Survival and cell culturability of biocontrol Pseudomonas fluorescens CHA0 in lysimeter effluent water and utilization of a deleterious genetic modification to study the impact of the strain on numbers of resident culturable bacteria

Little is known on the behavior of soil-inoculated biocontrol pseudomonads once they are transported to deeper soil layers and/or groundwater levels after a heavy rain. This issue was investigated in inoculated microcosms containing lysimeter effluent water, and experimental conditions mimicking a worse-case scenario for potential bacterial dissemination were chosen. First, the survival of the polyketide-producing biocontrol strain Pseudomonas fluorescens CHA0-Rif was studied for 175 days at two inoculation levels in unamended and nutrient-amended lysimeter effluent water, and its impact on numbers of resident culturable bacteria was determined. Cell numbers of CHA0-Rif declined to 3-4 log cells ml−1 (at high inoculum level) or reached the detection limit or below (at low inoculum level) by day 175, without generating significant numbers of non-culturable cells. At high inoculum level, strain CHA0-Rif resulted durably (from day 50 to 175) in higher numbers of the total resident culturable bacteria when compared with the uninoculated control. This effect, which did not take place at low inoculum level or when nutrients had been added, contrasts with the transient ecological impact of the strain on rhizosphere bacterial populations in previous studies. Neither 2,4-diacetylphloroglucinol nor pyoluteorin were found in the water using HPLC, and inoculation with CHA0-Rif had no effect on the percentages of the total culturable aerobic bacteria sensitive to either antimicrobial polyketide on day 20. Second, the impact of CHA0-Rif on numbers of resident culturable bacteria was compared with that of CHA0-Rif(pME3424). Plasmid pME3424 carries an extra copy of the strain's rpoD gene (encoding sigma factor σ70). CHA0-Rif(pME3424) disappeared within 50 days in the water, but had the same impact as CHA0-Rif on the total number of resident culturable bacteria. This suggests that the impact of CHA0-Rif took place at the early stages of the experiment and was probably linked to the release of nutrients by introduced cells during inoculant declin

Genetic diversity and biocontrol potential of fluorescent pseudomonads producing phloroglucinols and hydrogen cyanide from Swiss soils naturally suppressive or conducive to Thielaviopsis basicola-mediated black root rot of tobacco

Pseudomonas populations producing the biocontrol compounds 2,4-diacetylphloroglucinol (Phl) and hydrogen cyanide (HCN) were found in the rhizosphere of tobacco both in Swiss soils suppressive to Thielaviopsis basicola and in their conducive counterparts. In this study, a collection of Phl+ HCN+Pseudomonas isolates from two suppressive and two conducive soils were used to assess whether suppressiveness could be linked to soil-specific properties of individual pseudomonads. The isolates were compared based on restriction analysis of the biocontrol genes phlD and hcnBC, enterobacterial repetitive intergenic consensus (ERIC)-PCR profiling and their biocontrol ability. Restriction analyses of phlD and hcnBC yielded very concordant relationships between the strains, and suggested significant population differentiation occurring at the soil level, regardless of soil suppressiveness status. This was corroborated by high strain diversity (ERIC-PCR) within each of the four soils and among isolates harboring the same phlD or hcnBC alleles. No correlation was found between the origin of the isolates and their biocontrol activity in vitro and in planta. Significant differences in T. basicola inhibition were however evidenced between the isolates when they were grouped according to their biocontrol alleles. Moreover, two main Pseudomonas lineages differing by the capacity to produce pyoluteorin were evidenced in the collection. Thus, Phl+ HCN+ pseudomonads from suppressive soils were not markedly different from those from nearby conducive soils. Therefore, as far as biocontrol pseudomonads are concerned, this work yields the hypothesis that the suppressiveness of Swiss soils may rely on the differential effects of environmental factors on the expression of key biocontrol genes in pseudomonads rather than differences in population structure of biocontrol Pseudomonas subcommunities or the biocontrol potential of individual Phl+ HCN+ pseudomonad strain

Prevalence of fluorescent pseudomonads producing antifungal phloroglucinols and/or hydrogen cyanide in soils naturally suppressive or conducive to tobacco black root rot

Certain soils from Morens, Switzerland, are naturally suppressive to Thielaviopsis basicola-mediated black root rot of tobacco, and fluorescent pseudomonads are involved in this suppressiveness. Here, we compared two conducive, one moderately suppressive and one suppressive soil from Morens. Disease levels on tobacco after heavy T. basicola inoculation varied from 29% to 85% for the two conducive soils, 10% to 78% for the moderately suppressive soil and 11% to 42% for the suppressive soil, depending on time of the year. In the absence of T. basicola inoculation, disease levels were between 0% and 40% and varied also in time. Fluorescent pseudomonads were isolated from the rhizosphere and roots of tobacco subjected to T. basicola inoculation and characterized for production of the biocontrol metabolites 2,4-diacetylphloroglucinol (Phl) and HCN. No difference in population size was found between the suppressive and the conducive soils for total, Phl+ and HCN+ fluorescent pseudomonads colonizing the rhizosphere or roots of tobacco. Yet, the percentage of Phl+ isolates was significantly higher (30-32% vs. 6-11%) in the rhizosphere and roots for plants grown in the suppressive soil compared with the moderately suppressive and conducive soils. Different restriction profiles for phlD, one of the Phl biosynthetic genes, were often found when analyzing Phl+ isolates colonizing the same plant. Most phlD alleles were recovered from both suppressive and conducive soils, except one allele found only in root isolates from the suppressive soi

