Yvan Moënne-Loccoz

The rhizosphere: a playground and battlefield for soilborne pathogens and beneficial microorganisms

The rhizosphere is a hot spot of microbial interactions as exudates released by plant roots are a main food source for microorganisms and a driving force of their population density and activities. The rhizosphere harbors many organisms that have a neutral effect on the plant, but also attracts organisms that exert deleterious or beneficial effects on the plant. Microorganisms that adversely affect plant growth and health are the pathogenic fungi, oomycetes, bacteria and nematodes. Most of the soilborne pathogens are adapted to grow and survive in the bulk soil, but the rhizosphere is the playground and infection court where the pathogen establishes a parasitic relationship with the plant. The rhizosphere is also a battlefield where the complex rhizosphere community, both microflora and microfauna, interact with pathogens and influence the outcome of pathogen infection. A wide range of microorganisms are beneficial to the plant and include nitrogen-fixing bacteria, endo- and ectomycorrhizal fungi, and plant growth-promoting bacteria and fungi. This review focuses on the population dynamics and activity of soilborne pathogens and beneficial microorganisms. Specific attention is given to mechanisms involved in the tripartite interactions between beneficial microorganisms, pathogens and the plant. We also discuss how agricultural practices affect pathogen and antagonist populations and how these practices can be adopted to promote plant growth and health.

Plant growth-promoting rhizobacteria and root system functioning

The rhizosphere supports the development and activity of a huge and diversified microbial community, including microorganisms capable to promote plant growth. Among the latter, plant growth-promoting rhizobacteria (PGPR) colonize roots of monocots and dicots, and enhance plant growth by direct and indirect mechanisms. Modification of root system architecture by PGPR implicates the production of phytohormones and other signals that lead, mostly, to enhanced lateral root branching and development of root hairs. PGPR also modify root functioning, improve plant nutrition and influence the physiology of the whole plant. Recent results provided first clues as to how PGPR signals could trigger these plant responses. Whether local and/or systemic, the plant molecular pathways involved remain often unknown. From an ecological point of view, it emerged that PGPR form coherent functional groups, whose rhizosphere ecology is influenced by a myriad of abiotic and biotic factors in natural and agricultural soils, and these factors can in turn modulate PGPR effects on roots. In this paper, we address novel knowledge and gaps on PGPR modes of action and signals, and highlight recent progress on the links between plant morphological and physiological effects induced by PGPR. We also show the importance of taking into account the size, diversity, and gene expression patterns of PGPR assemblages in the rhizosphere to better understand their impact on plant growth and functioning. Integrating mechanistic and ecological knowledge on PGPR populations in soil will be a prerequisite to develop novel management strategies for sustainable agriculture.

Nonpathogenic Fusarium oxysporum strain Fo47 induces resistance to Fusarium wilt in tomato.

Nonpathogenic Fusarium oxysporum strain Fo47 controls the incidence of Fusarium wilt. Four bioassays in which a strain of the pathogen F. oxysporum f. sp. lycopersici and Fo47 were not in direct contact were developed to evaluate whether Fo47 could induce resistance to Fusarium wilt in tomato plants. Fo47 and the pathogen were separated either physically or in time. Bio-assays were carried out under hydroponic conditions (two bioassays), in potting mix, or in autoclaved soil. Strain Fo47 protected tomato against Fusarium wilt in all four bioassays. Inoculation with Fo47 increased chitinase, β-1,3-glucanase, and β-1,4-glucosidase activity in plants, confirming the ability of Fo47 to induce resistance in tomato. This report is the first to demonstrate that a nonpathogenic strain of F. oxysporum can induce resistance to Fusarium wilt in tomato plants. This result has important practical implications for biocontrol of tomato diseases under commercial conditions.

Biological control of Pythium ultimum by Stenotrophomonas maltophilia W81 is mediated by an extracellular proteolytic activity

