Endophytic colonization of olive roots by the biocontrol strain Pseudomonas fluorescens PICF7

Confocal microscopy combined with three-dimensional olive root tissue sectioning was used to provide evidence of the endophytic behaviour of Pseudomonas fluorescens PICF7, an effective biocontrol strain against Verticillium wilt of olive. Two derivatives of the green fluorescent protein (GFP), the enhanced green and the red fluorescent proteins, have been used to visualize simultaneously two differently fluorescently tagged populations of P. fluorescens PICF7 within olive root tissues at the single cell level. The time-course of colonization events of olive roots cv. Arbequina by strain PICF7 and the localization of tagged bacteria within olive root tissues are described. First, bacteria rapidly colonized root surfaces and were predominantly found in the differentiation zone. Thereafter, microscopy observations showed that PICF7-tagged populations eventually disappeared from the root surface, and increasingly colonized inner root tissues. Localized and limited endophytic colonization by the introduced bacteria was observed over time. Fluorescent-tagged bacteria were always visualized in the intercellular spaces of the cortex region, and no colonization of the root xylem vessels was detected at any time. To the best of our knowledge, this is the first time this approach has been used to demonstrate endophytism of a biocontrol Pseudomonas spp. strain in a woody host such as olive using a nongnotobiotic system.     

Sequence and role in virulence of the three plasmid complement of the model tumor-inducing bacterium Pseudomonas savastanoi pv. savastanoi NCPPB 3335

Pseudomonas savastanoi pv. savastanoi NCPPB 3335 is a model for the study of the molecular basis of disease production and tumor formation in woody hosts, and its draft genome sequence has been recently obtained. Here we closed the sequence of the plasmid complement of this strain, composed of three circular molecules of 78,357 nt (pPsv48A), 45,220 nt (pPsv48B), and 42,103 nt (pPsv48C), all belonging to the pPT23A-like family of plasmids widely distributed in the P. syringae complex. A total of 152 coding sequences were predicted in the plasmid complement, of which 38 are hypothetical proteins and seven correspond to putative virulence genes. Plasmid pPsv48A contains an incomplete Type IVB secretion system, the type III secretion system (T3SS) effector gene hopAF1, gene ptz, involved in cytokinin biosynthesis, and three copies of a gene highly conserved in plant-associated proteobacteria, which is preceded by a hrp box motif. A complete Type IVA secretion system, a well conserved origin of transfer (oriT), and a homolog of the T3SS effector gene hopAO1 are present in pPsv48B, while pPsv48C contains a gene with significant homology to isopentenyl-diphosphate delta-isomerase, type 1. Several potential mobile elements were found on the three plasmids, including three types of MITE, a derivative of IS801, and a new transposon effector, ISPsy30. Although the replication regions of these three plasmids are phylogenetically closely related, their structure is diverse, suggesting that the plasmid architecture results from an active exchange of sequences. Artificial inoculations of olive plants with mutants cured of plasmids pPsv48A and pPsv48B showed that pPsv48A is necessary for full virulence and for the development of mature xylem vessels within the knots; we were unable to obtain mutants cured of pPsv48C, which contains five putative toxin-antitoxin genes.     

Endophytic colonization and biocontrol performance of Pseudomonas fluorescens PICF7 in olive (Olea europaea L.) are determined neither by pyoverdine production nor swimming motility

Pseudomonas fluorescens PICF7 is an indigenous inhabitant of olive (Olea europaea L.) rhizosphere, able to display endophytic lifestyle in roots, to induce a wide range of defence responses upon colonization of this organ and to exert effective biological control against Verticillium wilt of olive (VWO) (Verticillium dahliae). We aimed to evaluate the involvement of specific PICF7 phenotypes in olive root colonization and VWO biocontrol effectiveness by generating mutants impaired in swimming motility (fliI) or siderophore pyoverdine production (pvdI). Besides, the performance of mutants with diminished in vitro growth in potato dextrose agar medium (gltA) and cysteine (Cys) auxotrophy was also assessed. Results showed that olive root colonization and VWO biocontrol ability of the fliI, pvdI and gltA mutants did not significantly differ from that displayed by the parental strain PICF7. Consequently, altered in vitro growth, swimming motility and pyoverdine production contribute neither to PICF7 VWO suppressive effect nor to its colonization ability. In contrast, the Cys auxotroph mutant showed reduced olive root colonization capacity and lost full biocontrol efficacy. Moreover, confocal laser scanning microscopy revealed that all mutants tested were able to endophytically colonize root tissue to the same extent as wild‐type PICF7, discarding these traits as relevant for its endophytic lifestyle.     

