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FP6 - 1687
2E-BCAS IN CROPS - Enhancement and Exploitation of Soil Biocontrol Agents for Bio-Constraint Management in Crops |
| Source: | Annual Progress Report January 2005 |
Executive Summary
Objectives
The main objective of the project is to improve the efficacysome of the already available or the most promising biocontrol microorganisms (such as Fusarium, Trichoderma or Coniothyrium sp.) in vegetable crops. particularly cabbage. tomato, carrots arid lettuce. or the biological control of some diseases, such as those caused by Sclerotinia Fusarium or Pythium spp,. or some parasitic and perennial weeds such as Orobanche sp. and Cirsium arvense.
The studies of the genetic and physiological enhancement strategies, the ecological fitness of the agents, the production. formulation and application methods, the integration with other organisms and with control methods, and assessing their quality arid the risk of release into the environment will allow to improve the efficacy of fungal biocontrol agents, and to use them more widely at the European level, giving new important tools to support the production of safer and healthier foods.
Activities and results
The project has been organized in a series of nine interactive work packages (WPs). All of these,. with the exception of WP7 (Assessment of field efficacy), started in the first year of the project. The following activities for each WP were performed during the reporting period (month 1-12):
WP1 Efficacy enhancement through the knowledge of genetic characters
This WP aimed at characterising genes and gene products associated with biological control to enable better prediction and reliability of activity of these agents in the environment.
The studies on differential gene expression of an effective biocontrol antagonistic strain of F. oxysporum with a mutant which has lost its bioontrol capacity during tomato infection were started. Ihis allows genes and functions involved in the determination of the biocontrol capacity to be identified.
Considering it is known that the antagonistic mechanism of Trichoderma requires the degradation of cell wall of the fungal host by various fungal cell wall degrading enzymes the examination of changes in gene expression during interaction with the pathogenic fungus Sclerotinia sclerotiorum was started by building cDNA libraries from Trichoderma grown on cell walls or sclerotia of the pathogen
To identify changes in gene expression by the biocontrol agent C. minitans during infection of its host S. sclerotiorum, especially during early stages, C. minitans was inoculated onto sclerotia and cDNA libraries were produced at key infection stages. Genes differentially expressed started to he identified and cloned. To identify pathogenicity genes REMI (restriction enzyme mediated integration) and ATMT (Agrobacterium tumefaciens - mediated transformation) of C. minitans were developed.
To obtain strains that are hyperviruient towards Orubanche due to the insufficient virulence of all organisms isolated, the Nep1 gene was engineered into Fusarium arthrosporoides. Furthermore, the construction of a universal high expression cassette based on the highly successful cassette used with NEP1 was started.
In order to study the role of novel genes possibly associated to pathogcnieitv on plant pathogenic fungi, different Trichoderma mutant strains were obtained and characterized molecularly and physiologically. Trichoderma mutants deficient (obtained by targeted knock out) for genes involved in signal transduction do not display an improved biocontrol activity against Pythium ultimm and Rhizoctonia solani on tomato plants. Trichoderma mutants overexpressing avr genes were tested in vivo to study their antagonistic activity against P. ultimm; the mutation did not enhance the biocontrol activity of Trichoderma against this pathogenic fungus but it did against Alternaria spp.
WP 2: Physiological enhancement
This WP is devoted to improving the biocontrol ability and the usefulness of biocontrol agents by stimulating the biocontrol process, selecting strains overproducing compounds toxic to the target pests and having enhanced sporulation/aggressiveness.
During the antagonist-pathogen and antagonist-plant interactions specific genes are activated. A fungal biocontrol agent 'detects' the production of inducers of mycoparasitism released from the pathogen or the plant. Ihe identification and the characterization of both proteins and small molecules produced during the complex interactions antagonistic Fungi-plant and antagonistic fungi-pathogenic fungi were started.
In order to identify and characterize molecules involved in the interactions between Trichoderma and others pathogens different culture filtrates (CFs) were obtained growing strain P1 in Salt Medium containing cell walls or biomass of the pathogenic fungi P. ultmum. R. solani and S. sclecrotiorum. Enzymatic assays were performed to study the biological activity of these CFs. and in vitro and in vivo assays of their fractions bigger and smaller than 3kDa. CFs were also obtained by the interaction betwcen Trichoderma and plant (tomato) that identified an enhanced inducing activity on endochitinase and N-acetyl-glucaminidase.
To enhance the virulence and the efficacy of bioherbicides by selecting variants of the weed pathogen that overproduce and excrete the amino acids that are inhibitory to the target plant. determination of amino acids or combinations of amino acids that are most inhibitory to the growth and development of the target weed was started on parasitic weeds.
In order to obtain a comprehensive culture collection of fungal pathogens perennial weeds for further development as biocontrol agents field surveys were undertaken in different regions of Russia. Seventy new isolates have been added to the existing culture collection whose aggressiveness evaluation is in progress.
Studies on the production of toxic metabolites were started on stains of potential phytotoxic microorganisms, in particular Septoria cirsium, Ascochyta sonchi and Phomopsis cirsii, pathogens of Cirsium arvense and Soncus arvensis. Optimization of growth condition, preliminary extraction and purification of metabolites, amid biological assays to evaluate the herbicidal potential were carried out.
WP 3: Ecological fitness
Ihe main aim of this WP is to assess the ecological fitness of the biocontrol agents, that is the population dynamics of biocontrol agents in different soil types under different conditions of temperature and water content, and the rhizosphere competence of these biocontrol agents.
The study of the population dynamics of a Fusarium oxysporum mutant has been completed in several soils of different physico-chemical properties. showing that the strain established very well in the 4 different soil types when introduced at high concentration.
