Responses by iron-efficient and inefficient oat cultivars to inoculation with siderophore-producing bacteria in a calcareous soil

Rhizosphere bacteria may enhance plant uptake of Fe by producing siderophores that chelate sparingly soluble Fe³⁺ in calcareous soils. To evaluate the extent to which plants benefit from colonization of the roots by prolific siderophore-producing bacteria, we inoculated two oat cultivars with six strains of bacteria that produced high concentrations of siderophores under Felimiting conditions in vitro. Oat cv Coker 227, an Fe-efficient cultivar, which produces the phytosiderophore avenic acid, and cv TAM 0-312, and Fe-inefficient cultivar, which does not produce the phytosiderophore, were grown in a calcareous soil (Weswood silt loam) on a light bench in the laboratory. Half of the plants were fertilized with a nutrient solution containing 5 mM Fe and half with a nutrient solution containing no Fe. After 6 weeks of growth, we compared colonization of the roots by the inoculant bacteria and the dry weight and Fe content of roots and shoots. Three species of Pseudomonas colonized the roots of both oat cultivars in high numbers (10⁶ cells g^-1 root dry weight), whereas the remaining bacteria colonized the roots in substantially lower numbers (10⁴ cells g^-1 root dry weight). Plants fertilized with 5 mM Fe were larger and supported greater numbers or rhizosphere bacteria per gram of root than plants not supplied with Fe. Comparisons of the Fe content and dry weight of roots and shoots revealed few significant differences between inoculated and uninoculated plants, or among the plants inoculated with the different strains of siderophore-producing bacteria. The differences that were observed revealed no consistent response to inoculation. We conclude that inoculation of the roots of the two oat cultivars with bacteria that produce high concentrations of siderophores in response to an Fe deficiency had little or no effect on Fe acquisition by the plants.     

玉米根际细菌中PGPR的筛选及初步鉴定

通过离体拮抗试验和检测菌株产生铁载体的情况从玉米根际细菌中筛选出 8株拮抗菌和2 6株铁载体产生菌 ,其中 DC0 1、DC0 9、DC2 2、PY313既具有真菌抗性又能产生铁载体 ,并对部分菌株进行了初步鉴定     

Characterization of functional traits of two fluorescent pseudomonads isolated from basidiomes of ectomycorrhizal fungi

Some functional traits of Pseudomonas fluorescens 92 and BBc6, two strains isolated, respectively, from the basidiome of the ectomycorrhizal fungi Suillus grevillei and Laccaria laccata, were evaluated. A rifampicin-resistant mutant of P. fluorescens 92 (P. fluorescens 92R1) showed a significant in vivo plant growth promotion effect on cucumber plants. Quantitative analysis of enzymatic and physiological activities on different substrates showed that P. fluorescens 92 produced about a three times higher level of avicelase than BBc6, while equivalent amounts of β-glucosidase were produced by both strains. Satisfactory levels of neutral phosphomonoesterase and medium levels of acid phosphomonoesterase were produced by both. Only P. fluorescens BBc6 produced a very low amount of phosphodiesterase. Both strains produced high amounts of IAA and siderophores. Both strains showed on an iron deficient medium a very high antagonistic activity against the phytopathogenic fungus Heterobasidion annosum. Purification of fluorescent siderophores by copper-chelate chromatography showed that P. fluorescens 92 produced one pyoverdin (Pf92) and BBc6 two pyoverdins (PfBI and PfBII). A good inhibitory activity against mycelial growth of H. annosum was also observed when using the pyoverdines purified by affinity chromatography. Further purification by reverse phase high pressure liquid chromatography produced multiple fractionation of the three pyoverdins. Analysis of the reverse-phase purified pyoverdines by electronspray ionization mass spectrometry gave for pyoverdin Pf92 the mass value of 1213.8 and for both PfBI and PfBII the mass value of 1305.7. The presence of iron-chelating forms and sodium adducts were also evidenced.     

