Visualization of interactions between a pathogenic and a beneficial Fusarium strain during biocontrol of tomato foot and root rot

The soilborne fungus Fusarium oxysporum f. sp. radicis-lycopersici causes tomato foot and root rot (TFRR), which can be controlled by the addition of the nonpathogenic fungus F. oxysporum Fo47 to the soil. To improve our understanding of the interactions between the two Fusarium strains on tomato roots during biocontrol, the fungi were labeled using different autofluorescent proteins as markers and subsequently visualized using confocal laser scanning microscopy. The results were as follows. i) An at least 50-fold excess of Fo47over F. oxysporum f. sp. radicis-lycopersici was required to obtain control of TFRR. ii) When seedlings were planted in sand infested with spores of a single fungus, Fo47 hyphae attached to the root earlier than those of F. oxysporum f. sp. radicis-lycopersici. iii) Subsequent root colonization by F. oxysporum f. sp. radicis-lycopersici was faster and to a larger extent than that by Fo47. iv) Under disease-controlling conditions, colonization of tomato roots by the pathogenic fungus was significantly reduced. v) When the inoculum concentration of Fo47 was increased, root colonization by the pathogen was arrested at the stage of initial attachment to the root. vi) The percentage of spores of Fo47 that germinates in tomato root exudate in vitro is higher than that of the pathogen F. oxysporum f. sp. radicis-lycopersici. Based on these results, the mechanisms by which Fo47 controls TFRR are discussed in terms of i) rate of spore germination and competition for nutrients before the two fungi reach the rhizoplane; ii) competition for initial sites of attachment, intercellular junctions, and nutrients on the tomato root surface; and iii) inducing systemic resistance.     

Colonization of tomato root by pathogenic and nonpathogenic Fusarium oxysporum strains inoculated together and separately into the soil

In soil, fungal colonization of plant roots has been traditionally studied by indirect methods such as microbial isolation that do not enable direct observation of infection sites or of interactions between fungal pathogens and their antagonists. Confocal laser scanning microscopy was used to visualize the colonization of tomato roots in heat-treated soil and to observe the interactions between a nonpathogenic strain, Fo47, and a pathogenic strain, Fol8, inoculated onto tomato roots in soil. When inoculated separately, both fungi colonized the entire root surface, with the exception of the apical zone. When both strains were introduced together, they both colonized the root surface and were observed at the same locations. When Fo47 was introduced at a higher concentration than Fol8, it colonized much of the root surface, but hyphae of Fol8 could still be observed at the same location on the root. There was no exclusion of the pathogenic strain by the presence of the nonpathogenic strain. These results are not consistent with the hypothesis that specific infection sites exist on the root for Fusarium oxysporum and instead support the hypothesis that competition occurs for nutrients rather than for infection sites.     

Tomato root colonization by fluorescent-tagged pathogenic and protective strains of Fusarium oxysporum in hydroponic culture differs from root colonization in soil

The colonization process of tomato roots inoculated separately or/and simultaneously by a pathogenic Fusarium oxysporum f. sp. lycopersici strain Fol8 and the protective F. oxysporum strain Fo47, genetically tagged with the red and green fluorescent protein genes, respectively, was studied in a hydroponic culture. Plants were coinoculated with Fol8 and Fo47 at two conidial concentration ratios of 1/1 and 1/100, in which biological control was not effective or effective, respectively. First observation of fungi on root was possible 48 h after inoculation at a high inoculum level and 5 days post inoculation at the lower concentration of inoculum. The pattern of root colonization was similar for both strains with the initial development of hyphal network on the upper part of taproot, followed by the growth of hyphae towards the elongation zone, lateral roots and root apices. Finally, the whole elongation zone and root apex were invaded by both strains but no specific infection sites were observed. When coinoculated, both strains could grow very closely or even at the same spot on the root surface. At the nonprotective ratio, Fol8 was the successful colonizer, but application of Fo47 at a concentration 100 times >Fol8 delayed vessel colonization by the pathogen.     