Lytic enzymes induced by Pseudomonas fluorescens and other biocontrol organisms mediate defence against the anthracnose pathogen in mango

Talc-based bioformulations containing cells of Pseudomonas fluorescens, Bacillus subtilis and Saccharomyces cerevisiae were evaluated for their potential to attack the mango (Mangifera indica L.) anthracnose pathogen Colletotrichum gloeosporioides Penz. under endemic conditions. The preharvest aerial spray was given at fortnightly and monthly intervals. The plant growth-promoting rhizobacteria Pseudomonas fluorescens (FP7) amended with chitin sprayed at fortnightly intervals gave the maximum induction of flowering, a yield attribute in the preharvest stage, consequently reduced latent symptoms were recorded at the postharvest stage. An enormous induction of the defence-mediating lytic enzymes chitinase and β-1,3-glucanase was recorded in colorimetric assay and the expression of discrete bands in native PAGE analysis after FP7 + chitin treatment. The enhanced expression of defence-mediating enzymes may collectively contribute to suppress the anthracnose pathogen, leading to improved yield attributes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Survival, root colonisation and biocontrol capacities of Pseudomonas fluorescens F113 LacZY in dry alginate microbeads

Cells of Pseudomonas fluorescens F113 LacZY were encapsulated in alginate and their survival and ability to colonise sugar beet were evaluated. To assess survival, the formulation, composed of dry alginate microbeads of 300- to 700-μm diameter, was stored 1 year at 28±2 and 4±2°C and then tested against pathogenic fungi Pythium ultimum and Rhizoctonia solani in in vitro inhibition experiments. The same material was also used as inoculant for protection of sugar beet against Py. ultimum in microcosm experiments. The results obtained indicated that, although drying alginate beads resulted in a significant reduction of bacterial viability, the use of microbeads enabled a satisfactory level of root colonisation and protection, at least under microcosm conditions. The capability of the encapsulated cells to produce the antifungal metabolite 2,4-diacetylphloroglucinol (Phl) was not significantly affected by 12 months storage. Journal of Industrial Microbiology & Biotechnology (2001) 27, 337–342. Received 07 September 2000/ Accepted in revised form 08 May 2001     

Synergistic interaction between the type III secretion system of the endophytic bacterium Pantoea agglomerans DAPP-PG 734 and the virulence of the causal agent of olive knot Pseudomonas savastanoi pv. savastanoi DAPP-PG 722

The endophytic bacterium Pantoea agglomerans DAPP-PG 734 was previously isolated from olive knots caused by infection with Pseudomonas savastanoi pv. savastanoi DAPP-PG 722. Whole-genome analysis of this P. agglomerans strain revealed the presence of a Hypersensitive response and pathogenicity (Hrp) type III secretion system (T3SS). To assess the role of the P. agglomerans T3SS in the interaction with P. savastanoi pv. savastanoi, we generated independent knockout mutants in three Hrp genes of the P. agglomerans DAPP-PG 734 T3SS (hrpJ, hrpN, and hrpY). In contrast to the wildtype control, all three mutants failed to cause a hypersensitive response when infiltrated in tobacco leaves, suggesting that P. agglomerans T3SS is functional and injects effector proteins in plant cells. In contrast to P. savastanoi pv. savastanoi DAPP-PG 722, the wildtype strain P. agglomerans DAPP-PG 734 and its Hrp T3SS mutants did not cause olive knot disease in 1-year-old olive plants. Coinoculation of P. savastanoi pv. savastanoi with P. agglomerans wildtype strains did not significantly change the knot size, while the DAPP-PG 734 hrpY mutant induced a significant decrease in knot size, which could be complemented by providing hrpY on a plasmid. By epifluorescence microscopy and confocal laser scanning microscopy, we found that the localization patterns in knots were nonoverlapping for P. savastanoi pv. savastanoi and P. agglomerans when coinoculated. Our results suggest that suppression of olive plant defences mediated by the Hrp T3SS of P. agglomerans DAPP-PG 734 positively impacts the virulence of P. savastanoi pv. savastanoi DAPP-PG 722.     