Screening of bacterial isolates from various European soils for in vitro antagonistic activity towards Rhizoctonia solani and Fusarium oxysporum: Site-dependent composition and diversity revealed

A cultivation-based approach was used to determine the in vitro antagonistic potential of soil bacteria towards Rhizoctonia solani AG3 and Fusarium oxysporum f. sp. lini (Foln3). Four composite soil samples were collected from four agricultural sites with previous documentation of disease suppression, located in France (FR), the Netherlands (NL), Sweden (SE) and the United Kingdom (UK). Similarly, two sites from Germany (Berlin, G-BR; and Braunschweig, G-BS) without documentation of disease suppression were sampled. Total bacterial counts were determined by plating serial dilutions from the composite soil samples onto R2A, AGS and King's B media. A total of 1,788 isolates (approximately 100 isolates per medium and site) was screened for antifungal activity, and in vitro antagonists (327 isolates) were found amongst the dominant culturable bacteria isolated from all six soils. The overall proportion of antagonists and the number of isolates with inhibitory activity against F. oxysporum were highest in three of the suppressive soils (FR, NL and SE). Characterization of antagonistic bacteria revealed a high phenotypic and genotypic diversity. Siderophore and protease activity were the most prominent phenotypic traits amongst the antagonists. The composition and diversity of antagonists in each soil was site-specific. Nevertheless, none of the antimicrobial traits of bacteria potentially contributing to soil suppressiveness analyzed in this study could be regarded as specific to a given site.     

Phenotypic structure of Pseudomonas populations is altered under elevated pCO2 in the rhizosphere of perennial grasses

The increasing atmospheric CO₂ content (pCO₂) is likely to modify the ecosystem functioning including rhizosphere bacteria that are directly dependent on rhizodeposition. This may include alteration of Pseudomonas populations that display phenotypic traits in relation with plant fitness. In the present study, 1228 Pseudomonas strains were isolated from the non-rhizosphere soil, rhizosphere soil and root fractions of perennial grassland systems: Lolium perenne and Molinia coerulea. Both plants were grown under ambient (36 Pa) and elevated (60 Pa) pCO₂ in the Swiss Free Air CO₂ Enrichment (FACE) system. Pseudomonas spp. were tested for their ability to produce auxin, siderophores and hydrogen cyanide, and to dissimilate nitrate. No effect of root proximity and elevated pCO₂ was observed on the proportions of auxin producers. For L. perenne and M. coerulea, siderophore and hydrogen cyanide Pseudomonas producers were stimulated in the root fraction. In contrast lower frequencies of nitrate reducers were observed in the root fraction compared to non-rhizosphere soil. The frequencies of siderophore producers and nitrate dissimilating strains were higher, and those of hydrogen cyanide producers lower, under elevated pCO₂ for L. perenne. This alteration of the phenotypic structure of Pseudomonas guild in the root fraction is discussed in relation with the physico-biochemical modifications of the rhizosphere condition via rhizodeposition and environmental changes occurring under elevated pCO₂.     

Simple microplate assays to measure iron mobilization and oxalate secretion by ectomycorrhizal tree roots

The ability of ectomycorrhizal (ECM) fungi to mobilize soil inorganic nutrients has been well documented for a diversity of species. However, most of these studies were performed with tree seedlings or fungal cultures, making them hardly comparable. We propose here three microplate assays to compare iron chelation, free iron uptake and oxalate production by ECMs freshly sampled in mature forest ecosystems. These assays proved to be sensitive enough to detect significant differences between two common ECM types. Lactarius subdulcis was less efficient than Xerocomus sp. for accessing to free or complexed iron, but produced 100 times more oxalate. These preliminary results open the way to study the contribution of ECM communities to nutrient cycling in forest ecosystems.     

Use of chrome azurol S reagents to evaluate siderophore production by rhizosphere bacteria

Summary Siderophores produced by rhizosphere bacteria may enhance plant growth by increasing the availability of Fe near the root or by inhibiting the colonization of roots by plant pathogens or other harmful bacteria. To examine the populations of siderophore-producing bacteria colonizing the roots of two grass species that differed in their susceptibility to Fe deficiency, we inoculated serial dilutions of root samples onto chrome azurol S (CAS) agar and several other selective and non-selective culture meida. CAS agar effectively differentiated bacteria that were capable of excreting large amounts of siderophore, but the composition of the medium limited its usefulness for ecological studies. A large proportion (71–79%) of the bacterial population that grew on a non-selective medium (tryptic soy agar) failed to grow on CAS agar, and several isolates that showed no sign of siderophore production on CAS agar produced siderophore in liquid culture. Similar populations of siderophore-producing bacteria were observed on roots of St. Augustine grass, which frequently exhibits Fe chlorosis, and bermuda grass, which does not. Roots of both grasses were colonized by bacteria that produced siderophore in vitro at concentrations ranging from 100 to 230 M. The CAS assay solution was also used to compare siderophore production by Pseudomonas fluorescens Q6, an isolate from bermuda grass, and by P. putida B 10, a plant growth-promoting pseudomonad. P. fluorescens Q6 produced 2.4 times more siderophore in vitro than P. putida B 10.     