Development of a strain-specific real-time PCR assay for the detection and quantification of the biological control agent Fo47 in root tissues

Being able to identify specifically a biological control agent at the strain level is not the only requirement set by regulations (EC)1107/2009, it is also necessary to study the interactions of the agent with the plant and the pathogen in the rhizosphere. Fo47 is a soil-borne strain of Fusarium oxysporum which has the capacity to protect several plant species against the pathogenic formae speciales of F. oxysporum inducing wilts. A strain-specific sequence-characterized amplified region marker has been designed which makes it possible to distinguish Fo47 from other strains of F. oxysporum. In addition, a real-time PCR assay has been developed to quantify Fo47 in root tissues. The proposed assay has been validated by following the dynamics of root colonization of tomato plants grown in soil infested with Fo47. Results showed that with the method it is possible to quantify Fo47 in roots in the absence or presence of the pathogen and in the absence or in presence of the native microbial communities.     

Use of ELISA and GUS‐transformed strains to study competition between pathogenic and non‐pathogenic Fusarium oxysporum for root colonization

To characterize the ability of different strains of Fusarium oxysporum to colonize roots, and to analyze competition for root colonization between pathogenic and non‐pathogenic strains of F. oxysporum, it was necessary to develop specific labelling techniques for quantification of root colonization. Two methods were selected: the production of polyclonal antibodies, and the use of GUS‐transformed strains of F. oxysporum. The polyclonal antibodies recognized infected plants, and gave a minimum reaction with healthy plants, but were not specific for individual strains of F. oxysporum. These antibodies enabled total density of F. oxysporum to be assessed on roots, by ELISA. Metabolic activity of the root population of GUS‐marked strains was assessed by measuring the glucuronidase activity. Strains showed a diversity in their ability to colonize roots: patterns of root colonization were similar, but the intensity and the speed of colonization differed according to the plant—fungus combination used. Results demonstrated competition between the pathogenic and the non‐pathogenic strains for root colonization. In the presence of the non‐pathogenic strain Fo 47, the competition seems to be reciprocal, affecting both the pathogen and non‐pathogenic strain. Other non‐pathogenic strains reduced root colonization by the pathogenic strain, but some strains did not reduce the metabolic activity of the pathogen, suggesting that different mechanisms are involved in the interaction between pathogenic and non‐pathogenic F. oxysporum.     

Colonization of flax roots and early physiological responses of flax cells inoculated with pathogenic and nonpathogenic strains of Fusarium oxysporum

Fusarium oxysporum includes nonpathogenic strains and pathogenic strains that can induce necrosis or tracheomycosis in plants. The objective of this study was to compare the abilities of a pathogenic strain (Foln3) and a nonpathogenic strain (Fo47) to colonize flax roots and to induce early physiological responses in flax cell culture suspensions. Both strains colonized the outer cortex of the root; however, plant defense reactions, i.e., the presence of wall appositions, osmiophilic material, and collapsed cells, were less frequent and less intense in a root colonized by Foln3 than by Fo47. Early physiological responses were measured in flax cell suspensions confronted with germinated microconidia of both strains. Both pathogenic (Foln3) and nonpathogenic strains (Fo47) triggered transient H(2)O(2) production in the first few minutes of the interaction, but the nonpathogenic strain also induced a second burst 3 h postinoculation. Ca(2+) influx was more intense in cells inoculated with Fo47 than in cells inoculated with Foln3. Similarly, alkalinization of the extracellular medium was higher with Fo47 than with Foln3. Inoculation of the fungi into flax cell suspensions induced cell death 10 to 20 h postinoculation, with a higher percentage of dead cells observed with Fo47 than with Foln3 beginning at 14 h. This is the first report showing that early physiological responses of flax cells can be used to distinguish pathogenic and nonpathogenic strains of the soil-borne fungus F. oxysporum.     

