Suppression of root-knot disease by Pseudomonas fluorescens CHA0 in tomato: importance of bacterial secondary metabolite, 2,4-diacetylpholoroglucinol

The antimicrobial metabolites 2,4-diacetylphloroglucinol (2,4-DAPG) and pyoluteorin contribute to the ability of Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soil-borne pathogens. P. fluorescens strain CHA0 and its derivatives CHA89 (antibiotics-deficient) and CHA0/pME3424 (antibiotics overproducing) were investigated as potential biocontrol agents against Meloidogyne javanica the root-knot nematode. Exposure of root-knot nematode to culture filtrates of P. fluorescens under in vitro conditions significantly reduced egg hatch and caused substantial mortality of M. javanica juveniles. Nutrient broth yeast extract (NBY) medium amended with 2% (w/v) glucose or 1 mM EDTA markedly repressed hatch inhibition activity of the strain CHA0 but not that of CHA0/pME3424 or CHA89. On the other hand, NBY medium amended with glucose significantly enhanced nematicidal activity of the strain CHA0/pME3424. Neither glucose nor EDTA had an influence on the nematicidal activity of the strains CHA0 and CHA89. Under in vitro conditions, antibiotic overproducing strain CHA0/pME3424 and CHA0 expressed phl‘-’lacZ reporter gene but strain CHA89 did not. Expression of the reporter gene reflects actual production of DAPG. In general, CHA0/pME3424 expressed reporter gene to a greater extent compared to its wild type counterpart CHA0. Regardless of the bacterial strains, reporter gene expression was markedly enhanced when NBY medium was amended with glucose but EDTA had no such effect. A positive correlation between the degree of juvenile mortality and extent of phl‘-’lacZ reporter gene expression was also observed in vitro. Strain CHA0 produced zones of 4-6 mm on MM medium containing gelatin while strain CHA0/pME3424 and CHA89 did not. When MM medium containing gelatin was amended with 2% glucose of 1 mM EDTA size of haloes produced by the strain CHA0 reduced to 2 mm. Under glasshouse conditions aqueous cell suspension of the strains CHA0 or CHA0/pME3424 at various inoculum levels (10⁷, 10⁸ or 10⁹ cfu ml⁻¹) significantly reduced root-knot development. CHA89 caused significant reduction in galling when applied at 10⁹ cfu ml⁻¹. To better understand the mechanism of nematode suppression, split root bioassay was performed. Split-root experiments, that guarantee a spatial separation of inducing agent and a challenging pathogen, showed that soil treatment of one half of the root system with cell suspension of CHA0 or CHA0/pME3424 resulted in a significant systemic induced resistance leading to reduction of M. javanica infection of tomato roots in the non-baterized nematode treated half. The results clearly suggest that the antibiotic 2,4-DAPG from P. fluorescens CHA0 act as the inducing agents of systemic resistance in tomato roots. Populations of CHA0 and its derivatives declined progressively by 10-fold between first and fourth harvests (0-21 days after inoculation). However, bacterial populations increased at final harvest (28 days after application).     

Biocontrol strain Pseudomonas fluorescens CHA0 and its genetically modified derivative with enhanced biocontrol capability exert comparable effects on the structure of a Sinorhizobium meliloti population in gnotobiotic systems

The impact of biocontrol strain Pseudomonas fluorescens CHA0 and of its genetically modified, antibiotic-overproducing derivative CHA0/pME3424 on a reconstructed population of the plant-beneficial Sinorhizobium meliloti bacteria was assessed in gnotobiotic systems. In sterile soil, the final density of the reconstructed S. meliloti population decreased by more than one order of magnitude in the presence of either of the Pseudomonas strains when compared to a control without addition of P. fluorescens. Moreover, there was a change in the proportion of each individual S. meliloti strain within the population. Plant tests also revealed changes in the nodulating S. meliloti population in the presence of strains CHA0 or CHA0/pME3424. In both treatments one S. meliloti strain, f43, was significantly reduced in its root nodule occupancy. Analysis of alfalfa yields showed a slight but statistically significant increase in shoot dry weight when strain CHA0 was added to the reconstructed S. meliloti population whereas no such effect was observed with CHA0/pME3424.     