A new DGGE protocol targeting 2,4-diacetylphloroglucinol biosynthetic gene phlD from phylogenetically contrasted biocontrol pseudomonads for assessment of disease-suppressive soils

In the rhizosphere, biocontrol pseudomonads producing 2,4-diacetylphloroglucinol (Phl) can protect plants from soil-borne pathogens. DGGE of phlD has been proposed to monitor these bacteria, but two distinct protocols were needed for analysis of both the ‘Pseudomonas fluorescens' species complex and the strains from rrs restriction group ARDRA-1. Here, a single DGGE protocol performed on 668-bp GC-clamp-containing phlD amplicons was effective with both types of pseudomonads, and 36 reference biocontrol strains from the ‘P. fluorescens' complex or group ARDRA-1 gave a total of 11 distinct DGGE bands. phlD amplicons with at least two to seven nucleotidic differences could be discriminated, and the discrimination level was similar to that of phlD restriction analysis with four enzymes. Multiple phlD-DGGE bands were obtained when studying rhizosphere soil containing indigenous phlD+ pseudomonads, and phlD diversity was higher when DGGE was implemented after incubation of tobacco rhizosphere extracts in semi-selective medium (MPN approach) in comparison with approaches based on direct analysis of rhizosphere DNA extracts or assessment of phlD+colonies. phlD-DGGE profiles differed for a soil suppressive and a soil conducive to black root rot of tobacco, and each soil yielded new phlD sequences. In conclusion, this DGGE protocol was useful for monitoring indigenous rhizosphere consortia of phlD+ pseudomonad

Calystegine degradation capacities of microbial rhizosphere communities of Zea mays (calystegine-negative) and Calystegia sepium (calystegine-positive)

Calystegines are tropane alkaloids produced by the roots of a few plant species. A bioassay was developed to identify roots with a microbial rhizosphere community capable of calystegine degradation (i.e. MCD roots). In a field survey, the proportion of MCD roots of Zea mays (calystegine-negative) varied from 20 to 80%. In field experiments, the proportions of MCD roots of Z. mays and Calystegia sepium (calystegine-positive) grown in a particular plot were similar to each other but varied with time and, overall, were higher than those of Z. mays roots from adjacent plots free of C. sepium. In autoclaved soil, no root of C. sepium or Z. mays plants propagated as seeds was MCD, indicating that calystegine-degrading microorganisms were not seed-borne. However, MCD roots were found as early as 1 day after planting of rhizomes of C. sepium in autoclaved soil or planting of axenic seedlings of either plant in natural soil microcosms. In total, microorganisms capable of degrading calystegines were harboured not only in the rhizosphere of the calystegine-producing plant but also in that of the calystegine-negative plant and probably in bulk soi

Effect of long-term vineyard monoculture on rhizosphere populations of pseudomonads carrying the antimicrobial biosynthetic genes phlD and/or hcnAB

The impact of repeated culture of perennial plants (i.e. in long-term monoculture) on the ecology of plant-beneficial bacteria is unknown. Here, the influence of extremely long-term monocultures of grapevine (up to 1603 years) on rhizosphere populations of fluorescent pseudomonads carrying the biosynthetic genes phlD for 2,4-diacetylphloroglucinol and/or hcnAB for hydrogen cyanide was determined. Soils from long-term and adjacent short-term monoculture vineyards (or brushland) in four regions of Switzerland were baited with grapevine or tobacco plantlets, and rhizosphere pseudomonads were studied by most probable number (MPN)-PCR. Higher numbers and percentages of phlD+ and of hcnAB+ rhizosphere pseudomonads were detected on using soil from long-term vineyards. On focusing on phlD, restriction fragment length polymorphism profiling of the last phlD-positive MPN wells revealed seven phlD alleles (three exclusively on tobacco, thereof two new ones). Higher numbers of phlD alleles coincided with a lower prevalence of the allele displayed by the well-studied biocontrol strain Pseudomonas fluorescens F113. The prevalence of this allele was 35% for tobacco in long-term monoculture soils vs. >60% in the other three cases. We conclude that soils from long-term grapevine monocultures represent an untapped resource for isolating novel biocontrol Pseudomonas strains when tobacco is used as bai

Autecology of the biocontrol strain Pseudomonas fluorescens CHA0 in the rhizosphere and inside roots at later stages of plant development

