EBR1 genomic expansion and its role in virulence of Fusarium species.

Genome sequencing of Fusarium oxysporum revealed that pathogenic forms of this fungus harbour supernumerary chromosomes with a wide variety of genes, many of which likely encode traits required for pathogenicity or niche specialization. Specific transcription factor gene families are expanded on these chromosomes including the EBR1 family (Enhanced Branching). The significance of the EBR1 family expansion on supernumerary chromosomes and whether EBR1 paralogues are functional is currently unknown. EBR1 is found as a single copy in F.graminearum and other fungi but as multiple paralogues in pathogenic F.oxysporum strains. These paralogues exhibit sequence and copy number variation among different host-specific strains and even between more closely related strains. Relative expression of the EBR1 paralogues depends on growth conditions and on the presence of the single EBR1 gene in the core genome. Deletion of EBR1 in the core genome in different F.oxysporum strains resulted in impaired growth, reduced pathogenicity and slightly reduced biocontrol capacities. To identify genes regulated by EBR1, the transcriptomes of wild-type and Δebr1 strains were compared for both F.oxysporum and F.graminearum. These studies showed that in both species, EBR1 regulates genes involved in general metabolism as well as virulence.

The endophytic strain Fusarium oxysporum Fo47: a good candidate for priming the defense responses in tomato roots.

The protective Fusarium oxysporum strain Fo47 is effective in controlling Fusarium wilt in tomato. Previous studies have demonstrated the role of direct antagonism and involvement of induced resistance. The aim of the present study was to investigate whether priming of plant defense responses is a mechanism by which Fo47 controls Fusarium wilt. An in vitro design enabled inoculation of the tap root with Fo47 and the pathogenic strain (Fol8) at different locations and different times. The expression levels of six genes known to be involved in tomato defense responses were quantified using reverse-transcription quantitative polymerase chain reaction (qPCR). Three genes-CHI3, GLUA, and PR-1a-were overexpressed in the root preinoculated with Fo47, and then challenged with Fol8. The genes GLUA and PR-1a were upregulated in cotyledons after inoculation of Fo47. Fungal growth in the root was assessed by qPCR, using specific markers for Fo47 and Fol8. Results showed a reduction of the pathogen growth in the root of the tomato plant preinoculated with Fo47. This study demonstrated that priming of tomato defense responses is one of the mechanisms of action of Fo47, which induces a reduced colonization of the root by the pathogen.

Degradation of aromatic compounds through the ß-ketoadipate pathway is required for pathogenicity of the tomato wilt pathogen Fusarium oxysporum f. sp. lycopersici.

Plant roots react to pathogen attack by the activation of general and systemic resistance, including the lignification of cell walls and increased release of phenolic compounds in root exudate. Some fungi have the capacity to degrade lignin using ligninolytic extracellular peroxidases and laccases. Aromatic lignin breakdown products are further catabolized via the ß-ketoadipate pathway. In this study, we investigated the role of 3-carboxy-cis,cis-muconate lactonizing enzyme (CMLE), an enzyme of the ß-ketoadipate pathway, in the pathogenicity of Fusarium oxysporum f. sp. lycopersici towards its host, tomato. As expected, the cmle deletion mutant cannot catabolize phenolic compounds known to be degraded via the ß-ketoadipate pathway. In addition, the mutant is impaired in root invasion and is nonpathogenic, even though it shows normal superficial root colonization. We hypothesize that the ß-ketoadipate pathway in plant-pathogenic, soil-borne fungi is necessary to degrade phenolic compounds in root exudate and/or inside roots in order to establish disease.

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.

The nuclear protein Sge1 of Fusarium oxysporum is required for parasitic growth.

Dimorphism or morphogenic conversion is exploited by several pathogenic fungi and is required for tissue invasion and/or survival in the host. We have identified a homolog of a master regulator of this morphological switch in the plant pathogenic fungus Fusarium oxysporum f. sp. lycopersici. This non-dimorphic fungus causes vascular wilt disease in tomato by penetrating the plant roots and colonizing the vascular tissue. Gene knock-out and complementation studies established that the gene for this putative regulator, SGE1 (SIX Gene Expression 1), is essential for pathogenicity. In addition, microscopic analysis using fluorescent proteins revealed that Sge1 is localized in the nucleus, is not required for root colonization and penetration, but is required for parasitic growth. Furthermore, Sge1 is required for expression of genes encoding effectors that are secreted during infection. We propose that Sge1 is required in F. oxysporum and other non-dimorphic (plant) pathogenic fungi for parasitic growth.

Microbiological control of soil-borne phytopathogenic fungi with special emphasis on wilt-inducing Fusarium oxysporum.

