Processes of plant colonization by Methylobacterium strains and some bacterial properties

The pink-pigmented facultative methylotrophic bacteria (PPFMB) of the genus Methylobacterium are indespensible inhabitants of the plant phyllosphere. Using maize Zea mays as a model, the ways of plant colonization by PPFMB and some properties of the latter that might be beneficial to plants were studied. A marked strain, Methylobacterium mesophilicum APR-8 (pULB113), was generated to facilitate the detection of the methylotrophic bacteria inoculated into the soil or applied to the maize leaves. Colonization of maize leaves by M. mesophilicum APR-8 (pULB113) occurred only after the bacteria were applied onto the leaf surface. In this case, the number of PPFMB cells on inoculated leaves increased with plant growth. During seed germination, no colonization of maize leaves with M. mesophilicum cells occurred immediately from the soil inoculated with the marked strain. Thus, under natural conditions, colonization of plant leaves with PPFMB seems to occur via soil particle transfer to the leaves by air. PPFMB monocultures were not antagonistic to phytopathogenic bacteria. However, mixed cultures of epiphytic bacteria containing Methylobacterium mesophilicum or M. extorquens did exhibit an antagonistic effect against the phytopathogenic bacteria studied (Xanthomonas camprestris, Pseudomonas syringae, Erwinia carotovora, Clavibacter michiganense, and Agrobacterium tumifaciens). Neither epiphytic and soil strains of Methylobacterium extorquens, M. organophillum, M. mesophilicum, and M. fujisawaense catalyzed ice nucleation. Hence, they cause no frost injury to plants. Thus, the results indicate that the strains of the genus Methylobacterium can protect plants against adverse environmental factors.     

Root colonization and iron nutritional status of a Pseudomonas fluorescens in different plant species

Root colonization and induction of an iron stress regulated promoter for siderophore production by Pseudomonas fluorescens 2-79RLI was studied in vitro and in the rhizosphere of different plant species. P. fluorescens 2-79RLI was previously genetically modified with an iron regulated ice nucleation reporter, which allowed calibration of ice nucleation activity with siderophore production. Initial experiments examined ice nucleation activity and siderophore production under different growth conditions in vitro. These studies demonstrated that P. fluorescens 2-79RLI could utilize both Fe-citrate and Fe-phytosiderophore as iron sources, suggesting that production of these compounds by plants would increase iron availability for P. fluorescens 2-79RLI in the rhizosphere. Fe demand and Fe stress were further shown to be a function of nutrient availability and were reduced when carbon was limiting for growth. Subsequent experiments extended these observations to rhizosphere cells. Cells were sampled from the rhizosphere and the rhizoplane. Results of a soil microcosm experiment showed that Fe stress was reduced for P. fluorescens 2-79RLI in the barley rhizosphere as compared to the cells in the rhizosphere.of lupin. In lupin, relative Fe stress of P. fluorescens 2-79RLI was greater at the root tip than in the lateral root zone. In a second experiment comparing zucchini and bean, iron stress was greater for P. fluorescens 2-79RLI associated with zucchini than with bean. In a third experiment with rape plants under P deficient conditions, addition of soluble P was shown to increase Fe stress for P. fluorescens 2-79RLI located at the root tip, but not in the lateral root zone. This study showed that Fe stress of P. fluorescens 2-79RLI in the rhizosphere may be influenced by plant species, P source, root zone and localization of the cells within the rhizosphere.     