A spontaneous rifampicin-resistant mutant of the biocontrol agent Pseudomonas fluorescens CHA0 was released as soil inoculant in large outdoor lysimeters and its ability to colonise the roots of winter wheat, spring wheat (grown after Phacelia) and maize at the later stages of plant development was investigated by colony counts. The inoculant (i.e. CHA0-Rif) colonised the rhizosphere and the interior of the roots of both wheat varieties but CFUs at ripening were about 2 log (g root)−1 or lower. In contrast, the roots of maize were colonised poorly by the pseudomonad at flowering, but the latter was found at 3 or more log CFU (g root)−1 on and inside the roots in late ripening stage. Furthermore, CHA0-Rif was recovered at more than 5 log CFU (g root)−1 from the interior of several maize root samples. Whereas most cells of CHA0-Rif in soil were small and did not respond to Kogure's viability test, the pseudomonad was present as viable, unusually large (7 mm long) rods inside maize roots. In a microcosm experiment performed with similar sandy-loam soil, the CFUs of maize root-associated CHA0-Rif were higher where the shoots of the plant had been cut off, confirming that older and/or decaying maize roots represent a favourable niche for the inoculant. Overall, the results indicate that Pseudomonas inoculants have the potential to colonise the roots of certain crops (e.g. maize but not wheat for strain CHA0-Rif) at later stages of plant developmen

Cell culturability of Pseudomonas protegensCHA0 depends on soil pH

Pseudomonas inoculants may lose colony-forming ability in soil, but soil properties involved are poorly documented. Here, we tested the hypothesis that soil acidity could reduce persistence and cell culturability of Pseudomonas protegensCHA0. At 1 week in vitro, strain CHA0 was found as culturable cells at pH 7, whereas most cells at pH 4 and all cells at pH 3 were noncultured. In 21 natural soils of contrasted pH, cell culturability loss of P. protegensCHA0 took place in all six very acidic soils (pH < 5.0) and in three of five acidic soils (5.0 < pH < 6.5), whereas it was negligible in the neutral and alkaline soils at 2 weeks and 2 months. No correlation was found between total cell counts of P. protegensCHA0 and soil composition data, whereas colony counts of the strain correlated with soil pH. Maintenance of cell culturability in soils coincided with a reduction in inoculant cell size. Some of the noncultured CHA0 cells were nutrient responsive in Kogure's viability test, both in vitro and in soil. Thus, this shows for the first time that the sole intrinsic soil composition factor triggering cell culturability loss in P. protegensCHA0 is soil acidit

Cosmopolitan distribution of phlD-containing dicotyledonous crop-associated biocontrol pseudomonads of worldwide origin

In biocontrol fluorescent pseudomonads, phlD encodes a polyketide synthase required for the synthesis of the antifungal compound 2,4-diacetylphloroglucinol (Phl). Here, PCR-restriction fragment length polymorphism analysis was used to compare phlD alleles in 77 dicot-associated pseudomonads originating from various countries worldwide and 10 counterparts from a monocotyledonous host (wheat). The 16 restriction patterns obtained were mostly unrelated to geographic location or dicot host. Cluster analysis distinguished eight phlD clusters at a similarity level of 0.63. One cluster grouped 18 pseudomonads that produced also the antifungal polyketide pyoluteorin but could not assimilate D-galactose, D-galactonate lactone, D-sorbitol, L-arabinose, D-saccharate or D-xylose. These 18 pseudomonads, along with the eight pseudomonads from a second phlD cluster, were the only isolates that failed to deaminase 1-aminocyclopropane-1-carboxylate (ACC), a rare root growth promotion trait. Overall, assessment of phlD polymorphism, ACC deaminase activity and catabolic profiles pointed to a cosmopolitan distribution of Phl-producing biocontrol fluorescent pseudomonads of worldwide origin associated with dicotyledonous crop plant

Microbial diversity in soils suppressive to Fusarium diseases

Fusarium species are cosmopolitan soil phytopathogens from the division Ascomycota, which produce mycotoxins and cause significant economic losses of crop plants. However, soils suppressive to Fusarium diseases are known to occur, and recent knowledge on microbial diversity in these soils has shed new lights on phytoprotection effects. In this review, we synthesize current knowledge on soils suppressive to Fusarium diseases and the role of their rhizosphere microbiota in phytoprotection. This is an important issue, as disease does not develop significantly in suppressive soils even though pathogenic Fusarium and susceptible host plant are present, and weather conditions are suitable for disease. Soils suppressive to Fusarium diseases are documented in different regions of the world. They contain biocontrol microorganisms, which act by inducing plants’ resistance to the pathogen, competing with or inhibiting the pathogen, or parasitizing the pathogen. In particular, some of the Bacillus, Pseudomonas, Paenibacillus and Streptomyces species are involved in plant protection from Fusarium diseases. Besides specific bacterial populations involved in disease suppression, next-generation sequencing and ecological networks have largely contributed to the understanding of microbial communities in soils suppressive or not to Fusarium diseases, revealing different microbial community patterns and differences for a notable number of taxa, according to the Fusarium pathosystem, the host plant and the origin of the soil. Agricultural practices can significantly influence soil suppressiveness to Fusarium diseases by influencing soil microbiota ecology. Research on microbial modes of action and diversity in suppressive soils should help guide the development of effective farming practices for Fusarium disease management in sustainable agriculture