Colonization of Tomato Root by Pathogenic and Nonpathogenic Fusarium oxysporum Strains Inoculated Together and Separately into the Soil

In soil, fungal colonization of plant roots has been traditionally studied by indirect methods such as microbial isolation that do not enable direct observation of infection sites or of interactions between fungal pathogens and their antagonists. Confocal laser scanning microscopy was used to visualize the colonization of tomato roots in heat-treated soil and to observe the interactions between a nonpathogenic strain, Fo47, and a pathogenic strain, Fol8, inoculated onto tomato roots in soil. When inoculated separately, both fungi colonized the entire root surface, with the exception of the apical zone. When both strains were introduced together, they both colonized the root surface and were observed at the same locations. When Fo47 was introduced at a higher concentration than Fol8, it colonized much of the root surface, but hyphae of Fol8 could still be observed at the same location on the root. There was no exclusion of the pathogenic strain by the presence of the nonpathogenic strain. These results are not consistent with the hypothesis that specific infection sites exist on the root for Fusarium oxysporum and instead support the hypothesis that competition occurs for nutrients rather than for infection sites.     

Piriformospora indica,a plant-root-colonising fungus enhances growth and tolerance of wheat to seedlings damping-off caused by Fusarium oxysporum

In greenhouse experiments, we investigated the potentials of Piriformospora indica (Pi) to penetrate and colonise roots of wheat and to induce beneficial effects on growth as well as to reduce seedlings damping-off disease of wheat caused by the soil-borne fungus Fusarium oxysporum (Fo). By microscopy we observed the intracellular hyphae within root cortex cells of 7–14 day-old plants and chlamydospores within root cortex cells of 14–28 day-old plants and within root hair cells of 21–28 day-old plants. Moreover, diagnostic PCR based on the β-tubulin gene marker confirmed the presence of Pi in roots of inoculated plants. Also, we found that plants inoculated with Pi exhibited a better growth of roots and shoots as well as early flowering as compared with non-treated plants. Moreover, Pi conferred tolerance of wheat to seedlings damping-off i.e. by reducing the harmful effects of Fo on infected plants.     

Colonization of Flax Roots and Early Physiological Responses of Flax Cells Inoculated with Pathogenic and Nonpathogenic Strains of Fusarium oxysporum

Fusarium oxysporum includes nonpathogenic strains and pathogenic strains that can induce necrosis or tracheomycosis in plants. The objective of this study was to compare the abilities of a pathogenic strain (Foln3) and a nonpathogenic strain (Fo47) to colonize flax roots and to induce early physiological responses in flax cell culture suspensions. Both strains colonized the outer cortex of the root; however, plant defense reactions, i.e., the presence of wall appositions, osmiophilic material, and collapsed cells, were less frequent and less intense in a root colonized by Foln3 than by Fo47. Early physiological responses were measured in flax cell suspensions confronted with germinated microconidia of both strains. Both pathogenic (Foln3) and nonpathogenic strains (Fo47) triggered transient H₂O₂ production in the first few minutes of the interaction, but the nonpathogenic strain also induced a second burst 3 h postinoculation. Ca²⁺ influx was more intense in cells inoculated with Fo47 than in cells inoculated with Foln3. Similarly, alkalinization of the extracellular medium was higher with Fo47 than with Foln3. Inoculation of the fungi into flax cell suspensions induced cell death 10 to 20 h postinoculation, with a higher percentage of dead cells observed with Fo47 than with Foln3 beginning at 14 h. This is the first report showing that early physiological responses of flax cells can be used to distinguish pathogenic and nonpathogenic strains of the soil-borne fungus F. oxysporum.     

Role of chemotaxis toward fusaric acid in colonization of hyphae of Fusarium oxysporum f. sp. radicis-lycopersici by Pseudomonas fluorescens WCS365

Pseudomonas fluorescens WCS365 is an excellent competitive colonizer of tomato root tips after bacterization of seed or seedlings. The strain controls tomato foot and root rot (TFRR) caused by the phytopathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici. Under biocontrol conditions, fungal hyphae were shown to be colonized by WCS365 bacteria. Because chemotaxis is required for root colonization by WCS365 cells, we studied whether chemotaxis also is required for hyphae colonization. To that end, an in vitro assay was developed to study hyphae colonization by bacteria. The results indicated that cells of the cheA mutant FAJ2060 colonize hyphae less efficiently than cells of wild-type strain WCS365, when single strains were analyzed as well as when both strains were applied together. Cells of WCS365 show a chemotactic response toward the spent growth medium of F. oxysporum f. sp. radicis-lycopersici, but those of its cheA mutant, FAJ2060, did not. Fusaric acid, a secondary metabolite secreted by Fusarium strains, appeared to be an excellent chemo-attractant. Supernatant fluids of a number of Fusarium strains secreting different levels of fusaric acid were tested as chemo-attractants. A positive correlation was found between chemo-attractant activity and fusaric acid level. No chemotactic response was observed toward the low fusaric acid-producer FO242. Nevertheless, the hyphae of FO242 still were colonized by WCS365, suggesting that other metabolites also play a role in this process. The possible function of hyphae colonization for the bacterium is discussed.     

