Christian Steinberg

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.

Terminal restriction fragment length polymorphism analysis of ribosomal RNA genes to assess changes in fungal community structure in soils

Monitoring the structure and dynamics of fungal communities in soils under agricultural and environmental disturbances is currently a challenge. In this study, a terminal restriction fragment length polymorphism (T-RFLP) fingerprinting method was developed for the rapid comparison of fungal community structures. The terminal restriction fragment polymorphism of different regions of the small-subunit (SSU) ribosomal RNA (rRNA) gene was simulated by sequence comparison using 10 restriction enzymes, and analyzed among three different soils using fungal-specific primers. Polymerase chain reaction amplification of the 3 end of the SSU rRNA gene with the primer nu-SSU-0817-5 and with the fluorescently labelled primer nu-SSU-1536-3, and digestion of the amplicons with AluI and MboI were found to be optimal and were used in a standardized T-RFLP procedure. Both the number and the intensity of terminal restriction fragments detected by capillary gel electrophoresis were integrated in correspondence analyses. Three soils with contrasting physicochemical properties were differentiated according to the structure of their fungal communities. Assessment of the impact on the fungal community structure of the amendment of two soils with compost or manure confirmed the reproducibility and the sensitivity of the method. Shifts in the community structure were detected between non-amended and amended soil samples. In both soils, the shift differed with the organic amendment applied. In addition, the fungal community structures of the two soils were affected in a different way by the same organic amendment. The fingerprinting method provides a rapid tool to investigate the effect of various perturbations on the fungal communities in soils.

Evaluation of restriction analysis of polymerase chain reaction (PCR)-amplified ribosomal DNA for the identification of Fusarium species

Eighty-seven strains belonging to 18 species of Fusarium were characterized by restriction fragment length polymorphism (RFLP) analysis of ribosomal DNA (rDNA). The polymerase chain reaction (PCR) was used to amplify a fragment including internal transcribed spacers and variable domains of the 28S rDNA. Data from RFLP analysis of amplified rDNA using eight frequent-cutting restriction enzymes permitted the definition of 23 rDNA haplotypes. The combination of only four restriction enzymes was suaecient to resolve the 23 haplotypes. Each haplotype could be assigned to a single Fusarium species, except for two haplotypes which were common to two or three closely related species. Polymorphism was found within some Fusarium species. Grouping among Fusarium strains derived from restriction analysis was, on the whole, in agreement with other molecular and morphological classification criteria. Therefore, the PCR}RFLP method described in this paper provides a simple and rapid procedure for the differentiation of Fusarium strains at the species level.

Involvement of clay type and pH in the mechanisms of soil suppressiveness to fusarium wilt of flax

Abstract To assess the influence of clay minerals and soil pH on the degree of soil suppressiveness, 25% ( w w ) kaolinite, Ca-montmorillonite and illite were added to a fusarium wilt-conducive soil (loam, pH 4.0) and 3 values of soil pH (pH 4.0, 5.0 and 7.0) were obtained by liming. The soil-clay mixtures were sown with wheat; after 6 and 30 weeks, respectively, of wheat growth, soil samples were taken. Their levels of suppressiveness to fusarium wilt of flax were assessed and several characteristics of the soil microflora were determined. After 6 weeks of wheat growth, the addition of either montmorillonite or illite significantly increased the degree of soil suppressiveness to fusarium wilts, but only at pH 7. Neither liming alone nor the addition of kaolinite, with or without lime, had an effect. After another 6 months of wheat growth, the level of suppressiveness had increased in all amended soils. The strongest effect was obtained when kaolinite, montmorillonite or illite was added and the soil pH was raised to 7. The level of soil suppressiveness was correlated with physicochemical characteristics (texture, pH, exchangeable Ca and Mg and EDTA-extractable iron), and biological characteristics (population densities of bacteria, fluorescent pseudomonads and autochtonous Fusarium oxysporum, and the induced respiration of the soil microflora after glucose supply). The results are discussed in relation to the common hypotheses related to factors and mechanisms involved in soil suppressiveness to fusarium wilts.

Ribosomal DNA-targeted oligonucleotide probe and PCR assay specific for Fusarium oxysporum

A PCR assay and a hybridisation assay were developed for identification of Fusarium oxysporum. Based on sequence data from the 28S rDNA, two primers (PFO2 and PFO3) and one 20-mer probe (HFO1) were designed. These oligonucleotides were tested with 16 strains of F. oxysporum and 80 strains of 23 other Fusarium species or of eight species from other fungal genera. A PCR procedure for specific amplification of DNA from the F. oxysporum strains using primers PFO2 and PFO3 was set up. Under the conditions defined, no PCR product was obtained with these primers from the 80 other strains. The oligonucleotide probe HFO1 was labelled using a non-radioactive method and evaluated for specificity in dot blot hybridisation experiments with rDNA amplified by PCR. Under optimised conditions, the probe hybridised exclusively with DNA from F. oxysporum strains. Both PCR and hybridisation procedures were validated with 53 additional isolates of F. oxysporum representative of the populations present in four different soils. These specific assays appear to be reliable tools for the identification of F. oxysporum and may have various applications in ecological studies.