Persistence of a biocontrol Pseudomonas inoculant as high populations of culturable and non-culturable cells in 200-cm-deep soil profiles

Little is known about the ecology of soil inoculants used for pathogen biocontrol, biofertilization and bioremediation under field conditions. We investigated the persistence and the physiological states of soil-inoculated Pseudomonas protegens (previously Pseudomonas fluorescens) CHA0 (10⁸ CFU g^?1 surface soil) in different soil microbial habitats in a planted ley (Medicago sativa L.) and an uncovered field plot. At 72 days, colony counts of the inoculant were low in surface soil (uncovered plot) and earthworm guts (ley plot), whereas soil above the plow pan (uncovered plot), and the rhizosphere and worm burrows present until 1.2 m depth (ley plot) were survival hot spots (10⁵–10⁶ CFU g^?1 soil). Interestingly, strain CHA0 was also detected in the subsoil of both plots, at 10²–10⁵ CFU g^?1 soil between 1.8 and 2 m depth. However, non-cultured CHA0 cells were also evidenced based on immunofluorescence microscopy. Kogure's direct viable counts of nutrient-responsive cells showed that many more CHA0 cells were in a viable but non-culturable (VBNC) or a non-responsive (dormant) state than in a culturable state, and the proportion of cells in those non-cultured states depended on soil microbial habitat. At the most, cells in a VBNC state amounted to 34% (above the plow pan) and those in a dormant state to 89% (in bulk soil between 0.6 and 2 m) of all CHA0 cells. The results indicate that field-released Pseudomonas inoculants may persist at high cell numbers, even in deeper soil layers, and display a combination of different physiological states whose prevalence fluctuates according to soil microbial habitats.     

Development of a real-time PCR method to quantify the PGPR strain Azospirillum lipoferum CRT1 on maize seedlings

Azospirillum lipoferum CRT1 is a promising phytostimulatory PGPR for maize, whose effect on the plant is cell density-dependent. A nested PCR method is available for detection of the strain but does not allow quantification. The objective was to develop a real-time PCR method for quantification of A. lipoferum CRT1 in the rhizosphere of maize seedlings. Primers were designed based on a strain-specific RFLP marker, and their specificity was verified under qualitative and quantitative PCR conditions based on successful CRT1 amplification and absence of cross-reaction with genomic DNA from various rhizosphere strains. Real-time PCR conditions were then optimized using DNA from inoculated or non-inoculated maize rhizosphere samples. The detection limit was 60 fg DNA (corresponding to 19 cells) with pure cultures and 4 × 10⁴ CFU equivalents g⁻¹ lyophilized sample consisting of mixture of rhizosphere soil and roots. Inoculant quantification was effective down to 10⁴ CFU equivalents g⁻¹. Assessment of CRT1 rhizosphere levels in a field trial was in accordance with estimates from semi-quantitative PCR targeting another locus. This real-time PCR method, which is now available for direct rhizosphere monitoring of A. lipoferum CRT1 in greenhouse and field experiments, could be used as a reference for developing quantification tools for other Azospirillum inoculants.     

Rhizosphere and endophytic bacteria for induction of systemic resistance of banana plantlets against bunchy top virus

Bunchy top is one of the most important viral diseases affecting banana production worldwide. Several approaches have been made to control or combat the bunchy top virus disease in banana, transmitted by the banana aphid, Pentalonia nigronervosa Coq. No tactic is able to completely protect the plants against this viral infection. To ameliorate this problem, an attempt was made to use different bacterial strains, which could be inoculated to the roots of micropropagated plantlets to avoid the post transplanting problems. Virus indexed micropropagated plantlets of banana cv. Virupakshi (AAB) were subjected to root colonization followed by foliar spraying with three bacterial strains viz., Pseudomonas fluorescens strains Pf1, CHA0 and Bacillus subtilis strain EPB22 along with an un-inoculated control during primary and secondary hardening stage in the nursery and at the time of transplanting, 3rd, 5th and 7th month after planting in the field. Microbe inoculated plantlets showed improved vegetative growth, physiological attributes, PR—proteins and phenol contents besides reducing banana bunchy top disease (BBTD) incidence in the field. These results indicate that, transplant mixes amended with beneficial bacterial strains can enhance growth of banana plants, besides reducing the damage caused by banana bunchy top virus.     