Different, overlapping mechanisms for colonization of abiotic and plant surfaces by Pseudomonas putida

Mechanisms governing biofilm formation have generated considerable interest in recent years, yet comparative analyses of processes for bacterial establishment on abiotic and biotic surfaces are still limited. In this report we have expanded previous information on the genetic determinants required for colonization of plant surfaces by Pseudomonas putida populations and analyzed their correlation with biofilm formation processes on abiotic surfaces. Insertional mutations affecting flagellar genes or the synthesis and transport of the large adhesin LapA lead to decreased adhesion to seeds and biofilm formation on abiotic surfaces. The latter also causes reduced fitness in the rhizosphere. Decreased seed adhesion and altered biofilm formation kinetics are observed in mutants affected in heme biosynthesis and a gene that might participate in oxidative stress responses, whereas a mutant in a gene involved in cytochrome oxidase assembly is affected in the bacterium-plant interaction but not in bacterial establishment on abiotic surfaces. Finally, a mutant altered in lipopolysaccharide biosynthesis is impaired in seed and root colonization but seems to initiate attachment to plastic faster than the wild type. This variety of phenotypes reflects the complexity of bacterial adaptation to sessile life, and the partial overlap between mechanisms leading to biofilm formation on abiotic and biotic surfaces.     

Rhizoplane colonization of pea seedlings by Rhizobium leguminosarum and a deleterious root colonizing Pseudomonas sp. and effects on plant growth

Some pseudomonads produce a toxin that specifically inhibits winter wheat (Triticum aestivum L.) root growth and the growth of several microorganisms. The toxin does not inhibit pea (Pisum sativum) root growth, but the organisms are aggressive root colonizers and their effect on Rhizobium leguminosarum growth, colonization, and nodulation of peas was not known. Peas were grown in Leonard jars in the greenhouse. Pea roots were inoculated with R. leguminosarum, a toxin-producing Pseudomonas sp., both, or neither (control). The Pseudomonas sp. colonized pea roots more rapidly and in greater number than R. leguminosarum after ten days. In the presence of the Pseudomonas sp., the R. leguminosarum population on the rhizoplane was less at ten days. When the roots were inoculated with both R. leguminosarum and Pseudomonas sp., the number of nodules were greater than when R. leguminosarum was inoculated alone, but nodule dry weight and pea shoot biomass were similar to plants inoculated with only R. leguminosarum. Although these results need confirmation with non-sterile soil and field studies, these preliminary results indicate that peas will not be affected by wheat root-inhibitory rhizobacteria.     

Quantification of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens strains in the plant rhizosphere by real-time PCR

A real-time PCR SYBR green assay was developed to quantify populations of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing (phlD+) strains of Pseudomonas fluorescens in soil and the rhizosphere. Primers were designed and PCR conditions were optimized to specifically amplify the phlD gene from four different genotypes of phlD+ P. fluorescens. Using purified genomic DNA and genomic DNA extracted from washes of wheat roots spiked with bacteria, standard curves relating the threshold cycles (C(T)s) and copies of the phlD gene were generated for P. fluorescens strains belonging to genotypes A (Pf-5), B (Q2-87), D (Q8r1-96 and FTAD1R34), and I (FTAD1R36). The detection limits of the optimized real-time PCR assay were 60 to 600 fg (8 to 80 CFU) for genomic DNA isolated from pure cultures of P. fluorescens and 600 fg to 6.0 pg (80 to 800 CFU, corresponding to log 4 to 5 phlD+ strain CFU/rhizosphere) for bacterial DNA extracted from plant root washes. The real-time PCR assay was utilized to quantify phlD+ pseudomonads in the wheat rhizosphere. Regression analysis of population densities detected by real-time PCR and by a previously described phlD-specific PCR-based dilution endpoint assay indicated a significant linear relationship (P = 0.0016, r2 = 0.2). Validation of real-time PCR assays with environmental samples was performed with two different soils and demonstrated the detection of more than one genotype in Quincy take-all decline soil. The greatest advantage of the developed real-time PCR is culture independence, which allows determination of population densities and the genotype composition of 2,4-DAPG producers directly from the plant rhizospheres and soil.     