Effect of pseudobactin 358 production by Pseudomonas putida WCS358 on suppression of fusarium wilt of carnations by nonpathogenic Fusarium oxysporum Fo47.

Nonpathogenic Fusarium oxysporum Fo47b10 combined with Pseudomonas putida WCS358 efficiently suppressed fusarium wilt of carnations grown in soilless culture. This suppression was significantly higher than that obtained by inoculation of either antagonistic microorganism alone. The increased suppression obtained by Fo47b10 combined with WCS358 only occurred when Fo47b10 was introduced at a density high enough (at least 10 times higher than that of the pathogen) to be efficient on its own. P. putida WCS358 had no effect on disease severity when inoculated on its own but significantly improved the control achieved with nonpathogenic F. oxysporum Fo47b10. In contrast, a siderophore-negative mutant of WCS358 had no effect on disease severity even in the presence of Fo47b10. Since the densities of both bacterial strains at the root level were similar, the difference between the wild-type WCS358 and the siderophore-negative mutant with regard to the control of fusarium wilt was related to the production of pseudobactin 358. The production of pseudobactin 358 appeared to be responsible for the increased suppression by Fo47b10 combined with WCS358 relative to that with Fo47b10 alone.     

Effect of pseudobactin 358 production by Pseudomonas putida WCS358 on suppression of fusarium wilt of carnations by nonpathogenic Fusarium oxysporum Fo47.

Nonpathogenic Fusarium oxysporum Fo47b10 combined with Pseudomonas putida WCS358 efficiently suppressed fusarium wilt of carnations grown in soilless culture. This suppression was significantly higher than that obtained by inoculation of either antagonistic microorganism alone. The increased suppression obtained by Fo47b10 combined with WCS358 only occurred when Fo47b10 was introduced at a density high enough (at least 10 times higher than that of the pathogen) to be efficient on its own. P. putida WCS358 had no effect on disease severity when inoculated on its own but significantly improved the control achieved with nonpathogenic F. oxysporum Fo47b10. In contrast, a siderophore-negative mutant of WCS358 had no effect on disease severity even in the presence of Fo47b10. Since the densities of both bacterial strains at the root level were similar, the difference between the wild-type WCS358 and the siderophore-negative mutant with regard to the control of fusarium wilt was related to the production of pseudobactin 358. The production of pseudobactin 358 appeared to be responsible for the increased suppression by Fo47b10 combined with WCS358 relative to that with Fo47b10 alone.     

Biological Control of Fusarium oxysporum f.sp. asparagi by Applying Non-pathogenic Isolates of F . oxysporum

Root rot severity of asparagus plants grown in sterilized field soil inoculated with Fusarium oxysporum f . sp . asparagi (Foa) was reduced by more than 50% when the soil was precolo nized by each of 13 non - pathogenic (np) isolates of F. oxysporum originating from asparagus roots or field soils . In a greenhouse experiment , application of six np isolates to naturally infested field soil was followed by a 23 - 49% decrease of disease severity , depending on the isolate . One of them , Fo47 originating from Fusarium suppressive soil in France , was applied to field plots infested with Foa . Foa root rot was not suppressed in asparagus plants grown for 1 year in these plots . Pathogenic and np isolates extensively colonized the root surface and isolates of both types infected the roots of asparagus plants grown in sterilized field soil , with significant differences among the np isolates . Inoculation of sterilized field soil with np isolates reduced germination of Foa chlamydospores by 43 - 64% depending on the isolate used . It is concluded that np isolates of F. oxysporum can suppress asparagus root rot caused by Foa in naturally infested field soil . The differences for root colonization capacity among the np isolates imply that selection for this trait might reveal isolates that perform better under field conditions .     