Survival and cell culturability of biocontrol Pseudomonas fluorescens CHA0 in the rhizosphere of cucumber grown in two soils of contrasting fertility status

 The effect of cucumber roots on survival patterns of the biocontrol soil inoculant Pseudomonas fluorescens CHA0-Rif was assessed for 22 days in two non-sterile soils, using a combination of total immunofluorescence cell counts, Kogure's direct viable counts and colony counts on plates containing rifampicin. In Eschikon soil (high fertility status for cucumber), CHA0-Rif persisted as culturable cells in bulk soil and in the rhizosphere, but colony counts were lower than viable counts and total cell counts inside root tissues. The occurrence of viable but non-culturable (VBNC) cells inside root tissues (5 log cells g^–1 root) was unlikely to have resulted from the hydrogen peroxide treatment used to disinfect the root surface, as hydrogen peroxide caused the death of CHA0-Rif cells in vitro. In Siglistorf soil (low fertility status for cucumber), the inoculant was found mostly as non-culturable cells. Colony counts and viable counts of CHA0-Rif were similar, both in bulk soil and inside root tissues, whereas in the rhizosphere viable counts exceeded colony counts at the last two samplings (giving about 7 log VBNC cells g^–1). In conclusion, soil type had a significant influence on the occurrence of VBNC cells of CHA0-Rif, although these cells were found in root-associated habitats (i.e. rhizosphere and root tissues) and not in bulk soil. Received: 12 November 1999     

Soil DNA extraction and assessment of the fate of Mycobacterium chlorophenolicum strain PCP-1 in different soils by 16S ribosomal RNA gene sequence based most-probable-number PCR and immunofluorescence

Since Mycobacterium chlorophenolicum strain PCP-1 is not detectable in soil by selective plating, a specific tracking method was based on the polymerase chain reaction (PCR) using soil DNA as a target. A direct extraction protocol based on bead beating was adapted and used to obtain PCR-amplifiable DNA from five different soils. In one soil, the disruption of cells of PCP-1, of Pseudomonas fluorescens R2f and of Paenibacillus azotofixans P3L5, as well as of the indigenous bacteria increased with increasing bead beating times. After 4.5 min, lysis efficiency was about 90% or more in all cases. Total DNA yields varied between soils, from 2 to 35 μg g^–1. The purification steps needed to obtain amplifiable DNA were different per soil. To detect target DNA specifically in bacterial cells, a new indirect extraction protocol was developed, which efficiently dislodged bacterial cells from the soil matrix, and produced amplifiable DNA with high yield. To detect strain PCP-1 in soil, 16S ribosomal gene-based PCR combined with oligonucleotide hybridization was applied using a most-probable-number (MPN) set-up, whereas immunofluorescence was used for calibration. Strain PCP-1 was detected shortly after introduction into three soils at about the inoculum levels, as evidenced by both approaches. Both the direct and indirect DNA extraction methods yielded similar MPN estimates. The dynamics of M. chlorophenolicum PCP-1 was estimated in two soils over 14 days via MPN-PCR/oligonucleotide probing. PCP-1 showed good survival in both soils, and results obtained by MPN-PCR with directly and indirectly extracted DNA were internally consistent. Immunofluorescence cell enumerations supported the gross stability of PCP-1 in these two as well as in two additional soils. Received: 8 February 1996     

Promotion of plant growth and in situ degradation of phenol by an engineered Pseudomonas fluorescens strain in different contaminated environments

We show that Pseudomonas fluorescens strain P13, a plant growth-promoting bacterium, enhanced the growth of corn in uncontaminated soil but not in contaminated soil, perhaps because of its inability to reduce phytotoxicity. Another bacterial strain, Pseudomonas aeruginosa strain SZH16, showed in situ phenol-degrading activity and contained a plasmid loaded with a gene encoding for catechol 2, 3-dioxygenase, an important enzyme in the degradation pathway of aromatic compounds. We implanted this biodegradation ability into strain P13, using horizontal gene transfer techniques using strain SZH16 as the donor and P13 as the recipient, to generate a phenol-degrading transconjugant which obtained the effective plasmid from strain SZH16. Introduction of the transconjugant P13 strain into an artificially phenol-spiked soil promoted the growth of corn and in situ phenol degradation, and the increase in plant biomass correlated with the decrease in soil phenol content. Furthermore, the transconjugant P13 strain was also found to stimulate corn growth and reduce phenol concentration in water containing phenol and in historically contaminated field soils, indicating that the transconjugant strain could promote plant growth in both contaminated and uncontaminated environments. The transconjugant P13 strain was more efficient than either strain P13 or SZH16, and shows how plant growth-promoting bacteria which show no, or only limited, ability to degrade organic pollutants may be modified. This technique is attractive for many environmental remediation and agronomic applications.     