Novel Chlamydiales strains isolated from a water treatment plant

Chlamydiae are obligate intracellular bacteria infecting free-living amoebae, vertebrates and some invertebrates. Novel members are regularly discovered, and there is accumulating evidence supporting a very important diversity of chlamydiae in the environment. In this study, we investigated the presence of chlamydiae in a drinking water treatment plant. Samples were used to inoculate Acanthamoeba monolayers ( Acanthamoeba co-culture), and to recover autochthonous amoebae onto non-nutritive agar. Chlamydiae were searched for by a pan-chlamydia 16S rRNA gene PCR from both Acanthamoeba co-cultures and autochthonous amoebae, and phylotypes determined by 16S rRNA gene sequencing. Autochthonous amoebae also were identified by 18S rRNA gene amplification and sequencing. From a total of 79 samples, we recovered eight chlamydial strains by Acanthamoeba co-culture, but only one of 28 amoebae harboured a chlamydia. Sequencing results and phylogenetic analysis showed our strains belonging to four distinct chlamydial lineages. Four strains, including the strain recovered within its natural host, belonged to the Parachlamydiaceae ; two closely related strains belonged to the Criblamydiaceae ; two distinct strains clustered with Rhabdochlamydia spp.; one strain clustered only with uncultured environmental clones. Our results confirmed the usefulness of amoeba co-culture to recover novel chlamydial strains from complex samples and demonstrated the huge diversity of chlamydiae in the environment, by identifying several new species including one representing the first strain of a new family.     

Soil and plant water relations in a crested wheatgrass pasture: response to spring grazing by cattle

Summary Few field studies have attempted to relate effects of actual livestock grazing on soil and plant water status. The present study was initiated to determine the effects of periodic defoliations by cattle during spring on soil moisture and plant water status in a crested wheatgrass (Agropyron cristatum (L.) Gaertn. and A. desertorum (Fisch. ex Link) Schult.) pasture in central Utah. Soil moisture in the top 130 cm of the soil profile was depleted more rapidly in ungrazed plots than in grazed plots during spring and early summer. Soil moisture depletion was more rapid in grazed plots in one paddock after 1 July due to differential regrowth, but there was no difference in soil water depletion between plots in another paddock during the same period. This difference in soil water depletion between paddocks was related to a difference in date of grazing. Although more water had been extracted from the 60 cm to 130 cm depths in ungrazed plots by late September, cumulative soil moisture depletion over the entire 193 cm profile was similar in grazed and ungrazed plots. Prior to 1 July, grazing had no effect on predawn leaf water potentials as estimated by a pressure chamber technique; however, after 1 July, predawn leaf water potentials were lower for ungrazed plants. Midday leaf water potentials were lower for grazed plants before 1 July, but did not differ between grazed and ungrazed plants after 1 July. A 4- to 8-day difference in date of defoliation did not affect either predawn or midday leaf water potentials. The observed differences in water use patterns during spring and early-summer may be important in influencing growth and competitive interactions in crested wheatgrass communities that are subject to grazing by domestic livestock.     

增强的UV—B辐射对春小麦植株化学成分、真菌定殖和分解的影响

在UV－B辐射下,叶可溶性糖含量显著降低,叶可溶性蛋白含量和粗纤维含量以及茎粗纤维含量显著增加,而根粗纤维含量没有显著变化,在生长期接受UV－B辐射的叶和茎上,赭绿青霉和黑曲霉的定殖率显著增加,康宁木霉和出芽短梗霉的定殖率明显降低,而土曲霉的定殖率未受明显影响,这些叶和茎经过60d和100d的分解,分解率均显著增加,叶分解率与粗纤维含量和可溶性蛋白含量呈显著正相关,而与可溶性糖含量呈显著负相关,茎分解率与粗纤维含量呈显著正相关,在增强的UV－B辐射下,春小麦植株化学成分的变化,真菌定殖率的改变,分解率的增加,可能会导致麦田生态系统营养周转加快,土壤库中营养贮量增加。     

Tomato seed and root exudate sugars: composition, utilization by Pseudomonas biocontrol strains and role in rhizosphere colonization