Bioinformatics correctly identifies many type III secretion substrates in the plant pathogen Pseudomonas syringae and the biocontrol isolate P. fluorescens SBW25

The plant pathogen Pseudomonas syringae causes disease by secreting a potentially large set of virulence proteins called effectors directly into host cells, their environment, or both, using a type III secretion system (T3SS). Most P. syringae effectors have a common upstream element called the hrp box, and their N-terminal regions have amino acids biases, features that permit their bioinformatic prediction. One of the most prominent biases is a positive serine bias. We previously used the truncated AvrRpt2(81-255) effector containing a serine-rich stretch from amino acids 81 to 100 as a T3SS reporter. Region 81 to 100 of this reporter does not contribute to the secretion or translocation of AvrRpt2 or to putative effector protein chimeras. Rather, the serine-rich region from the N-terminus of AvrRpt2 is important for protein accumulation in bacteria. Most of the N-terminal region (amino acids 15 to 100) is not essential for secretion in culture or delivery to plants. However, portions of this sequence may increase the efficiency of AvrRpt2 secretion, delivery to plants, or both. Two effectors previously identified with the AvrRpt2(81-255) reporter were secreted in culture independently of AvrRpt2, validating the use of the C terminus of AvrRpt2 as a T3SS reporter. Finally, using the reduced AvrRpt2(101-255) reporter, we confirmed seven predicted effectors from P. syringae pv. tomato DC3000, four from P. syringae pv. syringae B728a, and two from P. fluorescens SBW25.     

Survival of the rhizosphere-competent biocontrol strain Pseudomonas fluorescens NBRI2650 in the soil and phytosphere

Pseudomonas fluorescens NBRI2650 was isolated after screening 360 bacterial strains from the rhizosphere of chickpea (Cicer arietinum L.) grown in fungal-disease-suppressive field soil. The strain was selected because of its high rhizosphere competence and ability to inhibit the growth of Fusarium oxysporum f.sp. ciceri, Rhizoctonia bataticola, and Pythium sp. under in vitro conditions. Survival and colonization of NBRI2650 in the phytosphere of chickpea, cotton (Gossypium hirsutum L.), cucumber (Cucumis sativus L.), and tomato (Lycopersicon seculentum Mill.) were monitored using a chromosomally located rifampicin-marked mutant P. fluorescens NBRI2650R. The strain showed variable ability to invade and survive in the phytosphere of different plants. Chickpea was used as a tester plant for further work, as it was not invaded by NBRI2650R. The interaction between NBRI2650R and F oxysporum fsp. ciceri was studied by both light microscopy and scanning electron microscopy. The lysis of the fungal cell wall by NBRI2650R was clearly demonstrated. Treatment of the chickpea seeds with NBRI2650R in prerelease experiments in the greenhouse using disease-conducive field soils from Jhansi and Kanpur resulted in increased plant growth and did not result in any perturbation of the indigenous microbial community that inhabited the rhizosphere of chickpea compared with nonbacterized seeds. Direct fermentation of diluted NBRI2650R on vermiculite without the need of expensive fermentors offers a reliable process for manufacturing bacterial inoculants in developing countries. Under field conditions, the horizontal and vertical movement of NBRI2650R was restricted to 30 and 60 cm, respectively, and the strain could not survive in the field during the 7 months before the chickpea could be planted for next cropping season. Field trials conducted at Jhansi, Kanpur, and Pantnagar resulted in higher grain yield increase in the bacteria-treated seed compared with the nonbacterized control. Seed and furrow treatment of the two chickpeas ('Radhey' and 'H-208') at Pantnagar resulted in significantly (P = 0.05) greater seedling mortality in nonbacterized seedlings compared with bacterized ones. The seed dry weight and yield for each variety were also significantly higher in bacterized seedlings than in nonbacterized ones. The population of NBRI2650R persisted throughout the growing season of chickpea in the range of 5.4-6.4 log10 CFU/g root.     