Inoculation of the redox effector Pseudomonas fluorescens C7R12 strain affects soil redox status at the aggregate scale

In order to investigate the redox condition in soil aggregates, an approach using the inoculation of the soil bacteria Pseudomonas fluorescens C7R12, which affects soil redox, was developed. First, redox modifications were studied at the colony level in pure culture on solid synthetic medium. Then, the effect of strain inoculation on the redox status of soil aggregates was studied under controlled conditions with millimeter size aggregates from a Mollisol. A redox micro-electrode was employed to determine redox intensity (redox potential, E), which ranged from 240±70 to 110±25 mV at the surface of non-inoculated (control) and inoculated aggregates after 3 days. Differential pulse polarography (DPP) was employed to assess redox capacity (amount of electro-active compounds), which was higher (7.17 nA/mV) in inoculated aggregates than in the control samples (3.04 nA/mV). Similarities between redox potential depth profile for P. fluorescens colonies on plates and for some inoculated aggregate profiles suggested a patch colonisation of the surface of the aggregates by the redox effector strain. By comparison with non-inoculated aggregates, the influence of the inoculated strain on the redox status of the aggregates was observed to increase with the amount of some electroactive compounds with potential values from about −110 and +15-40 mV. Lower mean values and the limitation of the redox potential range fluctuation along the profiles from surface to 220 μm depth were also characteristics of the inoculated aggregates.     

Mixtures of plant disease suppressive bacteria enhance biological control of multiple tomato pathogens

Plant growth-promoting rhizobacteria (PGPR) strains CHA0 (Pseudomonas fluorescens), IE-6 S⁺ (Pseudomonas aeruginosa) and 569Sm^r (Bradyrhizobium japonicum) were tested singly and in combinations for biological control against multiple tomato pathogens (root-infecting fungi and root-knot nematodes). Strains CHA0 and IE-6S⁺ inhibited in vitro growth of 569Sm^r while IE-6S⁺ suppressed CHA0. The bacterial species not only inhibited the radial growth of three root-infecting fungi, Macrophomina phaseolina, Fusarium solani and Rhizoctonia solani (AG 8), but also caused substantial mortality of Meloidogyne javanica juveniles. Used as a soil drench the three bacteria not only suppressed root-infecting fungi and root-knot nematodes but also enhanced growth of tomato plants both under glasshouse and field conditions. The suppressive effect was generally more pronounced when the bacteria were employed together. Strain IE-6S⁺ exhibited better rhizosphere colonization than CHA0 and 569Sm^r. Populations of CHA0 in the rhizosphere declined when the bacterium was used with either IE-6S⁺ and/or 569Sm^r, while populations of IE-6S⁺ in the rhizosphere were enhanced when used in combination with CHA0 and/or 569Sm^r. IE-6S⁺ was the only bacterium that colonized inner root tissues of tomato plants. When using an iron chelator to create iron deficiency in the soil, the biocontrol efficacy of the bacteria against F. solani and R. solani was enhanced while against M. phaseolina and M. javanica this activity remained unchanged. Only strain 569Sm^r gave significant suppression of M. phaseolina in both iron-deficient and iron-sufficient soils.     

Mycorrhizal fungi increase biocontrol potential of Pseudomonas fluorescens

Wheat roots are susceptible to colonisation by soil-borne pathogens, such as Gaeumannomyces graminis var. tritici (Ggt), which causes the globally important disease take-all, and mutualistic arbuscular mycorrhizal fungi (AMF). Certain rhizosphere fluorescent Pseudomonas strains have received much attention as potential biocontrol agents given their ability to produce antibiotics, such as 2,4-diacetylphloroglucinol (DAPG), that confer a measure of plant protection. Here we show that Pseudomonas fluorescens only produced DAPG in the presence of soluble carbon from soil containing either Ggt or AMF, and production increased by two orders of magnitude in response to both AMF and Ggt. Encouragement of mycorrhizal colonisation may therefore offer a sustainable strategy for protection against take-all.     