Identification of rice blast disease-suppressing bacterial strains from the rhizosphere of rice grown in Pakistan

Sixteen bacterial strains isolated from the roots and rhizosphere of rice plants growing in saline and non-saline soils from the Shorkot area of Pakistan were tested for their ability to promote plant growth and reduce the incidence of rice blast disease. When applied to the soil, many of the isolated rhizobacterial strains increased seedling growth and/or suppressed rice blast disease in greenhouse-grown plants of the cultivars Super Basmati and Azucena, but each cultivar responded to different subsets of the bacteria. In the cv Super Basmati, increased blast resistance was correlated with the production of siderophores by the rhizobacteria. Several strains inhibited the growth of the causative agent of rice blast disease, the fungal pathogen Magnaporthe grisea, in an in vitro dual culture assay. Direct bioantagonism was correlated with disease resistance in Super Basmati, but not in Azucena, and direct antagonism as a cause for the reduced disease incidence is also unlikely since no epiphytic colonisation of leaves was detected. Rhizosphere colonisation by the bacteria in plants grown in sterile sand was correlated with disease resistance in Super Basmati, but not in Azucena. As well as the differences in strains that protected each cv against blast disease, we also found that there were differences in the ability of some strains to protect plants against blast depending on soil type. Hence, there are complex interactions between rhizobacteria and rice plants with respect to biocontrol of rice blast disease, dependent upon both rice cv and soil type. The identity of strains that promoted high levels of disease protection, including three that performed well across all plant cultivars and growth conditions, was determined by 16S rRNA gene sequencing.     

Effect of the expression of Escherichia coli fhuA gene in Rhizobium sp. IC3123 and ST1 in planta: Its role in increased nodule occupancy and function in pigeon pea

The inability to utilize a fungal siderophore as source of iron nutrition by most of the rhizobial cultures isolated from pigeon pea, could be considered a negative fitness factor since hydroxamate siderophores are found in significant amounts in natural soils. Thus these cultures were engineered to use ferrichrome a prototype of hydroxamate type siderophore. Pigeon pea Rhizobium spp. IC3123 and ST1 harboring Escherichia coli fhuA gene, responsible for uptake of Fe³⁺-ferrichrome, were obtained by transformation with pGR1, a broad host range plasmid carrying the fhuA gene under the control of the lac promoter of E. coli. Expression of fhuA in transformed rhizobial strains IC3123::pGR1 and ST1::pGR1 was confirmed by the ability of the plasmid-bearing strains to utilize iron bound to ferrichrome. Inoculation of pigeon pea plants with fhuA expressing rhizobial strains in pot experiments showed a significant increase in plant growth as well as nodule density as compared to those inoculated with the parent as well as the empty vector-bearing strain. Inoculation of pigeon pea seedlings with IC3123::pGR1 and ST1::pGR1 led to marked increase in shoot fresh weight, nodule number per plant, chlorophyll content of leaves and effective nodule symbiosis when compared with plants inoculated with the parent strains IC3123 and ST1. The positive effect of IC3123::pGR1 and ST1::pGR1 treatment on plant growth was more significantly observed when ferrichrome producing Ustilago maydis, known to secrete ferrichrome, was co-inoculated along with the transformed rhizobia. The presence of fhuA gene in rhizobial strains also led to an increased survival and root colonization.     

A commercially available iron-chelating agent, Desferal, promotes Fe- and Al-mobilization in soils

The effects of deferoxamine mesylate (DFAM), a bacterial siderophore, on the availability of iron and aluminium in three different soils were investigated. The distinct ability of DFAM to mobilize both metals in soil solution indicate a possible importance of siderophores in connection with toxicological aspects as well as for weathering and soil forming processes.