Indigenous bradyrhizobia populations in tropical farm-lands

Using the plant-infection technique, and soybeans as test plants, counts of root nodule bacteria in seven farm-lands in Southern Nigeria were estimated. All sites including those without a recent legume cover history contained bradyrhizobia. Abundance, however correlated with the presence of a legume. Populations varied from 0.95 to 3.63 log₁₀ rhizobia per g soil.
In addition to the presence of a legume, soil moisture and pH appeared to influence the rhizobial distribution in these soils. Most of the nodules produced on the test plant were ineffective, indicating that the rhizobial presence alone does not guarantee adequate N₂ fixation in a farming set-up.     

Evaluation of the symbiotic nitrogen-fixing potential of soils by direct microbiological means

A method for estimating the nitrogen-fixing capacity of a population of rhizobia resident in soil is presented. legume test plants, growing under microbiologically-controlled conditions in test tubes packed with a vermiculite substrate moistened with a nitrogen-free plant nutrient solution, are inoculated directly with a suspension of the soil under examination. Rhizobia in the soil nodulate the test plants, and the amount of foliage dry matter produced in the 28 days after inoculation is regarded as an index of their effectiveness. An inoculum of at least 30, and preferably 100, rhizobia is needed to ensure that nitrogen fixation is not masked by delayed nodulation. The new method is tentatively described as the ‘whole-soil inoculation’ technique. Appraisals were made withTrifolium subterraneum L. andRhizobium trifolii and withMedicago sativa L. andR. meliloti. Soil-borne pathogens did not interfere with plant growth. The whole-soil inoculation technique was less tedious and time-consuming than an alternative method which involved extracting representative isolates from the soil and testing their effectiveness individually, and appeared to give more realistic values for the nitrogen-fixing capacity of the soil as a whole. Used in association with a field experiment, the whole-soil inoculation technique confirmed microbiologically that there had been an agronomic response to surface application of inoculant to poorly-nodulatedT. subterraneum pasture. It is submitted that this technique for determining the effectiveness of rhizobia in soil, combined with a plant-infection method for counting rhizobia, can be a reliable guide to the need for inoculation in the field.     

Methods of detection and estimation of rhizobia in soil

Summary The plant-infection technique for the estimation of rhizobia, in which small-seeded hosts are grown on agar within test-tubes, is applicable to soils with a moderate rhizobial population (in the order of at least 100/g). Account might have to be taken of skips (less diluted: negative, when more diluted are positive) likely to result, at least in part, from unfavourable conditions for rhizobial survival, multiplication or nodulation. Because of such effects, a sparse population (in the order of (10/g) may not be detected even without dilution (1 g soil per plant tube). Localisation of rhizobia in the soil is likely to be important in determining contact with the plant roots in the dilution count and in sampling from the field. Difficulties with sparsely populated soils can be partly overcome by carefully conducted direct sowings of sterilised seed, preferably in the confines of cores, either left in the field or brought back to the glasshouse.     

Lognormal Distribution of Epiphytic Bacterial Populations on Leaf Surfaces

Total populations of epiphytic bacteria and selected components thereof were determined on sets of 24 to 36 individual leaves (corn, rye) or leaflets (snap bean, soybean, tomato) of field-grown plants by washing and dilution plating. In general, levels of component populations (e.g., bacteria that are ice nucleation active) were quantitatively more variable from leaf to leaf within a set than were total epiphytic bacterial populations. Populations of a given component frequently varied by a factor of 100 to 1,000 within a set of leaves. Total bacterial populations usually varied by a factor of about 10. For each set of leaves, total and component epiphytic bacterial populations were found to approximate a lognormal distribution by the Shapiro-Wilk test for normality. Due to the lognormal distribution of epiphytic bacterial populations, estimates of population size based on the common practice of using bulked samples (wherein several leaves are washed together) will overestimate the population median by a factor of approximately 1.15σ². From the known probability distribution of bacterial populations, the frequency with which a given bacterial population size is met or exceeded on individual leaves can be estimated. If the bacterial component is phytopathogenic, the frequency estimates could be used to relate quantitatively pathogen populations and disease incidence.     

