Chantal Olivain

Biological Control of Plant Diseases: The European Situation

The most common approach to biological control consists of selecting antagonistic microorganisms, studying their modes of action and developing a biological control product. Despite progress made in the knowledge of the modes of action of these biological control agents (BCAs), practical application often fails to control disease in the fields. One of the reasons explaining this failure is that the bio-control product is used the same way as a chemical product. Being biological these products have to be applied in accordance with their ecological requirements. Another approach consists of induction of plant defence reactions. This can be done by application of natural substances produced by or extracted from microorganisms, plants, or algae. Since they do not aim at killing the pathogens, these methods of disease control are totally different from chemical control. Although promising, these methods have not been sufficiently implemented under field conditions. A third approach consists of choosing cultural practices that might decrease the incidence or severity of diseases. These methods include the choice of an appropriate crop rotation with management of the crop residues, application of organic amendments and the use of new technology such as the biological disinfestation of soils. Biological control practices need an integrative approach, and more knowledge than chemical control.

Implication of systemic induced resistance in the suppression of Fusarium wilt of tomato by Pseudomonas fluorescens WCS417r and by nonpathogenic Fusarium oxysporum Fo47.

Fluorescent pseudomonads and nonpathogenic Fusarium oxysporum have been shown to suppress fusarium wilts. This suppression has been related to both microbial antagonism and induced resistance.The aim of the present study was to assess the relative importance of systemic induced resistance in the suppression of fusarium wilt of tomato in commercial-like conditions by a reference strain of each type of microorganism (P. fluorescens WCS417r and nonpathogenic F. oxysporum Fo47). The spatial separation of the pathogen and the biocontrol strains excluded any possible microbial antagonism and implicated the involvement of the systemic induced resistance; whereas the absence of any separation between these microorganisms allowed the expression of both mechanisms. Since systemic induced resistance has often been associated with the synthesis of PR-proteins, their accumulation in tomato plants inoculated with WCS417r or with Fo47 was determined.The analysis of the results indicates that the suppression of fusarium wilt by P. fluorescens WCS417r was ascribed to systemic induced resistance without any detection of the PR-proteins tested (PR-1 and chitinases). In contrast, the suppression achieved by nonpathogenic F. oxysporum Fo47 appeared to be mainly ascribed to microbial antagonism but also to a lesser extent to systemic induced resistance. This induced resistance could be related to the accumulation of PR-1 and chitinases.The possible relationship between the ability of Fo47 to suppress fusarium wilt more efficiently than WCS417r and its ability to show both mechanisms is discussed.

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.

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

ABSTRACT 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

ABSTRACT 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.

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.

Recovery of Mutants Impaired in Pathogenicity After Transposition of Impala in Fusarium oxysporum f. sp. melonis

ABSTRACT The ability of transposon impala to inactivate genes involved in pathogenicity was tested in Fusarium oxysporum f. sp. melonis. Somatic excision of an impala copy inserted in the nitrate reductase-encoding niaD gene was positively selected through a phenotypic assay based on the restoration of nitrate reductase activity. Independent excision events were analyzed molecularly and shown to carry reinsertedimpala in more than 70% of the cases. Mapping of reinserted impala elements on large NotI-restriction fragments showed that impala transposes randomly. By screening 746 revertants on plants, a high proportion (3.5%) of mutants impaired in their pathogenic potential was recovered. According to the kinetics of wilt symptom development, the strains that were impaired in pathogenicity were clustered in three classes: class 1 grouped two strains that never induced Fusarium wilt symptoms on the host plant; class 2 and class 3 grouped 15 and 9 revertants which caused symptoms more than 50 and 30 days after inoculation, respectively. The first results demonstrate the efficiency of transposition in generating mutants affected in pathogenicity, which are usually difficult to obtain by classical mutagenesis, and open the possibility to clone the altered genes with impala as a tag.

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.

Degradation of aromatic compounds through the β-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 β-ketoadipate pathway. In this study, we investigated the role of 3-carboxy-cis,cis-muconate lactonizing enzyme (CMLE), an enzyme of the β-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 β-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 β-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.

USING STRAINS OF FUSARIUM OXYSPORUM TO CONTROL FUSARIUM WILTS: DREAM OR REALITY?

Soil-borne strains of F. oxysporum are involved in the mechanisms of soil suppressiveness to Fusarium wilts, and many attempts have been made to use strains of Fusarium oxysporum to control Fusarium diseases. The modes of action of the protective strains are diverse; they include direct antagonism, competition for nutrients, and indirect antagonism through induced resistance of the plant. The use of newer tools has enabled a reconsideration of these modes of action; e.g., competition for infection sites whose importance has been minimized, and to make progress in the understanding of the interactions between the plant and either pathogenic or protective strains of F. oxysporum. Even though the mechanisms of biocontrol of F. oxysporum are far from being understood, several processes of mass production have been developed to enable field application of the biocontrol strains. These strains possess a strong ecological fitness and establish in soil of different physico-chemical properties. Their introduction into the soil does not durably modify the structure of the soil-borne communities of fungi and bacteria, indicating that their use does not present any risk to the environment.