Peter A. H. M. Bakker

Deciphering the rhizosphere microbiome for disease-suppressive bacteria.

A common plant pathogen induces the growth of disease-suppressive microbes in local soil communities. Disease-suppressive soils are exceptional ecosystems in which crop plants suffer less from specific soil-borne pathogens than expected owing to the activities of other soil microorganisms. For most disease-suppressive soils, the microbes and mechanisms involved in pathogen control are unknown. By coupling PhyloChip-based metagenomics of the rhizosphere microbiome with culture-dependent functional analyses, we identified key bacterial taxa and genes involved in suppression of a fungal root pathogen. More than 33,000 bacterial and archaeal species were detected, with Proteobacteria, Firmicutes, and Actinobacteria consistently associated with disease suppression. Members of the γ-Proteobacteria were shown to have disease-suppressive activity governed by nonribosomal peptide synthetases. Our data indicate that upon attack by a fungal root pathogen, plants can exploit microbial consortia from soil for protection against infections.

Induced Systemic Resistance by Beneficial Microbes

Beneficial microbes in the microbiome of plant roots improve plant health. Induced systemic resistance (ISR) emerged as an important mechanism by which selected plant growth-promoting bacteria and fungi in the rhizosphere prime the whole plant body for enhanced defense against a broad range of pathogens and insect herbivores. A wide variety of root-associated mutualists, including Pseudomonas, Bacillus, Trichoderma, and mycorrhiza species sensitize the plant immune system for enhanced defense without directly activating costly defenses. This review focuses on molecular processes at the interface between plant roots and ISR-eliciting mutualists, and on the progress in our understanding of ISR signaling and systemic defense priming. The central role of the root-specific transcription factor MYB72 in the onset of ISR and the role of phytohormones and defense regulatory proteins in the expression of ISR in aboveground plant parts are highlighted. Finally, the ecological function of ISR-inducing microbes in the root microbiome is discussed.

Induced Systemic Resistance by Fluorescent Pseudomonas spp.

ABSTRACT Fluorescent Pseudomonas spp. have been studied for decades for their plant growth-promoting effects through effective suppression of soilborne plant diseases. The modes of action that play a role in disease suppression by these bacteria include siderophore-mediated competition for iron, antibiosis, production of lytic enzymes, and induced systemic resistance (ISR). The involvement of ISR is typically studied in systems in which the Pseudomonas bacteria and the pathogen are inoculated and remain spatially separated on the plant, e.g., the bacteria on the root and the pathogen on the leaf, or by use of split root systems. Since no direct interactions are possible between the two populations, suppression of disease development has to be plant-mediated. In this review, bacterial traits involved in Pseudomonas-mediated ISR will be discussed.

Biocontrol by Phenazine-1-carboxamide-Producing Pseudomonas chlororaphis PCL1391 of Tomato Root Rot Caused by Fusarium oxysporum f. sp. radicis-lycopersici

Seventy bacterial isolates from the rhizosphere of tomato were screened for antagonistic activity against the tomato foot and root rot-causing fungal pathogen Fusarium oxysporum f. sp. radicis-lycopersici. One isolate, strain PCL1391, appeared to be an efficient colonizer of tomato roots and an excellent biocontrol strain in an F. oxysporum/tomato test system. Strain PCL1391 was identified as Pseudomonas chlororaphis and further characterization showed that it produces a broad spectrum of antifungal factors (AFFs), including a hydrophobic compound, hydrogen cyanide, chitinase(s), and protease(s). Through mass spectrometry and nuclear magnetic resonance, the hydrophobic compound was identified as phenazine-1-carboxamide (PCN). We have studied the production and action of this AFF both in vitro and in vivo. Using a PCL1391 transposon mutant, with a lux reporter gene inserted in the phenazine biosynthetic operon (phz), we showed that this phenazine biosynthetic mutant was substantially decreased in both in vitro antifungal activity and biocontrol activity. Moreover, with the same mutant it was shown that the phz biosynthetic operon is expressed in the tomato rhizosphere. Comparison of the biocontrol activity of the PCN-producing strain PCL1391 with those of phenazine-1-carboxylic acid (PCA)producing strains P. fluorescens 2-79 and P. aureofaciens 30-84 showed that the PCN-producing strain is able to suppress disease in the tomato/F. oxysporum system, whereas the PCA-producing strains are not. Comparison of in vitro antifungal activity of PCN and PCA showed that the antifungal activity of PCN was at least 10 times higher at neutral pH, suggesting that this may contribute to the superior biocontrol performance of strain PCL1391 in the tomato/F. oxysporum system.