Phytodetoxification of TNT by transplastomic tobacco (<Emphasis Type="Italic">Nicotiana tabacum</Emphasis>) expressing a bacterial nitroreductase

Key message

Expression of the bacterial nitroreductase gene, nfsI, in tobacco plastids conferred the ability to detoxify TNT.

Abstract

The toxic pollutant 2,4,6-trinitrotoluene (TNT) is recalcitrant to degradation in the environment. Phytoremediation is a potentially low cost remediation technique that could be applied to soil contaminated with TNT; however, progress is hindered by the phytotoxicity of this compound. Previous studies have demonstrated that plants transformed with the bacterial nitroreductase gene, nfsI have increased ability to tolerate and detoxify TNT. It has been proposed that plants engineered to express nfsI could be used to remediate TNT on military ranges, but this could require steps to mitigate transgene flow to wild populations. To address this, we have developed nfsI transplastomic tobacco (Nicotiana tabacum L.) to reduce pollen-borne transgene flow. Here we have shown that when grown on solid or liquid media, the transplastomic tobacco expressing nfsI were significantly more tolerant to TNT, produced increased biomass and removed more TNT from the media than untransformed plants. Additionally, transplastomic plants expressing nfsI regenerated with high efficiency when grown on medium containing TNT, suggesting that nfsI and TNT could together be used to provide a selectable screen for plastid transformation.

     

Effect of crop rotation and soil cover on alteration of the soil microflora generated by the culture of transgenic plants producing opines

The culture of transgenic Lotus corniculatus plants producing opines, which are bacterial growth substrates, leads to the selection of rhizospheric bacteria able to utilize these substrates. We have investigated the fate of the opine-utilizing community over time under different experimental conditions following elimination of selective pressure exerted by the transgenic plants. These plants were removed from the soil, which was either left unplanted or replanted with wild-type L. corniculatus or wheat plants. The density of opine-utilizing bacteria in the fallow soils remained essentially unchanged throughout the experiment, regardless of the soil of origin (soil planted with wild-type or transgenic plants). When wild-type Lotus plants were used to replace their transgenic counterparts, only the bacterial populations able to utilize the opines were affected. Long-term changes affecting the opine-utilizing bacterial community on Lotus roots was dependent upon the opine studied. The concentration of nopaline utilizers decreased, upon replacement of the transgenic plants, to a level similar to that of normal plants, while the concentration of mannopine utilizers decreased to levels intermediate between transgenic and normal plants. These data indicate that: (i) the opine-utilizing bacterial populations can be controlled in the rhizosphere via plant-exudate engineering; (ii) the interaction between the engineered plants and their root-associated micro-organisms is transgene specific; and (iii) alterations induced by the cultivation of transgenic plants may sometimes be persistent. Furthermore, opine-utilizing bacterial populations can be controlled by crop rotation. Therefore, favouring the growth of a rhizobacterium of agronomic interest via an opine-based strategy appears feasible.     

Enumeration of iron-oxidizing bacteria by the membrane filter technique

This work presents a simple methodology to enumerate ferrous-iron-oxidizing bacteria in solution, easily applicable in bioleaching industrial plants, because it does not require expertise or specific equipment. The enumeration is based on bacterial concentration by microfiltration through a membrane filter. The filter containing the bacteria is placed on an agarose plate containing ferrous sulphate for bacterial growth. No difference was observed for the enumeration of Acidithiobacillus ferrooxidans ATCC 19859 when either 0.1 or 0.22 m pore size membrane filters were used. However, when the technique was applied to bacteria present in pregnant leaching solution, the smaller bacteria present in these solutions passed through the 0.22 m pore size membrane. Therefore the number of bacteria could be underestimated if they are monitored and filtered using a filter with pore size greater than 0.1 m. The limit of detection of this technique was one ferrous-iron-oxidizing bacterium in the filtered solutions.     

