, including morphological, auxanographic, biochemical and physiological properties. Three genomic species were delineated on a consensus assessment. One of these species corresponded to Photorhabdus luminescens, since strains were at least 50% related to the type strain of this species with delta Tm less than 7 degrees C. The two other species were novel genomic species II and III, which were less than 40% related to each other with delta Tm higher than 9 degrees C. A comparison of the complete 16S rDNA sequences of several representatives of genomic species II and genomic species III revealed that each of them formed a stable lineage independent of the cluster generated by P. luminescens strains. The genomic species differed in their maximum temperatures for growth. A correlation with the ecological origin of the bacterial samples was noticed. The heat-tolerant group I (maximum growth temperature 35-39 degrees C) corresponded to the symbionts of Heterorhabditis bacteriophora groups Brecon and HP88 and Heterorhabditis indica, nematodes living in warm and tropical countries, respectively. Group II (maximum growth temperature 33-35 degrees C) encompassed symbionts from Heterorhabditis megidis, Heterorhabditis zealandica and group NC1 of H. bacteriophora, nematodes isolated in temperate climates. Group III were bacteria isolated from human specimens. Two new species, Photorhabdus temperata sp. nov. (type strain CIP 105563T) and Photorhabdus asymbiotica sp. nov. (type strain ATCC 43950T), are proposed for genomic species II and III, respectively. Species I and II can be separated into sub-groups on the basis of high DNA-DNA relatedness (more than 80% DNA binding with delta Tm < 1.5 degrees C), 16S rDNA branching and phenotypic characters. Therefore, we propose that the two species P. luminescens and P. temperata should be subdivided into subspecies as follows: P. luminescens subsp. luminescens subsp. nov. (type strain ATCC 29999T), P. luminescens subsp. akhurstii subsp. nov. (type strain CIP 105564T), P. luminescens subsp. laumondii subsp. nov. (type strain CIP 105565T) and P. temperata subsp. temperata subsp. nov.

PCR-ribotyping of Xenorhabdus and Photorhabdus isolates from the Caribbean region in relation to the taxonomy and geographic distribution of their nematode hosts.

The genetic diversity of symbiotic Xenorhabdus and Photorhabdus bacteria associated with entomopathogenic nematodes was examined by a restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes (rDNAs). A total of 117 strains were studied, most of which were isolated from the Caribbean basin after an exhaustive soil sampling. The collection consisted of 77 isolates recovered from entomopathogenic nematodes in 14 Caribbean islands and of 40 reference strains belonging to Xenorhabdus and Photorhabdus spp. collected at various localities worldwide. Thirty distinctive 16S rDNA genotypes were identified, and cluster analysis was used to distinguish the genus Xenorhabdus from the genus Photorhabdus. The genus Xenorhabdus appears more diverse than the genus Photorhabdus, and for both genera the bacterial genotype diversity is in congruence with the host-nematode taxonomy. The occurrence of symbiotic bacterial genotypes was related to the ecological distribution of host nematodes.

Rapid identification of Medicago nodulating strains by using two oligonucleotide probes complementary to 16S rDNA sequences.

Symbiotic bacteria associated with the Medicago genus are separated into two closely related species named Sinorhizobium meliloti and Sinorhizobium medicae. To discriminate rapidly between these two bacterial species, two 15-base DNA probes, 16Smfs and 16Smed, were designed from the alignment of 16S rDNA sequences to differentiate S. meliloti from S. medicae. Their specificities were evaluated by dot-blot hybridization experiments on 25 reference strains representing 13 species of Rhizobium and Sinorhizobium, and by comparison with all 16S rDNA sequences available in the GenBank data base. No cross-reaction was found with 16Smed, which was thus considered species specific for S. medicae. By contrast, as expected according to the 16S rDNA sequence alignment, the labeled 16Smfs probe cross-hybridized with the DNAs of S. meliloti, Sinorhizobium fredii, and Sinorhizobium saheli but not with the DNA of S. medicae. Since S. saheli and S. fredii do not nodulate Medicago, 16Smed and 16Smfs can be routinely used to characterize the two Sinorhizobium species nodulating Medicago from pure cultures or from Medicago root nodules. Fifty strains isolated from eight annual Medicago species were then characterized by using colony hybridizations. Sinorhizobium meliloti was more frequently obtained (> 80% isolates) than was S. medicae. Both Sinorhizobium species seemed to be trapped by annual Medicago and no plant-host specificity was detected.

Fast and accurate identification of Xenorhabdus and Photorhabdus species by restriction analysis of PCR-amplified 16S rRNA genes.

Thirteen bacterial strains of Xenorhabdus and 14 strains of Photorhabdus originating from a wide range of geographical and nematode host sources were typed by analyzing 16S rRNA gene (rDNA) restriction patterns obtained after digestion of PCR-amplified 16S rDNAs. Eight tetrameric restriction endonucleases were examined. A total of 17 genotypes were identified, forming two heterogeneous main clusters after analysis by the unweighted pair-group method using arithmetic averages: group I included all Xenorhabdus species and strains, symbionts of Steinernema, whereas group II encompassed the Photorhabdus strains, symbionts of Heterorhabditis. To identify the four valid species of Xenorhabdus and unclassified strains and all the genotypes of Photorhabdus luminescens, three restriction enzymes are required: CfoI, AluI, and HaeIII. Our results, in substantial agreement with DNA-DNA pairing and 16S rDNA sequence data, indicate that amplified 16S rDNA restriction analysis is a simple and accurate tool for identifying entomopathogenic nematode bacterial symbionts.