Inoculation of wheat with Azospirillum brasilense and Pseudomonas fluorescens: Impact on the production and culturable rhizosphere microflora

Scientific evidence recognizes that the operation of a terrestrial ecosystem depends on soil microbial activity. Some Azospirillum strains produce plant growth regulators, increase the development of roots, and fix atmospheric nitrogen (N₂). Some Pseudomonas strains are capable of producing cytokinins and solubilizing organic phosphorus. A sustainability analysis requires a detailed knowledge of the interrelationships between the microorganisms added to the system and those present in the soil. This study examines the effect of three commercial inoculants Azospirillum brasilense Az1 and Az2 as well as Pseudomonas fluorescens Pf on biomass production, grain yield and rhizosphere colonization of wheat, combined with two levels of N-addition. Plate counts of rhizosphere soil showed that the inoculation and N-addition did not affect the number of P. fluorescens, whereas it significantly affected the number of Azospirillum. N-addition and inoculation did not change the communities of actinomycetes and bacteria but they changed the number of fungi at the rhizosphere of wheat plants. Community-level physiological profiles of carbon-source utilization of rhizosphere soil microbial communities were changed after inoculation with Az1, Az2 and Pf depending on the phenological stage of the crop. Although no significant responses were observed, in average, PGPB inoculation increased aerial biomass by 12%, root biomass by 40% and grain yield by 16%. These increases represent important earnings for the farmer and they may help to obtain a greater sustainability of the agroecosystems.     

Real-time polymerase chain reaction quantification of the transgenes for roundup ready corn and roundup ready soybean in soil samples

A method for quantification of recombinant DNA for Roundup Ready (RR) corn and RR soybean in soil samples is described. Soil DNA from experimental field samples was extracted using a soil DNA extraction kit with a modified protocol. For the detection and quantification of recombinant DNA of RR corn and RR soybean, a molecular beacon and two pairs of specific primers were designed to differentially target recombinant DNA in these two genetically modified crops. Soil DNA extracts were spiked with RR corn or RR soybean DNA, and recombinant DNA was quantified using real-time PCR with a molecular beacon. As few as one copy of RR corn genome or one copy of RR soybean genome was detected in the soil DNA extract.     

Survival of the biocontrol agents Coniothyrium minitans and Bacillus subtilis MBI 600 introduced into pasteurised, sterilised and non-sterile soils

The biocontrol agents Coniothyrium minitans and Bacillus subtilis MBI 600 were added separately to three soil types that had been either sterilised, pasteurised or left non-sterile. Applied as a conidial suspension of 1×10⁶ cfu g⁻¹ soil, C. minitans showed good survival in all sterilised, pasteurised and non-sterile soils, remaining at the numerical level at which it was applied for the duration of the 30 d experiment. Applied at a lower rate of 1×10³ cfu g⁻¹ soil, C. minitans proliferated in sterilised soil to numbers slightly over 1×10⁶ cfu g⁻¹ soil, whereas no increase was seen in pasteurised or non-sterile soils from this lower application rate. However, although C. minitans was not easily recovered on plates from non-sterile soil, it did survive at the lower numerical level in pasteurised soil, and was recoverable throughout the experiment at the rate at which it was applied. B. subtilis MBI 600 survived well following introduction as a cell suspension into sterilised soil at a rate of 1×10⁶ cfu g⁻¹ soil. Spores were formed rapidly and, after 14 d, the introduced microorganism survived in this form rather than as vegetative cells. However, in non-sterile soil, the introduced microorganism did not compete well and decreased in number, with spores being formed in low numbers. Survival of B. subtilis MBI 600 in pasteurised soil was variable, but resembled the survival seen in non-sterile soil more than that seen in sterilised soil. More B. subtilis MBI 600 spores were formed in pasteurised soil than in non-sterile soil, however, and may have been important for survival in pasteurised soil. In conclusion, this work has shown that the biocontrol agent C. minitans can survive well in soil irrespective of whether the soil has been pasteurised or not and shows good promise as a soil inoculant for control of Sclerotinia sclerotiorum. Although soil pasteurisation does improve establishment of B. subtilis MBI 600 compared to non-sterile soil, survival is relatively poor when applied as cells. The best survival of B. subtilis MBI 600 occurred as spores in sterilised soil, and spore applications to pasteurised soil in an integrated control strategy may allow sufficient establishment of the biocontrol agent to target pathogens causing damping-off.     