Effects of soil compaction on the microbial populations of melon and maize rhizoplane

Effects of soil compaction on the microbial populations of melon and maize rhizoplane were investigated in quantity and quality. The numbers of culturable bacteria and fluorescent pseudomonads on the rhizoplane were higher when plants were grown in more compacted soil and the relative increase was larger in fluorescent pseudomonads. Total bacterial counts, however, did not appear to be affected by soil compaction, resulting in the increase in the culturable bacteria among total counts in more compacted soil. The determination of extracellular enzymatic properties (pectinase, -glucosidase, -glucosidase and -galactosidase) of each 100 isolates from bulk soil and root samples suggested that the microbial populations on the rhizoplane, especially when plants were grown in highly, compacted soil, were composed of high ratios of bacteria with abilities to utilize root exudates efficiently. The microbial community structure estimated from the colony forming curves of bulk soil and root samples suggested that the microbial populations on the rhizoplane, especially when plants were grown in compacted soil, were likely to be composed of more r-strategists which were defined as those who formed colonies within 2 days.     

The population biology of bacterial viruses: why be temperate

A model of the interactions between populations of temperate and virulent bacteriophage with sensitive, lysogenic, and resistant bacteria is presented. In the analysis of the properties of this model, particular consideration is given to the conditions under which temperate bacteriophage can become established and will be maintained in bacterial populations. The effects of the presence of resistant bacteria and virulent phage on these "existence" conditions for temperate viruses are considered. It is demonstrated that under broad conditions temperate phage will be maintained in bacterial populations and will coexist with virulent phage. Extrapolating from this formal consideration of the population biology of temperate bacteriophage, a number of hypotheses for the conditions under which temperate, rather than virulent, modes of phage reproduction are to be anticipated and the nature of the selective pressures leading to the evolution and persistence of this "benign" type of bacterial virus are reviewed and critically evaluated. Two hypotheses for the "advantages of temperance" are championed: (1) As a consequence of the allelopathic effects of diffusing phage, in physically structured habitats, lysogenic colonies are able to sequester resources and, in that way, have an advantage when competing with sensitive nonlysogens. (2) Lysogeny is an adaptation for phage to maintain their populations in "hard times," when the host bacterial density oscillates below that necessary for phage to be maintained by lytic infection alone.     

Effects of streptomycin,cycloheximide, Fungizone,captan, carbofuran,cygon, and PCNB on soil microorganisms

Eight biocides were chosen to determine whether they had any effects on nontarget organisms in soil and to what extent they would reduce their target populations under laboratory experimental conditions. A simplified microcosm system was utilized in which reduced species arrays that included field populations of either only bacteria and fungi, or bacteria, fungi, and protozoa (no nematodes, arthropods, or plants) were inoculated into sterilized soil. In a second set of experiments, plants were grown in sterilized soil. A bactericide-streptomycin-four fungicides-cycloheximide, Fungizone (amphotericin B), captan, and PCNB (quintozene)-an acaricide-cygon-an insecticide-nematicide-carbofuran-and an insecticide-diazinon-were used. Each biocide had effects on nontarget organisms although the increases or decreases, with respect to the control, were of only limited duration. Reductions in target groups were typically of longer duration. Streptomycin, applied at 1 mg·g^–1 soil, did not decrease bacterial populations during the experimental incubation. At 3 mg·g^–1 soil, streptomycin decreased the numbers of bacteria that grew on tryptone agar, but also reduced active hyphae. Fungizone was the most effective of the 4 fungicides tested in reducing active hyphae. Increased bacterial populations were usually observed following fungal reductions. Carbofuran had the fewest effects on the test organisms (bacteria, fungi, and protozoa). Only an initial stimulation of bacterial and fungal populations was observed with cygon although it also increased NH₄ ⁺-N concentrations in soil during most of the incubation, as did streptomycin and cycloheximide. A transitory increase in fungal populations following a decrease in ciliate numbers was observed in the cygon with grazers treatments. Diazinon reduced all microbial populations and inorganic nitrogen concentrations measured. Cygon and PCNB decreased growth of blue grama plants, while streptomycin reduced shoot weights of blue grama. These results should be useful in assessing the effects of these biocides when applied to more complex systems.     