Determinants of Pseudomonas putida WCS358 involved in inducing systemic resistance in plants

SUMMARY Pseudomonas putida WCS358 is a plant growth-promoting rhizobacterium originally isolated from the rhizosphere of potato. It can suppress soil-borne plant diseases by siderophore-mediated competition for iron, but it has also been reported to result in induced systemic resistance (ISR) in Arabidopsis thaliana. Bacterial determinants of this strain involved in inducing systemic resistance in Arabidopsis were investigated using a Tn5 transposon mutant defective in biosynthesis of the fluorescent siderophore pseudobactin, a non-motile Tn5 mutant lacking flagella, and a spontaneous phage-resistant mutant lacking the O-antigenic side chain of the lipopolysaccharides (LPS). When using Pseudomonas syringae pv. tomato as the challenging pathogen, purified pseudobactin, flagella and LPS all triggered ISR. However, the mutants were all as effective as the parental strain, suggesting redundancy in ISR-triggering traits in WCS358. The Botrytis cinerea-tomato, B. cinerea-bean and Colletotrichum lindemuthianum-bean model systems were used to test further the potential of P. putida WCS358 to induce ISR. Strain WCS358 significantly reduced disease development in all three systems, indicating that also on tomato and bean WCS358 can trigger ISR. In both tomato and bean, the LPS mutant had lost the ability to induce resistance, whereas the flagella mutant was still effective. In bean, the pseudobactin mutant was still effective, whereas this mutant has lost its effectivity in tomato. In both bean and tomato, flagella isolated from the parental strain were not effective, whereas LPS or pseudobactin did induce systemic resistance.

Control of Fusarium Wilt of Radish by Combining Pseudomonas putida Strains that have Different Disease-Suppressive Mechanisms

ABSTRACT Biological control of soilborne plant pathogens in the field has given variable results. By combining specific strains of microorganisms, multiple traits antagonizing the pathogen can be combined and this may result in a higher level of protection. Pseudomonas putida WCS358 suppresses Fusarium wilt of radish by effectively competing for iron through the production of its pseudobactin siderophore. However, in some bioassays pseudobactin-negative mutants of WCS358 also suppressed disease to the same extent as WCS358, suggesting that an, as yet unknown, additional mechanism may be operative in this strain. P. putida strain RE8 induced systemic resistance against fusarium wilt. When WCS358 and RE8 were mixed through soil together, disease suppression was significantly enhanced to approximately 50% as compared to the 30% reduction for the single strain treatments. Moreover, when one strain failed to suppress disease in the single application, the combination still resulted in disease control. The enhanced disease suppression by the combination of P. putida strains WCS358 and RE8 is most likely the result of the combination of their different disease-suppressive mechanisms. These results demonstrate that combining biocontrol strains can lead to more effective, or at least, more reliable biocontrol of fusarium wilt of radish.

Interactions between plants and beneficial Pseudomonas spp.: exploiting bacterial traits for crop protection

Specific strains of fluorescent Pseudomonas spp. inhabit the environment surrounding plant roots and some even the root interior. Introducing such bacterial strains to plant roots can lead to increased plant growth, usually due to suppression of plant pathogenic microorganisms. We review the modes of action and traits of these beneficial Pseudomonas bacteria involved in disease suppression. The complex regulation of biological control traits in relation to the functioning in the root environment is discussed. Understanding the complexity of the interactions is instrumental in the exploitation of beneficial Pseudomonas spp. in controlling plant diseases.