Interactions between Pseudomonas fluorescens biocontrol agents and Glomus mosseae, an arbuscular mycorrhizal fungus, within the rhizosphere

Interactions between Pseudomonas fluorescens biocontrol agents and Glomus mosseae , an arbuscular mycorrhizal fungus, were studied. The biocontrol agents included the genetically modified strains CHA96 and CHA0 pME3424 which produced enhanced levels of antifungal compounds. Tomato ( Lycopersicum esculentum ) and leek ( Allium porrum ) host plants were grown in sterile Terra-Green (calcined attapulgite clay) with limited nutrients. Mycorrhizal activity was indicated by shoot dry weight and phosphorus content. In all experiments, plants grown in the presence of G. mosseae had a significantly higher shoot dry weight than those grown in the absence of G. mosseae . Colonisation and activity of G. mosseae was unaltered in the presence of P. fluorescens isolates and presence of G. mosseae increased the population of P. fluorescens in the rhizosphere.     

Effects of the tomato pathogen Fusarium oxysporum f. sp. radicis-lycopersici and of the biocontrol bacterium Pseudomonas fluorescens WCS365 on the composition of organic acids and sugars in tomato root exudate

The effects of the pathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici and of the bacterial biocontrol strain Pseudomonas fluorescens WCS365, and of both microbes, on the amounts and composition of root exudate components of tomato plants grown in a gnotobiotic stonewool substrate system were studied. Conditions were selected under which introduction of F. oxysporum f. sp. radicis-lycopersici caused severe foot and root rot, whereas inoculation of the seed with P. fluorescens WCS365 decreased the percentage of diseased plants from 96 to 7%. This is a much better disease control level than was observed in potting soil. Analysis of root exudate revealed that the presence of F. oxysporum f. sp. radicis-lycopersici did not alter the total amount of organic acids, but that the amount of citric acid decreased and that of succinic acid increased compared with the nontreated control. In contrast, in the presence of the P. fluorescens biocontrol strain WCS365, the total amount of organic acid increased, mainly due to a strong increase of the amount of citric acid, whereas the amount of succinic acid decreased dramatically. Under biocontrol conditions, when both microbes are present, the content of succinic acid decreased and the level of citric acid was similar to that in the nontreated control. The amount of sugar was approximately half that of the control sample when either one of the microbes was present alone or when both were present. Analysis of the interactions between the two microbes grown together in sterile tomato root exudate showed that WCS365 inhibited multiplication of F. oxysporum f. sp. radicis-lycopersici, whereas the fungus did not affect the number of CFU of the bacterium.     

GacS sensor domains pertinent to the regulation of exoproduct formation and to the biocontrol potential of Pseudomonas fluorescens CHA0