Impact of the biocontrol agent Trichoderma atroviride SC1 on soil microbial communities of a vineyard in northern Italy

The fungus Trichoderma atroviride SC1 is an experimental biocontrol agent (BCA) that is active against the fungus Armillaria mellea. Following the application of Trichoderma to the surface soil of a vineyard, we used a highly specific real-time PCR, previously validated for the analysis of soil microcosms, to monitor the populations of this fungus at different soil depths over several months. The quantification obtained using this molecular method was highly correlated with laboratory assays of colony-forming units. The native communities of bacteria and fungi in the soil were analyzed using automated ribosomal intergenic spacer analysis (ARISA), and transient changes were observed following the application of T. atroviride SC1 conidia. A principal component of variance analysis demonstrated that the introduction of T. atroviride SC1 had an effect on the soil microflora during the first two weeks following inoculation. However, at later dates, environmental conditions had a higher influence on the surveyed communities than the BCA application, as confirmed through the use of the Shannon index of biodiversity. Soil depth had a strong influence on the composition and biodiversity of fungal communities.     

Utilization of trehalose, benzoate, valerate, and seed and root exudates by genotypes of 2,4-diacetylphloroglucinol producing Pseudomonas fluorescens

Isolates of Pseudomonas fluorescens producing the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) are effective biocontrol agents against soilborne pathogens. A previous study showed that the superior (“premier”) root colonizer P. fluorescens Q8r1-96 (genotype D) utilized trehalose, benzoate and valerate as sole carbon sources but average colonizers Q2-87 (genotype B) and 1M1-96 (genotype L) did not. We tested the utilization of these three carbon sources by a collection of 55 2,4-DAPG-producing P. fluorescens strains from 17 genotypes and found no correlation between a strain's ability to utilize these carbon sources and superior rhizosphere competence on wheat and pea. Of the strains tested, 73%, 48% and 69% were able to utilize trehalose, benzoate and valerate as sole carbon sources, respectively. With some exceptions, we found a correlation between the utilization of these compounds and previous groupings of these strains by BOX-PCR; genotype D strains utilized all three compounds. Twenty-three strains grew efficiently on root and seed exudates from wheat and pea, with doubling times between 0.9 and 1.6 h generation⁻¹ and lag phases between 5 and 8 h, comparable to growth on glucose as a sole carbon source. Only 10 strains, including those with “premier” (Q8r1-96) and “average” (Q2-87) rhizosphere competence, showed slower growth in wheat root exudates, with lag phases between 16 and 22 h. Results were the same when soil was added to the culture medium. Growth of four strains in media containing glucose or wheat or pea seed exudates as a sole carbon source was not influenced by whether the bacterial cells used as inoculum were harvested from wheat seeds or broth culture. We conclude that the superior ability of some strains to colonize the roots of certain crops cannot be explained by the utilization of the carbon sources tested in our study.     

DNA- versus RNA-based denaturing gradient gel electrophoresis profiles of a bacterial community during replenishment after soil fumigation

We compared the responsiveness and sensitivity to soil fumigation of DNA- and RNA-based analyses of a bacterial community. We first established an improved RNA extraction method using DNA as an adsorption competitor, because it is extremely difficult to extract nucleic acids from clay-rich volcanic ash soil (Andisol), which adsorbs nucleic acids. This novel method facilitated RNA extraction from 500 mg of Andisol for molecular analyses. Then we monitored 16S rDNA PCR and 16S rRNA RT-PCR denaturing gradient gel electrophoresis (DGGE) profiles of samples collected from a chloropicrin (CP)-treated field over 2 months. The difference between untreated control and CP-treated plots was detected clearly both in DNA- and RNA-based DGGE profiles after treatment. The temporal changes in DGGE profiles, however, differed between DNA- and RNA-based analyses in CP-treated plots. RNA-based DGGE showed quicker and greater changes in the bacterial community after CP treatment than did DNA-based DGGE, which showed similar trends to RNA-based DGGE but with a time lag. The extent of decrease in the diversity index (H′) and the change in principal response curves was larger in RNA-based analyses. These results indicate that the rDNA PCR-DGGE method also detects DNA of microbes no longer alive after fumigation, and that rRNA provides a more responsive biomarker than rDNA.     