The role of tomato seed and root exudate sugars as nutrients for Pseudomonas biocontrol bacteria was studied. To this end, the major exudate sugars of tomato seeds, seedlings and roots were identified and quantified using high-performance liquid chromatographic (HPLC) analysis. Glucose, fructose and maltose were present in all studied growth stages of the plant, but the ratios of these sugars were strongly dependent on the developmental stage. In order to study the putative role of exudate sugar utilization in rhizosphere colonization, two approaches were adopted. First, after co-inoculation on germinated tomato seeds, the root-colonizing ability of the efficient root-colonizing P. fluorescens strain WCS365 in a gnotobiotic quartz sand-plant nutrient solution system was compared with that of other Pseudomonas biocontrol strains. No correlation was observed between the colonizing ability of a strain and its ability to use the major exudate sugars as the only carbon and energy source. Secondly, a Tn5lacZ mutant of P. fluorescens strain WCS365, strain PCL1083, was isolated, which is impaired in its ability to grow on simple sugars, including those found in exudate. The mutation appeared to reside in zwf, which encodes glucose-6-phosphate dehydrogenase. The mutant grows as well as the parental strain on other media, including tomato root exudate. After inoculation of germinated sterile tomato seeds, the mutant cells reached the same population levels at the root tip as the wild-type strain, both alone and in competition, indicating that the ability to use exudate sugars does not play a major role in tomato root colonization, despite the fact that sugars have often been reported to represent the major exudate carbon source. This conclusion is supported by the observation that the growth of mutant PCL1083 in vitro is inhibited by glucose, a major exudate sugar, at a concentration of 0.001%, which indicates that the glucose concentration in the tomato rhizosphere is very low.     

Evidence for a correlation between auxin production and host plant species among strains of Pseudomonas syringae subsp. savastanoi.

Auxin production by 131 strains of Pseudomonas syringae subsp. savastanoi was investigated with the aim of looking for correlations among this characteristic and the origin of the strains, the types of symptoms, and the host plant. Most of the P.syringae subsp. savastanoi strains, except those isolated from ash, produced auxin and harbored iaa genes. Among ash strains, which were pathogenic only on ash, only 2 out of 33 were found to produce auxin and to harbor iaa genes.     

Evidence for a correlation between auxin production and host plant species among strains of Pseudomonas syringae subsp. savastanoi.

Auxin production by 131 strains of Pseudomonas syringae subsp. savastanoi was investigated with the aim of looking for correlations among this characteristic and the origin of the strains, the types of symptoms, and the host plant. Most of the P.syringae subsp. savastanoi strains, except those isolated from ash, produced auxin and harbored iaa genes. Among ash strains, which were pathogenic only on ash, only 2 out of 33 were found to produce auxin and to harbor iaa genes.     

Diversity of bacteria growing in natural mineral water after bottling

Bacterial growth occurs in noncarbonated natural mineral waters a few days after filling and storage at room temperature, a phenomenon known for more than 40 years. Using the full-cycle rRNA approach, we monitored the development of the planktonic bacterial community in a noncarbonated natural mineral water after bottling. Seven 16S rRNA gene libraries, comprising 108 clones in total, were constructed from water samples taken at various days after bottling and from two different bottle sizes. Sequence analyses identified 11 operational taxonomic units (OTUs), all but one affiliated with the betaproteobacterial order Burkholderiales (6 OTUs) or the class Alphaproteobacteria (4 OTUs). Fluorescence in situ hybridization (FISH) was applied in combination with DAPI (4',6'-diamidino-2-phenylindole) staining, viability staining, and microscopic counting to quantitatively monitor changes in bacterial community composition. A growth curve similar to that of a bacterium grown in a batch culture was recorded. In contrast to the current perception that Gammaproteobacteria are the most important bacterial components of natural mineral water in bottles, Betaproteobacteria dominated the growing bacterial community and accounted for 80 to 98% of all bacteria detected by FISH in the late-exponential and stationary-growth phases. Using previously published and newly designed genus-specific probes, members of the betaproteobacterial genera Hydrogenophaga, Aquabacterium, and Polaromonas were found to constitute a significant proportion of the bacterial flora (21 to 86% of all bacteria detected by FISH). For the first time, key genera responsible for bacterial growth in a natural mineral water were identified by applying molecular cultivation-independent techniques.     