Fungicide effects on fungal community composition in the wheat phyllosphere.

The fungicides used to control diseases in cereal production can have adverse effects on non-target fungi, with possible consequences for plant health and productivity. This study examined fungicide effects on fungal communities on winter wheat leaves in two areas of Sweden. High-throughput 454 sequencing of the fungal ITS2 region yielded 235 operational taxonomic units (OTUs) at the species level from the 18 fields studied. It was found that commonly used fungicides had moderate but significant effect on fungal community composition in the wheat phyllosphere. The relative abundance of several saprotrophs was altered by fungicide use, while the effect on common wheat pathogens was mixed. The fungal community on wheat leaves consisted mainly of basidiomycete yeasts, saprotrophic ascomycetes and plant pathogens. A core set of six fungal OTUs representing saprotrophic species was identified. These were present across all fields, although overall the difference in OTU richness was large between the two areas studied.

Mycelial development of Fusarium oxysporum in the vicinity of tomato roots

The patterns of mycelial development (length and branching) of five strains of Fusarium oxysporum were measured in the vicinity of tomato and wheat roots. Two strains were of f. sp. lycopersici, two of f. sp. radicis-lycopersici and the fifth was a non-pathogenic strain currently used as a biocontrol agent. Hyphal extension and branching were measured microscopically using a CCD camera and an image analyser. Furthermore, the role of soluble root exudates, insoluble root materials, glucose supply, and nitrogen supply (casamino acids) on mycelial development was investigated. Each strain had its own pattern of development in the absence of a root. Fungal development of all five strains was stimulated in the vicinity of roots irrespective of plant species but there was no chemotropic response towards or away from the root. Both pathogenic and non-pathogenic F. oxysporum populations exhibited their own characteristic growth and development features which were not related to their pathogenicity. Growth stimulation was mainly related to the presence of organic nitrogen in the soluble exudates. It is proposed that signalling and recognition mechanisms between the host plant and the pathogenic or non-pathogenic strains are likely to occur on or in the root rather than external to the root.

Fusarium diversity in soil using a specific molecular approach and a cultural approach

Fusarium species are ubiquitous in soil. They cause plant and human diseases and can produce mycotoxins. Surveys of Fusarium species diversity in environmental samples usually rely on laborious culture-based methods. In the present study, we have developed a molecular method to analyze Fusarium diversity directly from soil DNA. We designed primers targeting the translation elongation factor 1-alpha (EF-1α) gene and demonstrated their specificity toward Fusarium using a large collection of fungi. We used the specific primers to construct a clone library from three contrasting soils. Sequence analysis confirmed the specificity of the assay, with 750 clones identified as Fusarium and distributed among eight species or species complexes. The Fusarium oxysporum species complex (FOSC) was the most abundant one in the three soils, followed by the Fusarium solani species complex (FSSC). We then compared our molecular approach results with those obtained by isolating Fusarium colonies on two culture media and identifying species by sequencing part of the EF-1α gene. The 750 isolates were distributed into eight species or species complexes, with the same dominant species as with the cloning method. Sequence diversity was much higher in the clone library than in the isolate collection. The molecular approach proved to be a valuable tool to assess Fusarium diversity in environmental samples. Combined with high throughput sequencing, it will allow for in-depth analysis of large numbers of samples.

Effects of nutritional sources on growth of one non-pathogenic strain and four strains of Fusarium oxysporum pathogenic on tomato

As part of an investigation into the factors influencing the colonization of the rhizosphere and the root tissues of host plant, by pathogenic and non-pathogenic strains of Fusarium oxysporum, the effect of a range of carbon (C) sources on the growth habits of five strains of F. oxysporum were compared. The strains used were two F. oxysporum f. sp. lycopersici (Fol strains), two F. oxysporum f. sp. radicis-lycopersici (Forl strains) pathogenic on tomato and strain Fo47, a non-pathogenic strain of F. oxysporum, currently used as a biocontrol agent to reduce severity of fusarium diseases on several crops. Radial extension rates on solid media were measured using soluble saccharides (glucose, xylose and D galacturonic acid), polysaccharides (carboxymethyl cellulose, xylan and pectin) and soluble and insoluble extracts of tomato roots as C sources. Growth parameters were estimated by fitting a logistic equation to biomass data recorded from liquid culture using the same C sources. The strains were characterized by means of the pattern of radial extension rates on the various C sources, and the two Forl strains were discriminated further from the other strains. The growth parameters were unique features of each F. oxysporum strains, whatever the C source. The pathogenic strains did not exhibit particular abilities in degrading the cell wall components. It was concluded that growth habits related to carbohydrate utilization are unique to each strain of F. oxysporum and that these traits are not related to pathogenicity.

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.