Effect of drought on three species of Rhizobium

Summary Three samples of soil were air dried and the reduction in population ofRhizobium meliloti, Rhizobium trifolii and a rhizobium of the ‘Lotus’ group was estimated by use of a plant-infection technique. The cells ofRhizobium trifolii proved to be more tolerant of the severe drought than did the cells of the other two species.     

Standardization of cage techniques to screen chickpeas for resistance to Helicoverpa armigera (Lepidoptera: Noctuidae) in greenhouse and field conditions

Because of variations in insect populations and staggered flowering of chickpea, Cicer arietinum L., genotypes, it is difficult to compare the genotypic performance across seasons and locations. We standardized a cage technique to screen chickpeas for resistance to Helicoverpa armigera (Hübner). Leaf feeding by the larvae was significantly lower on ICC 506 than on ICCC 37 when the seedlings were infested with 20 neonates per five plants at 15 d after seedling emergence or 10 neonates per three plants at the flowering stage. Maximum differences in pod damage were observed when the plants were infested with six third-instar larvae per three plants in the greenhouse, and with eight larvae per plant under field conditions. Larval weights were significantly lower on ICC 506 than on ICCC 37 across growth stages and infestation levels. At the podding stage, percentage of reduction in grain yield was significantly greater on ICCC 37 and Annigeri than on ICCV 2 and ICC 506. The no-choice test can be used to screen segregating breeding material and mapping populations for resistance to H. armigera. It also provides useful information on antibiosis mechanism of resistance to H. armigera.     

Expression of the entire polyhydroxybutyrate operon of <Emphasis Type="Italic">Ralstonia eutropha</Emphasis> in plants

Background

Previously we demonstrated that an entire bacterial operon (the PRN operon) is expressible in plants when driven by the Tomato -yellow-leaf-curl-virus (TYLCV) -derived universal vector IL-60.Petroleum-derived plastics are not degradable, and are therefore harmful to the environment. Fermentation of bacteria carrying operons for polyhydroxyalkanoates (PHAs) produces degradable bioplastics which are environmentally friendly. However, bacterial production of bioplastics is not cost-effective, and attention is turning to their production in plants. Such “green” plastics would be less expensive and environmentally friendly. Hence, attempts are being made to substitute petroleum-derived plastics with “green” plastics. However, transformation of plants with genes of operons producing bioplastics has deleterious effects. Transformation of plastids does not cause deleterious effects, however it is a complicated procedures.

Results

We have developed another TYLCV-based vector (SE100) and show that yet another bacterial operon (the phaCAB operon) when driven by SE100 is also expressed in plants. We employed the combination of SE100 and the phaCAB operon to drive the operon to the plastids and produce in plants a biodegradable plastic [polyhydroxybutyrate (PHB)].Here we indicate that the bacterial operon (phaCAB), when driven by the newly developed universal plant vector SE100 is directed to chloroplasts and produces in plants PHB, a leading PHA. The PHB-producing plants circumvent the need for complicated technical procedures.

Conclusion

The viral vector system SE100 facilitated the production of the bio-plastic poly-3-hydroxybutyrate. This was achieved by using the full pha-CAB operon indicating that TYLCV based system can transcribe and translate genes from bacterial operons controlled by a single cis element. Our data hints to the participation of the chloroplasts in these processes.