In vivo time-lapse documentation using confocal and multi-photon microscopy reveals the mechanisms of invasion into the Arabidopsis root vascular system by Fusarium oxysporum

Fusarium oxysporum, a major soil-borne fungal pathogen, causes vascular wilt, damping-off, and root rot diseases on over 100 cultivated plant species. Mechanisms of root colonization by F. oxysporum in Arabidopsis thaliana were studied through in planta 3-dimensional time-lapse documentation using confocal and multi-photon microscopy. Data from individual encounter sites were acquired repeatedly over a several day period without physical manipulation or retrieval from the growth chamber. In vivo observations were facilitated by transformation of F. oxysporum for constitutive cytoplasmic expression of the fluorescent protein ZsGreen, and host responses were monitored using autofluorescence or GFP-tagged endoplasmic reticulum. Penetration into the vascular system occurred primarily in the meristematic region of primary and lateral roots. Fungal hyphae may release phytotoxin(s) that compromise host cells not directly in contact with hyphae. This novel approach was essential for visualizing the dynamic interactions between F. oxysporum and A. thaliana from both the host and pathogen sides.     

Suppression of Fusarium Colonization of Cotton Roots and Fusarium Wilt by Seed Treatments with Gliocladium virens and Bacillus subtilis

In a growth chamber at 25 C, the fungal antagonist Gliocladium virens colonized tap roots and secondary roots of cotton in non-sterile soil after seed treatment with preparations of G. virens. Colonization of tap roots by G. virens increased over time, and decreased with root depth. Seed treatments with G. virens strains G-4 and G-6 and with Bacillus subtilis strains GB03 and GB07 reduced the colonization of tap roots and secondary roots of cotton seedlings by Fusarium spp. Under greenhouse conditions, the same seed treatments suppressed the incidence and severity of fusarium wilt of cotton in soil infested with Fusarium oxysporum f. sp. vasinfectum and Meloidogyne incognita. Gliotoxin, produced by 'Q-group' strains of G. virens, inhibited F. oxysporum f. sp. vasinfectum in vitro. The seed treatments with G. virens strain G-6 and B. subtilis strains GB03 and GB07 did not affect the reproduction of root-knot nematodes. The results of this study may help to explain why the treatment of cotton seed with biocontrol agents often results in more vigorous and higher yielding plants, and indicate that there is potential for using G. virens and B. subtilis as seed treatments to control fusarium wilt of cotton.     

Novel mode of resistance to Fusarium infection by a mild dose pre-exposure of cadmium in wheat.

Exposure of healthy wheat seeds (Triticum aestivum var Sonalika) to mild dose of cadmium (Cd(2+)) given as 50 microM CdCl(2) for 48 h and then washed off Cd(2+) offered resistance to the subsequent infection by Fusarium oxysporum inoculum. Seven days old seedlings having two primary leaves were aseptically inoculated with fungus, F. oxysporum (1 x 10(6)) spores. The seedlings pre-exposed to low level of Cd(2+) survived the Fusarium infection, while plantlets without Cd(2+) stress wilted and then perished due to Fusarium infection. The stress associated proteins induced by Cd(2+) (50 microM), F. oxysporum and by the co-stress (50 microM Cd(2+) and then with F. oxysporum) treatments were observed to be of same molecular weight (51 kDa). Antibody was raised against the purified Cd(2+)-stress associated protein (CSAP). Immuno-gold labeling of wheat seedling root tissue showed the presence of this CSAP in Cd(2+) pre-exposed and in co-stressed tissues and to be located predominantly on the inner linings of the cell membranes. We also observed that the anti-CSAP-antibody also labeled the root tissue of only Fusarium inoculated seedlings and the gold labeling was intensely located on the membrane. This cross-reaction of anti-CSAP suggests that Fusarium-induced stress protein (FISP) possibly has close homology to CSAP. We thus show for the first time the over expression of a high molecular mass protein by mild dose of Cd(2+) pre-exposure to wheat seeds which subsequently provided protection against Fusarium infection. This mode of resistance developed by an abiotic stress-causing agent against pathogen infection is novel.     