Sinorhizobium medicae sp. nov., isolated from annual Medicago spp.

The taxonomic position of isolates of a new genomic species (designated genomic species 2) obtained from several annual Medicago species and originating from different geographical locations was established through the results of phenotypic tests (including the results of auxanographic and biochemical tests and symbiotic properties) and 16S rRNA phylogenetic inferences. A comparison of the complete 16S rRNA sequence of a representative of genomic species 2 (strain A 321T [T = type strain]) with the 16S rRNA sequences of other members of the Rhizobiaceae and closely related taxa showed that genomic species 2 was phylogenetically related to Sinorhizobium meliloti, Sinorhizobium fredii, Sinorhizobium saheli, and Sinorhizobium teranga. The levels of sequence similarity and observed numbers of nucleotide substitutions in Sinorhizobium strains indicated that A 321T and S. meliloti exhibited the highest level of sequence similarity (99.7%), with four nucleotide substitutions and one deletion. The results of a numerical analysis based on data from 63 auxanographic and biochemical tests clearly separated genomic species 2 isolates from S. meliloti. Genomic species 2 isolates nodulated and fixed nitrogen with Medicago polymorpha, whereas S. meliloti isolates were ineffective and formed rudimentary nodules on this host plant. On the basis of phenotypic and 16S sequence analysis data, genomic species 2 isolates cannot be assigned to a previously described species. We propose that these isolates belong to a new species, Sinorhizobium medicae.

Evidence that two genomic species of Rhizobium are associated with Medicago truncatula.

Seventy-three isolates of rhizobia sampled from root nodules of Medicago truncatula were analyzed by restriction fragment length polymorphism (RFLP) of DNA regions amplified by the polymerase chain reaction (PCR) targeting the symbiotic plasmid (nifD-K, nodD1, and nodD2 genes) and the chromosome (16S rDNA plus intergenic spacer). Two genotypic groups were found, regardless of the DNA region targeted. These two groups were given the status of genomic species based on results of DNA/DNA hybridization.

An experimental set-up to study carbon, water, and nitrate uptake rates by hydroponically grown plants.

The experimental system described allows concomitant hourly measurements of CO2, H2O, and NO3 uptake rates by plants grown hydroponically in a greenhouse. Plants are enclosed in an airtight chamber through which air flows at a controlled speed. Carbon dioxide exchange and transpiration rates are determined from respective differences of concentrations of CO2 and water vapor of the air at the system inlet and outlet. This set-up is based on the "open-system" principle with improvements made on existing systems. For instance, propeller anemometers are used to monitor air flow rates in the chamber. From their signal it is possible to continuously adjust air speed to changing environmental conditions and plant activity. The air temperature inside the system therefore never rises above that outside. Water and NO3 uptake rates are calculated at time intervals from changes in the volume and the NO3 concentration of the nutrient solution in contact with the roots. The precise measurement of the volume of solution is achieved using a balance which has a higher precision than any liquid level sensors. Nitrate concentration is determined in the laboratory from aliquots of solution sampled at time intervals. A number of test runs are reported which validate the measurements and confirm undisturbed conditions within the system. Results of typical diurnal changes in CO2, H2O, and NO3 uptake rates by fruiting tomato plants are also presented.

Degradation of iprodione by a soil Arthrobacter-like strain.

A bacterial strain able to transform iprodione was isolated from a fast iprodione-degrading soil by enrichment procedures. Transformation was detected through 3,5-dichloroaniline production as measured by a rapid colorimetric method. The strain, MA6, was tentatively identified as an Arthrobacter sp. When it was incubated with MA6 in a minimum mineral medium (pH 6.5), iprodione (8.8 mumol/liter) was transformed into two major metabolites that were identified by high-performance liquid chromatography analysis: 3,5-dichlorophenylcarboximide (metabolite 1) and (3,5-dichlorophenylurea) acetic acid (metabolite 2), which was produced after ring cleavage of the former product. These products were synthesized in the laboratory and compared with metabolites 1 and 2 which were formed during iprodione degradation. Small quantities of 3,5-dichloroaniline also appeared in the bacterial culture but did not substantially increase between the first and second days of incubation. In contrast, in the sterile control medium, iprodione was spontaneously transformed into hydantoic acid and an iprodione isomer. Chemical and biological transformations of iprodione seem to occur through two different pathways. One biological degradation pathway is proposed.

Effect of transient oxic conditions on the composition of the nitrate-reducing community from the rhizosphere of Typha angustifolia.

