QLK5-1999-01065 Development of biocontrol agents for commercial application against post-harvest diseases of perishable foods

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Integrated Crop Protection & Biological Control : Quality of Life - 5.1.1 Sustainable Agriculture - Plant Systems

Contract No:	QLK5-1999-01065
Source:	Six Month Progress Report - December 2000 - Abstract

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Six Month Progress Report - December 2000 - Abstract

Objectives

The main aim of this project is to develop biocontrol agents for control of key post-harvest pathogens to a stage where these agents can be commercially exploited in close collaboration with the commercial partners. This will be achieved by the development of a number of microorganisms, which have already been demonstrated to be effective to a stage where registration for this use is possible. The following organisms will be used in a series of field scale trials: Pichia anomala, Epicoccum nigrum, Candida sake and Pantoea agglomerans

In order to achieve this key objective, the major objectives are:

• Molecular marking of the biological control agent strains for effective monitoring in field trials and for evaluating the impact of the biological control agents on the environment where they will be applied
• As the different biological control agents are in the developing process for commercial use, the main constraint is to quickly understand the potential modes of action, insisting on the antibiosis, in order to fulfil the registration procedure of these biopesticides
• Optimisation of quality of biological control agent production in liquid or solid fermentation for commercialisation and development of improved ecological fitness of biological control agents by endogenous and exogenous additives
• Formulation studies for developing effective biocontrol products
• Pilot scale evaluation of best formulations of biological control agents to control post-harvest diseases of perishables from organic and conventional crops, studying the best timing, doses and application systems.
• Improving the effectiveness of the biological control agents by combining these with physico-chemical treatments, heat and non toxic substances
• Evaluation of the impact of biological control agents on the environment where they will be applied in order to determinate their survival on the surfaces of the perishable products selected for study, their dispersion capacity and their effect on indigenous microflora
• In collaboration with commercial partners large scale field trials will be carried out with the best disease control system (biological control agents, physicochemical treatments and combinations of these) to evaluate and demonstrate the efficacy and feasibility of integration of these methods into actual commercial management systems.

Results and Milestones

During this first 6 months of the project work was started on WPI, WP2 and WP3. Although none of the milestones and deliverables have been reached yet, some interesting results have been obtained, as summarised below.

WPI Identification and monitoring of biological control agents. This involves the specific identification of the antagonistic strains using a RAPD method. Experiments have been run on a collection of P. anomala and P. agglomerans strains. For P. anomala, 87 primers have been tested without showing anv specific DNA fragment for the sole strain J121. For P. agglomerans, one specific DNA fragment was detected for the sole strain CPA-2 among 12 tested primers. In this context, design of SCAR primers and PCR amplification of this specific DNA fragment with SCAR primers has been carried out. At this point, SCAR primers have been found not to be specific. Further RAPD analysis is required for determining another specific DNA fragment. In addition, in relation to quantification of the population size of each antagonistic species of each antagonist strain after their application on fruits, a semi-selective medium has been developed for P. anomola J121 and C. sake CPA-1.

WP2 Studies of the mode of action of biological control agents The ability of P. anomala to inhibit spoilage moulds on cereals in simulated leaky, airtight storage was investigated after addition of different nutrients. After two weeks no significant difference between the control without additions and the different combinations of added glucose and glutamine could be seen. The production of volatile compounds was investigated using the four biocontrol agents. C. sakc, P. anomala and E. nigrum produced volatile compounds that inhibited radial growth, biomass and spore production of a number of different pathogens.

Since the various biocontrol agents are in the process of development for commercial use, the main constraint is to quickly analyse the potential modes of action, under conditions of antibiosis, in order to fullfill the registration procedures for such biopesticides. Streak assays as well as overlay assays on tryptic soy agar (TSA) and potato dextrose agar (PDA) demonstrated that P. agglomerans does not produce any components on these media that inhibit Penicillium digitatum or Penicillium italicum. P. agglomerans was also grown in liquid media (TSB, tryptic soy broth) and the superntant was tested for inhibiting components. There was no inhibition of the pathogens. It was also verified that P. agglomerans does not produce inhibiting components on the fruit itself. The pathogenicity and the effect of the antagonists were tested on oranges and apples before studying the induction of resistance. It was demonstrated that P. digitatum and P. italicum are pathogens of oranges and that P. agglomerans provides a local protection against these pathogens. It was also demonstrated that C. sake protects apples locally against the pathogens Rhizopus stolonifer, Penicillium expansum and Botrytis cinerea.

WP3 Ecophysiological studies for optimum production of ecologically fit inocula . Work has focused on ecophysiological studies of C. sake, E nigrum and P. anomala as well as to develop a commercial medium for the production of P. agglomerans. For both yeasts studied in this project, the lag phase increased and growth decreased when the water activity (a_w) of the growth medium decreased. Moreover, both yeasts were more sensitive when the a_w of the growth medium was modified with the ionic solute NaCl. Water potential and intracellular accumulation of polyols and sugars of C. sake cells has also been studied. The water potential of C. sake cells depended on the a_w of the growth media and also on the solute used to modify it. Glycerol and arabitol were the main compatible solutes accumulated in C. sake cells as response to lowered a_w of the growth medium. Radial growth and growth rates of E. nigrum spores also decreased with a lowered a_w of the growth medium. Temperature also influenced the growth rate and the lag phases significantly increased as the a_w of the medium and temperature decreased. An acceptable relationship between E. nigrum spore concentration and absorbance (415 nm) has been found. Some synthetic, commercial and by-products from the food industry have been tested as nitrogen and carbon sources at lab scale for P. agglomerans production. The studies have demonstrated that high levels of biomass of P. agglomerans can be produced using various nitrogen sources and inexpensive carbohydrates as feedstock.

Benefits and Beneficiaries

At this stage of the work benefits have not yet been established. It has been shown that the biocontrol agent P. agglomerans does not produce any inhibitory components in the growth media or on the surface of treated fruit.

Future Actions

Experiments will be continued along the lines indicated in the Technical Annex.