     

Effects of two different application methods of Burkholderia ambifaria MCI 7 on plant growth and rhizospheric bacterial diversity

In order to acquire a better understanding of the effects of the different delivery modes of bacterial inoculants on plant growth and on the community structure of rhizosphere bacterial populations, Burkholderia ambifaria MCI 7 (formerly B. cepacia MCI 7) was inoculated into the rhizosphere of maize plants by either seed adhesion or incorporation into soil. Plant growth was evaluated at different inoculum concentrations. The community structure of rhizosphere bacterial populations was evaluated by analysing the restriction patterns of the DNA coding for 16S rRNA amplified by polymerase chain reaction (PCR) (ARDRA) of 745 bacterial isolates. A number of diversity indices (richness, Shannon diversity, evenness and mean genetic distance) were calculated for each bacterial population isolated from control and treated plants according to the concept of the r/K strategy. Moreover, the analysis of molecular variance (AMOVA) method was applied to estimate the genetic differences among the various bacterial populations. Our results showed that the method of application can be an essential element in determining the effects of the inoculant on plant growth. In fact, when applied as a maize seed treatment, B. ambifaria MCI 7 promoted plant growth significantly; on the contrary, when incorporated into soil, the same strain reduced plant growth markedly. As far as the bacterial community structure is concerned, B. ambifaria MCI 7 affected the indigenous microflora of treated plants according to the application method: seed treatment brought about an abrupt decrease in bacterial diversity, whereas incorporation into soil increased bacterial diversity. Moreover, changes in bacterial diversity were limited to r-strategist bacteria. In conclusion, B. ambifaria MCI 7 can act as both a plant growth-promoting rhizobacterium and a deleterious rhizobacterium depending on the inoculation method.     

Interactive effects of herbivory and competition intensity determine invasive plant performance

Herbivory can reduce plant fitness, and its effects can be increased by competition. Though numerous studies have examined the joint effects of herbivores and competitors on plant performance, these interactive effects are seldom considered in the context of plant invasions. Here, we examined variation in plant performance within a competitive environment in response to both specialist and generalist herbivores using Chinese tallow as a model species. We combined tallow plants from native and invasive populations to form all possible pairwise combinations, and designated invasive populations as stronger neighbours and native populations as weaker neighbours. We found that when no herbivory was imposed, invasive populations always had higher total biomass than natives, regardless of their neighbours, which is consistent with our assumption of increased competitive ability. Defoliation by either generalist or specialist herbivores suppressed plant growth but the effects of specialists were generally stronger for invasive populations. Invasive populations had their lowest biomass when fed upon by specialists while simultaneously competing with stronger neighbours. The root/shoot ratios of invasive populations were lower than those of native populations under almost all conditions, and invasive plants were taller than native plants overall, especially when herbivores were present, suggesting that invasive populations may adopt an "aboveground first" strategy to cope with herbivory and competition. These results suggest that release from herbivores, especially specialists, improves an invader's performance and helps to increase its competitive ability. Therefore, increasing interspecific competition intensity by planting a stronger neighbour while simultaneously releasing a specialist herbivore may be an especially effective method of managing invasive plants.     

Bacterial diversity in three distinct sub-habitats within the pitchers of the northern pitcher plant, Sarracenia purpurea

Pitcher plants have been widely used in ecological studies of food webs; however, their bacterial communities are poorly characterized. Pitchers of Sarracenia purpurea contain several distinct sub-habitats, namely the bottom sediment, the liquid, and the internal pitcher wall. We hypothesized that those three sub-habitats within pitcher plants are inhabited by distinct bacterial populations. We used denaturing gradient gel electrophoresis and 16S rRNA gene sequencing to characterize bacterial populations in pitchers from three bogs. DGGE and sequencing revealed that in any given pitcher, the three sub-habitats contain significantly different bacterial populations. However, there was significant variability between bacterial populations inhabiting the same type of habitat in different pitchers, even at the same site. Therefore, no consistent set of bacterial populations was enriched in any of the three sub-habitats. All sub-habitats appeared to be dominated by alpha- and betaproteobacteria in differing proportions. In addition, sequences from the Bacteroidetes and Firmicutes were obtained from all three sub-habitats. We conclude that container aquatic habitats such as the pitchers of S.?purpurea possess a very high bacterial diversity, with many unique bacterial populations enriched in individual pitchers. Within an individual pitcher, populations of certain bacterial families may be enriched in one of the three studied sub-habitats.     