Pseudomonas fluorescens WCS374r-induced systemic resistance in rice against Magnaporthe oryzae is based on pseudobactin-mediated priming for a salicylic acid-repressible multifaceted defense response

Selected strains of nonpathogenic rhizobacteria can reduce disease in foliar tissues through the induction of a defense state known as induced systemic resistance (ISR). Compared with the large body of information on ISR in dicotyledonous plants, little is known about the mechanisms underlying rhizobacteria-induced resistance in cereal crops. Here, we demonstrate the ability of Pseudomonas fluorescens WCS374r to trigger ISR in rice (Oryza sativa) against the leaf blast pathogen Magnaporthe oryzae. Using salicylic acid (SA)-nonaccumulating NahG rice, an ethylene-insensitive OsEIN2 antisense line, and the jasmonate-deficient mutant hebiba, we show that this WCS374r-induced resistance is regulated by an SA-independent but jasmonic acid/ethylene-modulated signal transduction pathway. Bacterial mutant analysis uncovered a pseudobactin-type siderophore as the crucial determinant responsible for ISR elicitation. Root application of WCS374r-derived pseudobactin (Psb374) primed naive leaves for accelerated expression of a pronounced multifaceted defense response, consisting of rapid recruitment of phenolic compounds at sites of pathogen entry, concerted expression of a diverse set of structural defenses, and a timely hyperinduction of hydrogen peroxide formation putatively driving cell wall fortification. Exogenous SA application alleviated this Psb374-modulated defense priming, while Psb374 pretreatment antagonized infection-induced transcription of SA-responsive PR genes, suggesting that the Psb374- and SA-modulated signaling pathways are mutually antagonistic. Interestingly, in sharp contrast to WCS374r-mediated ISR, chemical induction of blast resistance by the SA analog benzothiadiazole was independent of jasmonic acid/ethylene signaling and involved the potentiation of SA-responsive gene expression. Together, these results offer novel insights into the signaling circuitry governing induced resistance against M. oryzae and suggest that rice is endowed with multiple blast-effective resistance pathways.

Understanding the involvement of rhizobacteria-mediated induction of systemic resistance in biocontrol of plant diseases

Specific strains of nonpathogenic rhizobacteria can induce systemic resistance that is effective against a range of plant pathogens. To exploit induced systemic resistance, detailed knowledge of the triggering bacterial traits involved and on signal transduction pathways in the plant is necessary. Possibilities to improve effectiveness of induced resistance by rhizobacterial strains are discussed.Key words: induced systemic resistance, lipopolysaccharides, Pseudomonas fluorescens, Pseudomonas putida, salicylic acid, siderophores, signaling pathways.

Microbial Antagonism at the Root Level Is Involved in the Suppression of Fusarium Wilt by the Combination of Nonpathogenic Fusarium oxysporum Fo47 and Pseudomonas putida WCS358

ABSTRACT Two biological control agents, nonpathogenic Fusarium oxysporum Fo47 and Pseudomonas putida WCS358, were evaluated for suppression of Fusarium wilt of flax grown in nutrient solution and for suppression of the population density and metabolic activity of the causal organism F. oxysporum f. sp. lini strain Foln3GUS on root surfaces. Due to the presence of an introduced gusA reporter gene construct in Foln3GUS, the pathogen expressed beta-glucuronidase activity that was related to its carbon metabolism. At a Fo47 to Foln3GUS inoculum ratio of 100:1, both the population density of the pathogen and the beta-glucuronidase activity on and in flax roots were reduced by the nonpathogenic strain, and Fusarium wilt was suppressed. At a Fo47 to Foln3GUS inoculum ratio of 10:1, Fo47 decreased the severity of Fusarium wilt to a smaller extent and it also reduced beta-glucuronidase activity without reducing the density of Foln3GUS on flax roots. At a nonpathogenic to pathogenic Fusarium strains ratio of 10:1, the addition of P. putida WCS358 further suppressed Fusarium wilt and the density of the pathogen at the root level, whereas a mutant of WCS358 deficient in pseudobactin production had no significant effect. Iron availability to WCS358 on flax roots, assessed by ice-nucleation activity conferred from a transcriptional fusion (pvd-inaZ) of an ice-nucleation reporter gene to an iron-regulated promoter, was sufficiently low to allow pseudobactin production. P. putida WCS358 did not reduce the severity of Fusarium wilt of flax when inoculated without Fo47, and it did not improve disease suppression achieved by high inoculum doses of Fo47 (a Fo47 to Foln3GUS ratio of 100:1). Together, these data provide evidence that (i) suppression of Fusarium wilt of flax by Fo47 is related to reductions in the population density and metabolic activity of the pathogen on the root surface; (ii) WCS358 can enhance the biological control activity of Fo47, but this enhancement depends on the population of Fo47 relative to the pathogen; and (iii) pseudobactin contributes to suppression of Fusarium wilt by the combination of Fo47 and WCS358 on roots in which conditions are conducive to pseudobactin production by the bacterium.