In the root-colonizing biocontrol strain CHA0 of Pseudomonas fluorescens, cell density-dependent synthesis of extracellular, plant-beneficial secondary metabolites and enzymes is positively regulated by the GacS/GacA two-component system. Mutational analysis of the GacS sensor kinase using improved single-copy vectors showed that inactivation of each of the three conserved phosphate acceptor sites caused an exoproduct null phenotype (GacS-), whereas deletion of the periplasmic loop domain had no significant effect on the expression of exoproduct genes. Strain CHA0 is known to synthesize a solvent-extractable extracellular signal that advances and enhances the expression of exoproduct genes during the transition from exponential to stationary growth phase when maximal exoproduct formation occurs. Mutational inactivation of either GacS or its cognate response regulator GacA abolished the strain's response to added signal. Deletion of the linker domain of the GacS sensor kinase caused signal-independent, strongly elevated expression of exoproduct genes at low cell densities. In contrast to the wild-type strain CHA0, the gacS linker mutant and a gacS null mutant were unable to protect tomato plants from crown and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici in a soil-less microcosm, indicating that, at least in this plant-pathogen system, there is no advantage in using a signal-independent biocontrol strain.     

Indole-3-acetaldoxime-derived compounds restrict root colonization in the beneficial interaction between Arabidopsis roots and the endophyte Piriformospora indica

The growth-promoting and root-colonizing endophyte Piriformospora indica induces camalexin and the expression of CYP79B2, CYP79B3, CYP71A13, PAD3, and WRKY33 required for the synthesis of indole-3-acetaldoxime (IAOx)-derived compounds in the roots of Arabidopsis seedlings. Upregulation of the mRNA levels by P. indica requires cytoplasmic calcium elevation and mitogen-activated protein kinase 3 but not root-hair-deficient 2, radical oxygen production, or the 3-phosphoinositide-dependent kinase 1/oxidative signal-inducible 1 pathway. Because P. indica-mediated growth promotion is impaired in cyp79B2 cyp79B3 seedlings, while pad3 seedlings-which do not accumulate camalexin-still respond to the fungus, IAOx-derived compounds other than camalexin (e.g., indole glucosinolates) are required during early phases of the beneficial interaction. The roots of cyp79B2 cyp79B3 seedlings are more colonized than wild-type roots, and upregulation of the defense genes pathogenesis-related (PR)-1, PR-3, PDF1.2, phenylalanine ammonia lyase, and germin indicates that the mutant responds to the lack of IAOx-derived compounds by activating other defense processes. After 6 weeks on soil, defense genes are no longer upregulated in wild-type, cyp79B2 cyp79B3, and pad3 roots. This results in uncontrolled fungal growth in the mutant roots and reduced performance of the mutants. We propose that a long-term harmony between the two symbionts requires restriction of root colonization by IAOx-derived compounds.     

Use of a lux-based procedure to rapidly visualize root colonisation by Pseudomonas fluorescens in the wheat rhizosphere

The bioluminescently marked Pseudomonas fluorescens strain 5RL, has been used previously to follow colonisation of soy bean roots (De Weger et al. [1991] Appl. Environ. Microbiol. 57:36-41). In the present paper the method has been further developed and optimized for wheat roots and it is used to get a quick overview of the colonisation patterns of many different root systems at the same time. Colonisation was followed on wheat plants grown in our gnotobiotic sand system (Simons et al., 1996. Mol Plant Microbe Interact 9: 600–607) and the following results were obtained. (i) A spatio-temporal analysis of the colonisation of wheat roots showed that 4 days after planting the highest bacterial activity was observed at the upper part of the root. After 6 days the high bacterial activity at the upper part was further increased, whereas spot-like activities were observed on the lower root parts, possibly due to micro-colonies. (ii) Bacterial mutations causing lack of motility or auxotrophy for amino acids resulted in impaired colonisation of the lower root parts, indicating that motility and prototrophy for the involved amino acid(s) are important factors for wheat root colonisation by strain 5RL. (iii) Coinoculation of strain 5RL with other wild type Pseudomonas strains on the root influenced the colonisation pattern observed for strain 5RL. Colonisation was not visually affected when the competing strain was a poor root coloniser, but was severely reduced when the competing strain was a good root coloniser. The results show that the spatio-temporal colonisation of wheat root by P. fluorescens strain 5RL and derivatives is similar to that of strain WCS365 on tomato. The advantage of the use of lux-marked strains is that the results are obtained much quicker than when conventional methods are used and that the result is supplied as an image of the colonisation pattern of many different roots.