Stenotrophomonas maltophilia strain W81, isolated from the rhizosphere of field-grown sugar beet, produced the extracellular enzymes chitinase and protease and inhibited the growth of the phytopathogenic fungus Pythium ultimum in vitro. The role of these lytic enzymes in the interaction between W81 and P. ultimum was investigated using Tn5 insertion mutants of W81 incapable of producing extracellular protease (W81M1), extracellular chitinase (W81M2) or the two enzymes (W81A1). Lytic enzyme activity was restored in W81A1 following introduction of a 15 kb cosmid-borne fragment of W81 genomic DNA. Incubation of P. ultimum in the presence of commercial purified protease or cell-free supernatants from cultures of wild-type W81, the chitinase-negative mutant W81M2 or the complemented derivative W81A1 (pCU800) resulted in hyphal lysis and loss of subsequent fungal growth ability once re-inoculated onto fresh plates. In contrast, commercial purified chitinase or cell-free supernatants from cultures of the protease-negative mutant WS1M1 or the chitinase- and protease-negative mutant W81A1 had no effect on integrity of the essentially chitin-free Pythium mycelium, and did not prevent subsequent growth of the fungus. In soil microcosms containing soil naturally infested by Pythium spp., strains W81, W81M2 and W81A1(pCU800) reduced the ability of Pythium spp. to colonize the seeds of sugar beet and improved plant emergence compared with the untreated control, whereas W81A1 and W21M1 failed to protect sugar beet from damping-off. Wild-type W81 and its mutant derivatives colonized the rhizosphere of sugar beet to similar extents, it was concluded that the ability of S. maltophilia W81 to protect sugar beet from Pythium -mediated damping-off was due to the production of an extracellular protease.

Biocontrol of soil-borne fungal plant diseases by 2,4-diacetylphloroglucinol-producing fluorescent pseudomonads with different restriction profiles of amplified 16S rDNA

Fluorescent pseudomonads producing the antimicrobial compound 2,4-diacetylphloroglucinol (Phl) are being studied extensively for use as biocontrol agents of soil-borne fungal diseases. Some of them can produce pyoluteorin (Plt) in addition to Phl, whereas others synthesise only Phl. Here, a collection of seven Phl+ Plt- pseudomonads, seven Phl+ Plt+ pseudomonads and seven Phl- biocontrol pseudomonads were compared for protection of plant roots against fungal pathogens. The seven Phl+ Plt+ pseudomonads were identical by restriction analysis of amplified spacer ribosomal DNA (spacer ARDRA), whereas the Phl+ Plt- pseudomonads and especially the Phl- biocontrol pseudomonads were quite diverse by spacer ARDRA. Collectively, the Phl+ Plt- pseudomonads proved superior to the Phl+ Plt+ pseudomonads and the Phl- biocontrol pseudomonads for protection of tomato against Fusarium crown and root rot (in rockwool microcosms) or cucumber against Pythium damping-off (in non-sterile soil microcosms). There was no correlation between protection in vivo and inhibition of the corresponding fungal pathogen on plates. However, there was a significant correlation between the amount of Phl produced on plates and protection of tomato against Fusarium crown and root rot, but not with protection of cucumber against Pythium damping-off. Interestingly, the minority of strains unable to produce HCN, an extracellular protease, or both, were among those unable to protect plants in both pathosystems. A seedling assay was developed to compare pseudomonads for suppression of Fusarium crown and root rot in vitro, and a significant correlation was found between disease severity in vitro and in vivo. Overall, results suggest that promising biocontrol pseudomonads may be identified based on the ability to produce Phl and/or specific ARDRA-based fingerprints.

Phylogeny of HCN synthase-encoding hcnBC genes in biocontrol fluorescent pseudomonads and its relationship with host plant species and HCN synthesis ability.

Hydrogen cyanide (HCN) is a broad-spectrum antimicrobial compound involved in biological control of root diseases by many plant-associated fluorescent pseudomonads. The HCN synthase is encoded by three biosynthetic genes (hcnA, hcnB, and hcnC), but little is known about the diversity of these genes in fluorescent Pseudomonas spp. and in other bacteria. Here, the partial hcnBC sequence was determined for a worldwide collection of biocontrol fluorescent Pseudomonas spp. Phylogenies based on hcnBC and deduced protein sequences revealed four main bacterial groups, but topological incongruences were found between hcnBC and rrs-based phylogenies, suggesting past lateral transfer of hcnBC among saprophytic root-colonizing pseudomonads. Three of the four groups included isolates from different countries and host plants. Yet, these groups corresponded to distinct, ecologically-adapted populations of HCN-producing biocontrol fluorescent pseudomonads, as indicated by high hcnBC distinctness ratio values and the differences in production levels of HCN in vitro found between groups. This is in accordance with previous results on catabolic properties and biocontrol abilities of these strains. HCN synthase gene diversity may thus reflect the adaptive radiation of HCN+ biocontrol fluorescent pseudomonads. Positive correlations were found between HCN production in vitro and plant protection in the cucumber/Pythium ultimum and tomato/Fusarium oxysporum f. sp. radicis-lycopersici pathosystems.

