Diversity of biocontrol fluorescent pseudomonads producing 2,4-diacetylphloroglucinol and hydrogen cyanide in disease suppressive soils

by Alban Ramette

Abstract

The antagonistic activity of certain indigenous fluorescent pseudomonads can reduce significantly the incidence of soil-borne diseases caused by fungal plant pathogens, as exemplified by the disease suppressiveness that occurs naturally in certain soils. Depending on whether or not crop monoculture is needed for the establishment of this soil property, soil suppressiveness is designated as induced or long-standing, respectively. Often, for both types of soil suppressiveness, biocontrol fluorescent pseudomonads antagonize soil-borne pathogens by producing the secondary metabolites 2,4-diacetylphloroglucinol(Phl) and/or hydrogen Cyanide (HCN). In induced suppressive soils where wheat is cropped continuously for several decades, large populations of root-colonizing Phl+ fluorescent pseudomonads are present, whereas they are barely detected in the conducive counterparts. Further, within those Phl+ populations, the diversity of one of the Phl biosynthetic genes (phlD) is low, suggesting that crop monoculture could select and enrich particular genotypes in the populations of PhP fluorescent pseudomonads.
The objective of this thesis was to determine the prevalence and diversity of populations of fluorescent pseudomonads in long-standing suppressive soils, postulatingthat the presence of different plant species mediated through crop rotation in those soils may favor a higher bacterial diversity than in induced suppressive soils.
In the first part of this thesis, the prevalence and diversity of Phl+ and HCN+ fluorescent pseudomonads were determined in long-standing suppressive soils in Morens, Switzerland, where suppression concerns black root rot of tobacco caused by pathogenic Thielaviopsis basicola strains. In both suppressive and conducive Morens soils, large population sizes of PhP and HCN+ fluorescent pseudomonads were found, and up to eight phlD alleles per soil were identified, regardless of the soil suppressiveness Status. Thus, it may be that Morens soil suppressiveness is due to the combination of high numbers and high diversity of PhP biocontrol fluorescent pseudomonads. Crop rotation, as practiced in long-standing suppressive soils, may provoke a higher disturbance of bacterial populations over time compared with monoculture, fostering bacterial diversification. In contrast, induced suppressive soils constitute a more stable environmentfor biocontrol populations, owing to the continuous presence of the same host plant. In addition it is possible that long-standing disease suppressiveness also entails specific environmental conditions promoting biocontrol gene expression, and coordination of this expression within the Community of biocontrolroot-associated fluorescent pseudomonads.
In a second part, the diversity ofphlD was examined for a collection of PhP biocontrol fluorescent pseudomonads from worldwide origin, to determine whether the high phlD diversity was unique to Morens soils. Remarkably, only two out of the 11 phlD alleles found in Morens were not found in the 16 alleles found at a worldwide scale. Surprisingly, no relationships were found between phlD polymorphism and (i) the origin of the isolates (i.e., host plant, geographic location), (ii) the pathosystem(s) in which they were found efficient, (iii) their abilities to utilize various carbonsources, (iv) their 1-aminocyclopropane-l-carboxylate deaminase (ACC) activity, which is implicated in root growth promotion, and (v) their qualitative and quantitative capacities to produce secondary metabolites such as Phl and extracellular proteases.
In a third part, the diversity of hcnBC, which along with hcnA codes for a HCN synthase, was determined for a bacterial collection mainly composed of PhP HCN+ biocontrol fluorescent pseudomonads. Gene and protein phylogenies inferred from phlD and hcnBC were mostly congruent with each other and with the phylogeny inferred from random amplified Polymorphie DNA-generated data, indicating that they may reflect the bacterial whole-genome evolution. Yet, they were not congruent with the 16S rRNA-basedphylogeny, suggesting the possibility of past lateral gene transfers at both phlD and hcnBC loci, and/or different evolutionary rates for proteincoding gene and ribosomal sequences. Further, for most of the isolates, no relationships were found between the polymorphism of hcnBC (or phlD) and their qualitative or quantitative abilities to produce HCN in vitro and also their biocontrol efficiency in different pathosystems. Therefore, the polymorphism analysis of phlD and hcnBC did not indicate any ecological differences and biocontrol abilities for most of the biocontrol pseudomonads examined.
In the fourth part, the evolutionary relationships between PhlD in biocontrol fluorescent pseudomonads and plant chalcone synthases involved in essential steps of defense mechanisms against pathogens were determined. Although some striking similarities at the level of the catalytic sites were evidenced between their protein sequences, detailed sequence and phylogenetic analyses revealed that those similarities could originate either from a convergent evolution of similar enzymatic functions, or from an early genetic transfer event that occurred before the divergence of monocots and dicots.
In the discussion, lines of evidence are provided that indicate that phlD and hcnBC behave as molecular clocks. Thus, the divergence time since descent from a common ancestor was estimated to be about 20 to 50 million years ago. This suggests that the diversification at the two loci in biocontrol fluorescent pseudomonads could have occurred concomitantly with the diversification oftheir host-plants.
In conclusion, although phlD and hcnBC polymorphism may reflect whole-genome evolution at a coarse level of resolution, no relationships were generally found between these polymorphisms and the origins or the biocontrol-relevant properties of biocontrol fluorescent pseudomonads. This could be explained by an ancient diversification at those loci, as indicated by estimated divergence times. Further, the results of this thesis suggest that cultural practices may have played a significant role in the shaping of this bacterial diversity in induced vs. longstanding suppressive soils. As the polymorphism of protein-coding genes implicated in biocontrol does not indicate the difference in properties between long-standing suppressive soils and their conducive counterparts, the development of other methods indicating the expression and regulation of those genes in communities of biocontrol fluorescent pseudomonads would be needed in future studies.

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