WP 4: Environmental impact of biocontrol agents
This WP is devoted to the assessment of the environmental impact of a variety of biocontrol agents by tracking their movement, assaying non-target effects and any changes in host range, especially after genetic or physiological modifications, and determine long term environmental persistence.
In order to identify molecular markers to recognize biocontrol agent strains after their release into the soil, genetic studies on one strain of F. oxysporum promising for biological control of Orobanche sp. was started using AFLP techniques. A number of bands considered to he unique for the studied strain were obtained.
In preliminary experiments F. oxysporum (strain Fo47) and Pseudomonas fluorescens (strain AG) were added to two diffferent soils. studying the populations dynamics of the introduced BCAs and the effect of their introduction on the structure of the microbial communities Results showed that the introduction of the biological control agents changed the structure of the microbial communities.
In order obtain a deeper understanding of the impact of added Trichoderma on resident microbial communities, mutants of T. atroviride P1 and T. hazianum T22 strains were obtained, using constructs containing the glucose-oxidase encoding goxA gene. To obtain a reporter system to study biocontrol mechanism in vivo, in both fungi a mutant gene of green fluorescent protein (GFP) was placed under the inducible nag1 and ech42 promoters. Both fungi did not produce fluorescence during growth on glucose while evident fluorescence was produced during growth on Botrytis cinerrea walls.
The determination of the effect of the introduction of C. minitans into soil on the soil in microbial population over time was started using a genetically modified strain of this fungus in lab experiments, by inoculating three different soil types. Early indications show that the level of C. minitans in all three soil types remains constant over a three month period.
Studies on the morphological character, molecular fingerprinting and physiological specialization/host range of different strains of Phomopsis spp. were started, in order to determine that no undesirable off-target effects could arise after introduction in the environment of those strains for biological control of Cirsium arvense.
Formulated strains of F. oxysporum ( FOXY ) transformed with gus or gfp and hygB and F. arthrosporiodies (FARTH) with gus and hygB were used to study the pathogen spread in soil, the interactions with other soil microbes, and the effect on emergent Orobanche shoots. The density of the transformed FARTH increased 2-3 fold from 2 to 10 weeks, whereas both the modified FOXY remained unchanged for the same period
WP 5 Production and formulation
This WP has the important aim of developing appropriate technologies for the production and formulation of the fungal biocontrol agents.
Eleven fungal strains provided by partners were used for the assessment of the best solid media for their growth. Ten different growth media were tested, using rye, wheat or rice as main ingredient. Some strains showed a very intensive conidia development, being able to produce a number of conodia per gram of dry weight as high as an internal standard strain, or even higher.
Furher studies were carried out on an antagonistic F. oxysporum strain in order to show if the amendment of cheap nutrient supplements could additionally increase the conidia yield in respect to a standard substrate, and millet was used as optimal basic standard substrate. Preliminary experiments showed that some additives could provide about 25% higher conidia production.
WP 6: Application methods
The main objective of the WP is to determine the best methods of application of the biocontrol agents.
To verify the efficiency of distribution of microbial particles by the irrigation systems, and evaluate their potential as biocontrol agents suppliers, an experimental drip irrigation system has been realized in a greenhouse. Nine different dripper lines, chosen among the more suitable for irrigating vegetable crops, with filters and valves were used, with a dosing pump to supply the desired amount of conidia in the pipe, and with a general controller that allows to manage any phase of the experiments, in terms of amount of water supplied, amount of microbes injected in the system, or duration of irrigation.
Considering that mycelium fragments of Phomopsis cirsii are infective, the relationship between blending time and fragment length and survival of the fragments was assessed. In order to evaluate the potential of applying the cut mycelium through nozzle or irrigation equipment but avoiding clogging.
To select the best kind of application of potential mycoherbicides Cirsium arvense studies were carried out comparing the efficacy of soil and leaf application of different inoculum types of F. oxysporum and Stagonospora cirsii. The virulence of the latter was evaluated in field conditions using different conidial and mycelial inoculum.
WP 8: Integration
The main objective of the WP is the enhancement of biocontrol efficacy by combining living biocontrol agents with other biocontrol agents, natural substances with specific enzymatic or toxic action that result in improved biocontrol activity against crop-pathogenic fungi or weeds.
In Trichoderma several genes are known to be associated with pathogenicity on host fungi, such as genes encoding for cell wall degrading enzymes (CWDEs). These enzymes are strongly synergistic with synthetic fungicides of different classes and our purpose was to obtain mixtures of powerful CWDEs produced by Trichoderma. Different growth conditions were tested, in which Trichoderma grew in various liquid substrates with different carbon and nitrogen sources, and the enzyme profiles of the culture to select specific enzyme mixtures with enhanced biocontrol activity were tested.
WP 9: Assessment of crop quality
The main obiective of the whole WP is to ascertain if the soil application of microbial agents can have an influence on the quality of vegetable crops. In particular, in the first year it was necessary to set up or to adapt some procedures to evaluate the main biochemical parameters characterizing the nutritional quality of tomato. This will allow to compare the content of antioxidant components (vitamin C, lycopene and phenolic compounds), sugars. organic acids and mineral content of tomato berries obtained using biocontrol methods vs those obtained using traditionalI methods of crop protection.
Plans for using and disseminating the knowledge
Research papers. Seven research papers in which the EU contribution was acknowledged were published during 2004, six of which were accepted by International Journals.
Posters. Two posters showing the activities carried out within the project were displayed at International Conferences.
Conference presentations.. Five oral presentations were given at International and National congresses during which the research activities carried out by partners in the first year of the project or the general aims of the project were presented.
Project brochure. A brochure of the project has been prepared in 5,000 copies and delivered to each partners, to be distributed at scientific conferences, symposia, meetings or workshops.
© Copyright 2006 Policy Statements
Updated
by CPL Press:
03/07/2007
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