Plant diseases induced by soil-borne plant pathogens are among the most difficult to control. In the absence of effective chemical control methods, there is renewed interest in biological control based on application of populations of antagonistic micro-organisms. In addition to Pseudomonas spp. and Trichoderma spp., which are the two most widely studied groups of biological control agents, the protective strains of Fusarium oxysporum represent an original model. These protective strains of F. oxysporum can be used to control wilt induced by pathogenic strains of the same species. Exploring the mechanisms involved in the protective capability of these strains is not only necessary for their development as commercial biocontrol agents but raises many basic questions related to the determinism of pathogenicity versus biocontrol capacity in the F. oxysporum species complex. In this paper, current knowledge regarding the interaction between the plant and the protective strains is reviewed in comparison with interactions between the plant and pathogenic strains. The success of biological control depends not only on plant-microbial interactions but also on the ecological fitness of the biological control agents.

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.

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.

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.

Wanted: pathogenesis-related marker molecules for Fusarium oxysporum.

Although Fusarium oxysporum pathogens cause severe wilts in about 80 botanical species, the mechanisms of pathogenicity and symptom induction are poorly understood. Knowledge about the genetic and biochemical pathways involved in the pathogenesis of F. oxysporum would be invaluable in getting targets for both fungicide development and search for biocontrol agents. In this respect, we described the main approaches that have been developed to identify some mechanisms underlying the pathogenesis of F. oxysporum. During the last decades, the potential functions triggering of F. oysporum pathogenicity have mainly been investigated by comparing soilborne pathogenic strains with nonpathogenic ones with regards to the analysis of the pre- and infection stages and of the resulting plant-fungus interactions. The relatively recent progress in the molecular biology of this fungus has allowed complementary approaches to be developed in order to identify key factors involved in F. oxysporum pathogenicity. Screening mutants of F. oxysporum for loss of virulence led to the successful identification of some pathogenesis-related factors, such as hydrophobicity or attachment of germlings. Taken together, the strategies described above support the idea that changes in fungal metabolism is also of importance in triggering of F. oxysporum pathogenesis.

Recovery of Fusarium oxysporum Fo47 Mutants Affected in Their Biocontrol Activity After Transposition of the Fot1 Element.

ABSTRACT To investigate the biocontrol mechanisms by which the antagonistic Fusarium oxysporum strain Fo47 is active against Fusarium wilt, a Fot1 transposon-mediated insertional mutagenesis approach was adopted to generate mutants affected in their antagonistic activity. Ninety strains in which an active Fot1 copy had transposed were identified with a phenotypic assay for excision and tested for their biocontrol activity against F. oxysporum f. sp. lini on flax in greenhouse experiments. Sixteen strains were affected in their capacity to protect flax plants, either positively (more antagonistic than Fo47) or negatively (less antagonistic). The molecular characterization of these mutants confirms the excision of Fot1 and its reinsertion in most of the cases. Moreover, we demonstrate that other transposable elements such as Fot2, impala, and Hop have no transposition activity in the mutant genomes. The phenotypic characterization of these mutants shows that they are affected neither in their in vitro growth habit nor in their competitiveness in soil compared with wild-type strain Fo47. These results show that mutants are not impaired in their saprophytic phase and suggest that the altered biocontrol phenotype should likely be expressed during the interaction with the host plant.

Colonization of tomato root by a non-pathogenic strain of Fusarium oxysporum.

A strain of non-pathogenic Fusarium oxysporum Schlecht. emend, Snyd. & Hans. has been selected for its capacity to reduce the incidence of Fusarium wilt of tomato. Among the possible modes of action of this strain, competition with the pathogen for the colonization of the root surface and tissues has been proposed. In order to study the pattern of root colonization, young Lycopersicon etculentum Miller (tomato) plants grown in a nutrient solution were inoculated by a suspension of F. oxysporum microconidia and processed at time-intervals for microscopic observations. The fungal strain was transformed with the Gus reporter gene to facilitate the observations. Within 24 h of inoculation the root surface was colonized by a dense network of hyphae, with the exception of the apex, which was colonized only after 48 h. A few hyphae were observed penetrating into the epidermis, leading to the internal colonization of the root cortex. This colonization was always discontinuous, since defence reactions of the plant limited the extension of the fungus. The barrier formed by thickenings and coilings of the cell walls and hypertrophied cells was most frequently observed in the external cortex and, sometimes, deeper in the internal cortex, close to the vessels which were never colonized. Typical defence reactions such as wall appositions, intercellular plugging and intracellular osmiophilic deposits, were frequently observed. This is the first report, based on microscopic observations, of the capacity of a non-pathogenic strain of F. oxysporum to colonize roots of tomato.