Soil colloids-bound plasmid DNA: Effect on transformation of E. coli and resistance to DNase I degradation

The adsorption and binding of plasmid p34S DNA on four different colloidal fractions from a Brown soil and clay minerals in the presence of various Ca²⁺ concentrations, the ability of bound DNA to transform competent cells of CaCl₂-treated Escherichia coli, and the resistance of bound DNA to degradation by DNase I were studied. DNA adsorption on soil colloids and clay minerals was promoted in the presence of Ca²⁺. Kaolinite exhibited the highest adsorption affinity for DNA among the examined soil colloids and clay minerals. In comparison with organo-mineral complexes (organic clays) and fine clays (<0.2 μm), DNA was tightly adsorbed by H₂O₂-treated clays (inorganic clays) and coarse clays (0.2-2 μm). The transformation efficiency of bound DNA increased with increasing concentrations of Ca²⁺ at which soil colloid or clay mineral-DNA complexes were formed. DNA bound by kaolinite showed the lowest transformation efficiency, and especially no transformants were observed with kaolinite-DNA complex prepared at 5-100 mM Ca²⁺. Compared to organic clays and fine clays, DNA bound on inorganic clays and coarse clays showed a lower capacity to transform E. coli at different Ca²⁺ concentrations. The presence of soil colloids and minerals provided protection to DNA against degradation by DNase I. Montmorillonite, organic clays and fine clays showed stronger protective effects for DNA than inorganic clays and coarse clays. The protection mechanisms as well as the differences in transforming efficiency of plasmid DNA molecules bound on various soil colloidal particles are discussed. The information obtained in this study is of fundamental significance for the understanding of the horizontal dissemination of recombinant DNA and the fate of extracellular DNA in soil environments.     

Quantifying potato pathogen DNA in soil

An improved method for the direct extraction of DNA from soil involving processing of a relatively large sample (60 g) was developed. The accurate and reliable detection and quantification of the soil-borne potato pathogens Colletotrichum coccodes (black dot), Rhizoctonia solani (black scurf) and Spongospora subterranea (powdery scab) following inoculation of soils was demonstrated. With this method, low levels of target DNA (30–40 pg DNA/g soil) could be detected in field soils. DNA recovery was proportionate across a wide range of inoculum (R² > 0.86) and there was no effect of soil type on the recovery of C. coccodes. The method was used to assess levels of naturally occurring pathogen DNA in 122 soil samples obtained from commercial potato fields.     

Pseudomonas spp. isolates with high phosphate-mobilizing potential and root colonization properties from agricultural bulk soils under no-till management

Seven phosphate-mobilizing pseudomonads were isolated, identified, and characterized in terms of their biofertilizer potential and root-colonizing properties. Pseudomonas protegens (ex-fluorescens) CHA0 was used for comparative purposes. Four isolates (LF-MB1, LF-P1, LF-P2, and LF-P3) clustered with members of the “Pseudomonas fluorescens complex,” whereas the other three (LF-MB2, LF-V1, and LF-V2) clustered with members of the “Pseudomonas putida/Pseudomonas aeruginosa complex.” Assays in buffered liquid growth medium supplemented with tricalcium phosphate enabled the separation of the isolates into two groups: group A (LF-P1, LF-P2, LF-P3, and LF-V1) solubilized P from 151 up to 182?μg?mL^?1, and group B (LF-MB1, LF-MB2, and LF-V2) solubilized less than 150?μg?P?mL^?1. All isolates displayed acid and alkaline phosphatase activities. With the exception of LF-MB2, all isolates were able to degrade phospholipids from lecithin. Additionally, all isolates exhibited extracellular protease activity, and four isolates produced hydrogen cyanide, two traits that are related to biocontrol of phytopathogens. To study root colonization in non-sterile soil, isolates were doubly tagged with gfp and a tetracycline resistance cassette. After 15?days of competition with the indigenous bacterial flora, all tagged isolates colonized soybean roots at counts ranging from 7.6?×?10⁵ to 1.7?×?10⁷?CFU?g^?1. The results indicate that there are already efficient phosphate-mobilizing pseudomonads adapted to agricultural bulk soils under no-till management in Argentina and thus having excellent potential for use as biofertilizers.