Effect of phenotypic plasticity on epiphytic survival and colonization by Pseudomonas syringae.

The bacterial epiphyte Pseudomonas syringae MF714R was cultured on agar or in broth or collected from colonized leaves; it was then inoculated onto greenhouse-grown bean plants incubated in a growth chamber at low relative humidity or in the field or onto field-grown bean plants. Cells cultured in liquid medium survived the least well after inoculation of leaf surfaces under all conditions. Cells cultured in solid medium exhibited the highest percent survival and desiccation tolerance in the growth chamber but generally survived less well in the field than did cells harvested from plants. Cells harvested from plants and inoculated onto plants in the field usually exhibited the highest percent survival, started to increase in population earlier, and reached a higher number than did cells cultured in vitro. Differences in field survival were apparently not attributable to differential UV tolerance. The observed effects of phenotypic plasticity on epiphytic survival and colonization should be considered in risk assessment studies, in studies of bacterial epidemiology, and in the use of microbial antagonists for biological pest control.     

Biocontrol activity and root colonization by Pseudomonas corrugata strains CCR04 and CCR80 against Phytophthora blight of pepper

Previously, we selected Pseudomonas corrugata strains CCR04 and CCR80 as rhizobacteria suppressive to Phytophthora blight of pepper caused by Phytophthora capsici. In this study, we investigated soil microbial activity in pepper plants root-drenched with strains CCR04 and CCR80 in relation to their biocontrol activity, root colonization by using bacterial population counts and scanning electron microscopy, biofilm formation and cell motility as well as cell sensitivity to hydrogen peroxide (H₂O₂). As a result, strains CCR04 and CCR80 more effectively suppressed disease expression in pepper plants through root colonization than did Paenibacillus polymyxa AC-1 (positive control), Escherichia coli DH5α (negative control) or MgSO₄ solution (untreated control). Strains CCR04 and CCR80 had efficient biofilm formation and cell motility (swimming and swarming activities) abilities and responded to certain tested compounds (amino acids, organic acids and sugars), which can be found in root exudates. Strains CCR04 and CCR80 and the positive control strain AC-1 were relatively insensitive to H₂O₂, a reactive oxidative species at concentration up to 20 mM, unlike the negative control strain DH5α. Taken together, these results suggest that P. corrugata CCR04 and CCR80 can effectively inhibit P. capsici infection of pepper plants through successful colonization of plant roots. This bacterial colonization may be facilitated by the biofilm formation ability and cell motility in addition to reduced sensitivity to H₂O₂ and probably the production of antimicrobial compounds. These findings highlight the potential of strains CCR04 and CCR80 as biocontrol agents for the management of Phytophthora blight of pepper.     

Pseudomonas cepacia colonization and infection in intensive care units.

Pseudomonas cepacia has become a prominent epidemic nosocomial pathogen over the past 15 years. Between December 1982 and September 1983 it was isolated from 29 patients in two intensive care units (ICUs) at one hospital. Twelve infections--five bacteremias, four pneumonias and three urinary tract infections--occurred. Most of the isolates (25/29) were from the respiratory tract, and most (23/29) had the same antibiogram as the only environmental isolate, which was cultured from a contaminated ventilator thermometer, a previously unrecognized source of nosocomial infection. The ventilator thermometers were calibrated in a bath whose water had not been changed for months and contained P. cepacia. Despite elimination of this reservoir, P. cepacia was eradicated from the ICUs only after intensive infection control efforts were instituted.     

Endophytic colonization of plant roots by nitrogen-fixing bacteria

Plant and Soil - Nitrogen-fixing bacteria are able to enter into roots from the rhizosphere, particularly at the base of emerging lateral roots, between epidermal cells and through root hairs. In...