Within a nitrate-reducing bacterial community, a niche differentiation between denitrifying and nitrate ammonifying bacteria may be determinated by a complex of environmental parameters, such as the availability of carbon, nitrate, and oxygen. Hence, oxygen- and carbon-releasing aerenchymatous plants may affect the composition of the nitrate-reducing community in waterlogged sediment. The composition of the nitrate-reducing community in the rhizosphere of the aerenchymatous plant species Typha angustifolia was compared with the community in nonrhizospheric sediment. All three functional groups (NO2 (-) accumulators, N2O producers, and presumed NH4 (+) producers) were present at both sites with an ratio of 36:45:12 and 43:22:18 for nonrhizospheric and rhizospheric sediments, respectively. Most of the isolated were gram-negative, and approximately 50% of these strains demonstrated an obligatory oxidative metabolism.In the absence of nitrate, Enterobacteriaceae (belonging to the NO2 (-) accumulating group) became dominant during enrichment of bacteria from the rhizosphere of T. angustifolia in a chemostat with glycerol (20 mM) as substrate, both under strictly anoxic and transient oxic conditions. Addition of nitrate to the chemostats led to the predominance of denitrifying pseudomonads, irrespective of the presence or absence of oxygen. However, in the presence of nitrate under anoxic conditions, enterobacteria persisted in the medium together with pseudomonads.It was concluded that oxidative bacteria such as pseudomonads are the better competitors for limiting amounts of glycerol, provided oxygen or nitrate is present. In the absence of these electron acceptors, fermentative bacteria become dominant.

Increased albumin excretion rate during a standardized exercise-test in diabetics with lowered blood filterability.

Albumin excretion rate in urine is a marker of early, reversible stages of diabetic nephropathy. Does abnormal blood rheology represent an additional risk factor in this multifactorial process? We investigated a possible link between red cell filterability and microalbuminuria during an exercise test (exercise is supposed to improve the detection of excessive microalbuminuria). 77 diabetics (27 females, 50 males, age: 15-60 yr) underwent a 20 min inframaximal progressively increasing workload on cycloergometer, rising heart rate up to 200 minus the age. Filterability of whole blood and washed red cells were measured on 5 microns polycarbonate sieves reused after ultrasonic cleaning. Whole blood filterability was found to be impaired in 35 subjects (group A) and normal in 41 (group B). Groups A and B were matched for age, sex, blood pressure, glycemic equilibrium, and duration of disease. Microalbuminuria was higher in A at rest (39.79 +/- 13.83 micrograms/min vs 12.9 +/- 3.21, p less than 0.01) and after exercise (91.80 +/- 20.79 vs 42.23 +/- 7.85, p less than 0.01). The slopes of regression lines between resting Microalbuminuria and blood pressure were greater in group A than in group B (p less than 0.01). No relationship between microalbuminuria and washed red cell filterability was detected. This study confirms on a larger scale a previous report of our team. Some hemorheologic disorders detectable with whole blood filterability (but not with washed red cell filtration) are associated with an increase in resting and postexercise microalbuminuria.

Phenotypic drift inBradyrhizobium japonicum populations after introduction into soils as established by numerical analysis.

The degree of phenotypic variation of the bacterial strains USDA 125-Sp, USDA 138 and USDA 138-SmBradyrhizobium japonicum a long time after introduction was studied in three experimental fields. A total of 54 phenotypic characters were analyzed by constructing a dendrogram based on an hierarchic classification. Strong similarities (92.6, 94 and 95%) were found between the isolates introduced into soil 8, 10 and 13 years ago and between their respectiveB. japonicum parental clones. The dendrogrammic analysis detected a small amount of phenotypic drift, however, between soil isolates and parental clones belonging to the same serogroup (selective effects were found to have generated 0 to 3.9% variation for the USDA 125-Sp inoculum introduced 8 years ago, and 3.2-3.5% after 10 and 13 years, respectively, for the USDA 138 and USDA 138-Sm bacterial inocula) and within the serogroup 125 soil isolates (2.7%). We found a similar evolution of serogroup 125 isolates when compared with parental clones conserved on slant agar at 4°C. When a drift was observed, the isolates from soil presented a lower activity for several enzymes and lower diversity compared with the parental clones.

Stability of Bradyrhizobium japonicum Inoculants after Introduction into Soil.

Bradyrhizobium japonicum USDA 125-Sp, USDA 138, and USDA 138-Sm had been used as inoculants for soybean (Glycine max (L.) Merr.) in soils previously free of B. japonicum. At 8 to 13 years after their release, these strains were reisolated from soil samples. A total of 115 isolates were obtained through nodules, and seven colonies were obtained directly by a serological method. The stability of the inoculants was confirmed by comparing the reisolated cultures with their respective parental strains which had been preserved by being lyophilized or stored on a yeast extract-mannitol agar slant at 4 degrees C. Comparisons were made on morphological and serological characters, carbon compound utilization (8 tested), intrinsic antibiotic resistance (9 tested), and enzymatic activity (19 tested). Mucous and nonmucous isolates of serogroup 125 were analyzed for symbiotic effectiveness and restriction fragment hybridization with a DNA probe. Our data suggest that the B. japonicum inoculants have survived for up to 13 years in the soils without significant mutation except for two reisolates with a slightly increased kanamycin resistance level.