Composts containing fluorescent pseudomonads suppress fusarium root and stem rot development on greenhouse cucumber

Three composts (Ball, dairy, and greenhouse) were tested for the ability to suppress the development of Fusarium root and stem rot (caused by Fusarium oxysporum f. sp. radicis-cucumerinum) on greenhouse cucumber. Dairy and greenhouse composts significantly reduced disease severity (P = 0.05), while Ball compost had no effect. Assessment of total culturable microbes in the composts showed a positive relationship between disease suppressive ability and total population levels of pseudomonads. In vitro antagonism assays between compost-isolated bacterial strains and the pathogen showed that strains of Pseudomonas aeruginosa exhibited the greatest antagonism. In growth room trials, strains of P. aeruginosa and nonantagonistic Pseudomonas maculicola, plus 2 biocontrol strains of Pseudomonas fluorescens, were tested for their ability to reduce (i) survival of F. oxysporum, (ii) colonization of plants by the pathogen, and (iii) disease severity. Cucumber seedlings grown in compost receiving P. aeruginosa and P. fluorescens had reduced disease severity index scores after 8 weeks compared with control plants without bacteria. Internal stem colonization by F. oxysporum was significantly reduced by P. aeruginosa. The bacteria colonized plant roots at 1.9 × 10(6) ± 0.73 × 10(6) CFU·(g root tissue)-1 and survival was >107 CFU·(g compost)-1 after 6 weeks. The locus for 2,4-diacetylphloroglucinol production was detected by Southern blot analysis and confirmed by PCR. The production of the antibiotic 2,4-diacetylphloroglucinol in liquid culture by P. aeruginosa was confirmed by thin layer chromatography. These results demonstrate that composts containing antibiotic-producing P. aeruginosa have the potential to suppress diseases caused by Fusarium species.     

Adhesion of Fusarium oxysporum conidia to tomato roots

The occurence of a binding process between Fusarium oxysporum conidia and the surface of tomato roots was demonstrated in vitro by using a quantitative assay and a serial washing procedure. The number of conidia bound per root unit increased with increasing the concentration of the spores in solution until binding reached saturation. The attachment could be described accurately in terms of the Langmuir adsorption isotherm, indicating the existence of a single class of specific, high-affinity adherence sites on the root surface. No differences were detected in the extent of binding of several strains of F. oxysporum differing either in pathogenicity or in host range. Site-specific binding of F. oxysporum conidia may be important in securing the fungal spores at the root surface, after which germination and other processes required for colonization can proceed.     

Interactions in the tomato rhizosphere of two Pseudomonas biocontrol strains with the phytopathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici

The fungus Fusarium oxysporum f. sp. radicis-lycopersici causes foot and root rot of tomato plants, which can be controlled by the bacteria Pseudomonas fluorescens WCS365 and P. chlororaphis PCL1391. Induced systemic resistance is thought to be involved in biocontrol by P. fluorescens WCS365. The antifungal metabolite phenazine-1-carboxamide (PCN), as well as efficient root colonization, are essential in the mechanism of biocontrol by P. chlororaphis PCL1391. To understand the effects of bacterial strains WCS365 and PCL1391 on the fungus in the tomato rhizosphere, microscopic analyses were performed using different autofluorescent proteins as markers. Tomato seedlings were inoculated with biocontrol bacteria and planted in an F. oxysporum f. sp. radicis-lycopersici-infested gnotobiotic sand system. Confocal laser scanning microscope analyses of the interactions in the tomato rhizosphere revealed that i) the microbes effectively compete for the same niche, and presumably also for root exudate nutrients; ii) the presence of either of the two bacteria negatively affects infection of the tomato root by the fungus; iii) both biocontrol bacteria colonize the hyphae extensively, which may represent a new mechanism in biocontrol by these pseudomonads; and iv) the production of PCN by P. chlororaphis PCL1391 negatively affects hyphal growth and branching, which presumably affects the colonization and infecting ability of the fungus.