Performance of Thirteen Clinical Rules to Distinguish Bacterial and Presumed Viral Meningitis in Vietnamese Children

Background and Purpose

Successful outcomes from bacterial meningitis require rapid antibiotic treatment; however, unnecessary treatment of viral meningitis may lead to increased toxicities and expense. Thus, improved diagnostics are required to maximize treatment and minimize side effects and cost. Thirteen clinical decision rules have been reported to identify bacterial from viral meningitis. However, few rules have been tested and compared in a single study, while several rules are yet to be tested by independent researchers or in pediatric populations. Thus, simultaneous test and comparison of these rules are required to enable clinicians to select an optimal diagnostic rule for bacterial meningitis in settings and populations similar to ours.

Methods

A retrospective cross-sectional study was conducted at the Infectious Department of Pediatric Hospital Number 1, Ho Chi Minh City, Vietnam. The performance of the clinical rules was evaluated by area under a receiver operating characteristic curve (ROC-AUC) using the method of DeLong and McNemar test for specificity comparison.

Results

Our study included 129 patients, of whom 80 had bacterial meningitis and 49 had presumed viral meningitis. Spanos''s rule had the highest AUC at 0.938 but was not significantly greater than other rules. No rule provided 100% sensitivity with a specificity higher than 50%. Based on our calculation of theoretical sensitivity and specificity, we suggest that a perfect rule requires at least four independent variables that posses both sensitivity and specificity higher than 85–90%.

Conclusions

No clinical decision rules provided an acceptable specificity (>50%) with 100% sensitivity when applying our data set in children. More studies in Vietnam and developing countries are required to develop and/or validate clinical rules and more very good biomarkers are required to develop such a perfect rule.     

Expression of a Thermostable Bacterial Cellulase in Transgenic Tobacco Plants

The bacterial gene of the thermostable endo--1,4-glucanase (cellulase) was shown to retain its activity and substrate specificity when expressed in transgenic tobacco plants. The leader peptide of the carrot extensin was efficient in transferring the bacterial enzyme into the apoplast. The expression of the bacterial cellulase gene leads to changes in the plant tissue morphology. In the transgenic plant lines, regeneration of primary shoots from callus occurred at the three to five times higher cytokinin (6-BAP) concentration than in control plants. The transgenic plants that expressed the bacterial gene exhibited increased bushiness and altered leaf shape. The transgenic plants developed can be used as models for studying the cellulases role and function in plants.     

Effect of aflatoxin-contaminated groundnut cake on rhizosphere microflora of some vegetable plants

Plant and Soil - Amendment of groundut cake stimulated bacterial and fungal populations in rhizospheres of Rumex, okra, and clusterbean. Maximum rhizosphere effect was noticed when the plants were...     

Microfluidic devices for studying bacterial taxis,drug testing and biofilm formation

Some bacteria have coevolved to establish symbiotic or pathogenic relationships with plants, animals or humans. With human association, the bacteria can cause a variety of diseases. Thus, understanding bacterial phenotypes at the single-cell level is essential to develop beneficial applications. Traditional microbiological techniques have provided great knowledge about these organisms; however, they have also shown limitations, such as difficulties in culturing some bacteria, the heterogeneity of bacterial populations or difficulties in recreating some physical or biological conditions. Microfluidics is an emerging technique that complements current biological assays. Since microfluidics works with micrometric volumes, it allows fine-tuning control of the test conditions. Moreover, it allows the recruitment of three-dimensional (3D) conditions, in which several processes can be integrated and gradients can be generated, thus imitating physiological 3D environments. Here, we review some key microfluidic-based studies describing the effects of different microenvironmental conditions on bacterial response, biofilm formation and antimicrobial resistance. For this aim, we present different studies classified into six groups according to the design of the microfluidic device: (i) linear channels, (ii) mixing channels, (iii) multiple floors, (iv) porous devices, (v) topographic devices and (vi) droplet microfluidics. Hence, we highlight the potential and possibilities of using microfluidic-based technology to study bacterial phenotypes in comparison with traditional methodologies.