Pseudomonas fluorescens and closely‐related fluorescent pseudomonads as biocontrol agents of soil‐borne phytopathogens

Many strains of Pseudomonas fluorescens show potential for biological control of phytopathogens especially root pathogens. In taxonomic terms, several of them are indeed P. fluorescens sensu stricto, while others belong in fact to neighbouring species of the ‘P. fluorescens’ complex or to ill‐defined related species within the fluorescent Pseudomonas spp. These bacteria have become prominent models for rhizosphere ecological studies and analysis of bacterial secondary metabolism, and in recent years knowledge on their plant‐beneficial traits has been considerably enhanced by widening the focus beyond the case of phytopathogen‐directed antagonism. Current genomic analyses of rhizosphere competence and biocontrol traits will likely lead to the development of novel tools for effective management of indigenous and inoculated P. fluorescens biocontrol agents and a better exploitation of their plant‐beneficial properties for sustainable agriculture.

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

Abstract 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 soil.

Potential of a 16S rRNA-based taxonomic microarray for analyzing the rhizosphere effects of maize on Agrobacterium spp. and bacterial communities.

ABSTRACT Bacterial diversity is central to ecosystem sustainability and soil biological function, for which the role of roots is important. The high-throughput analysis potential of taxonomic microarray should match the breadth of bacterial diversity. Here, the power of this technology was evidenced through methodological verifications and analysis of maize rhizosphere effect based on a 16S rRNA-based microarray developed from the prototype of H. Sanguin et al. (Environ. Microbiol. 8:289-307, 2006). The current probe set was composed of 170 probes (41 new probes in this work) that targeted essentially the Proteobacteria. Cloning and sequencing of 16S rRNA amplicons were carried out on maize rhizosphere and bulk soil DNA. All tested clones that had a perfect match with corresponding probes were positive in the hybridization experiment. The hierarchically nested probes were reliable, but the level of taxonomic identification was variable, depending on the probe set specificity. The comparison of experimental and theoretical hybridizations revealed 0.91% false positives and 0.81% false negatives. The microarray detection threshold was estimated at 0.03% of a given DNA type based on DNA spiking experiments. A comparison of the maize rhizosphere and bulk soil hybridization results showed a significant rhizosphere effect, with a higher predominance of Agrobacterium spp. in the rhizosphere, as well as a lower prevalence of Acidobacteria, Bacteroidetes, Verrucomicrobia, and Planctomycetes, a new taxon of interest in soil. In addition, well-known taxonomic groups such as Sphingomonas spp., Rhizobiaceae, and Actinobacteria were identified in both microbial habitats with strong hybridization signals. The taxonomic microarray developed in the present study was able to discriminate and characterize bacterial community composition in related biological samples, offering extensive possibilities for systematic exploration of bacterial diversity in ecosystems.

Comparison of rhizobacterial community composition in soil suppressive or conducive to tobacco black root rot disease.

Work on soils suppressive to Thielaviopsis basicola-mediated tobacco black root rot has focused on antagonistic pseudomonads to date. The role of non-Pseudomonas rhizosphere populations has been neglected, and whether they differ in black root rot-suppressive versus -conducive soils is unknown. To assess this possibility, tobacco was grown in a suppressive and a conducive soil of similar physicochemical properties, and rhizobacterial community composition was compared using a 16S rRNA taxonomic microarray. The microarray contains 1033 probes and targets 19 bacterial phyla. Among them, 398 probes were designed for Proteobacteria, Firmicutes, Actinomycetes, Cyanobacteria and Bacteroidetes genera/species known to include strains relevant for plant protection or plant growth promotion. Hierarchical clustering as well as principal component analysis of microarray data discriminated clearly between black root rot-suppressive and -conducive soils. In contrast, T. basicola inoculation had no impact on rhizobacterial community composition. In addition to fluorescent Pseudomonas, the taxa Azospirillum, Gluconacetobacter, Burkholderia, Comamonas and Sphingomonadaceae, which are known to comprise strains with plant-beneficial properties, were more prevalent in the suppressive soil. Mycobacterium, Bradyrhizobium, Rhodobacteraceae, Rhodospirillum and others were more prevalent in the conducive soil. For selected taxa, microarray results were largely corroborated by quantitative PCR and cloning/sequencing. In conclusion, this work identified novel bacterial taxa that could serve as indicators of disease suppressiveness in soil-quality assessments, and it extends the range of bacterial taxa hypothesized to participate in black root rot suppression.