WORK PACKAGE WP3

WP 3.1. In-vitro and in-vivo studies of liquid mixtures as pesticides against root and foliar pathogenic microorganisms

The in–vitro tests will be performed with different methods so that there will be reliability and the possibility of drawing as safe conclusions as possible:

• By studying the inhibition zones by the method of wells or paper trays, where after coating the target microorganism on a suitable solid medium (eg nutrient agar for bacteria, potato dextrose agar for yeasts)the liquid antimicrobial (liquid mixture of olive polyphenol extracts and pomegranate and/or orange juice residues) will be placed in the center of the plate and the incubation zone (mm) around the antimicrobial after incubation will be measured.
• By studying the MIC-Minimum Inhibitory Concentration (MIC) and the Minimum Lethal Concentration (MLC) on a suitable liquid growth medium (eg nutrient broth for bacteria, potato dextrose broth for fungal yeasts)with increasing concentration of liquid antimicrobial substance.

Specifically, the effectiveness against phytopathogenic microorganisms will be studied, especially soil and foliar phytopathogenic fungi such as Verticillium dahliae, Fusarium oxysporum, Botrytis cinerea, Rhizoctonia solani, Eutypa lata, Monilia laxa, Pyrenochaeta lycopersici, Phoma punicae, Aspergillus niger and Gaumanomyces graminis.

Following the completion of the in vitro evaluation of the extract mixtures, the most effective mixtures for each phytopathogenic microorganism will be selected for further evaluation in order to limit the treatment number of the in vivo experiments.

For the in vivo evaluation of the effectiveness of the selected mixtures of olive polyphenol extracts and pomegranate and/or orange juice residues against phytopathogenic fungi (to be inoculated in tomato plants), the effect of fungi on the growth of tomato plants will be evaluated. Especially, the ability of the polyphenol mixtures to reduce the growth and activity of phytopathogenic fungi will be evaluated by measurements on height, fresh and dry weight of the aboveground and underground parts, as well as the number of flowers and fruits of the tomato plants. The aim of the experiment will be to determine the most effective mixture that, at the same time, will not be phytotoxic to the crop.

An important aim of this subsection is to propose one (or more) of the co-produced liquid mixtures as an optimal natural pesticide for controlling soil phytopathogens and one (or more) as an optimal natural pesticide for controlling foliage phytopathogens.

ACTIONS WITHIN THE WORKING PACKAGE AND RESULTS

In the first phase of the study, the in vitro (Petri-dishes experiment) antimicrobial activity of polyphenols against important phytopathogenic fungi, such as Verticillium dahliae and Fusarium oxysporum, which cause adromycosis disease on tomato and Botrytis cinerea, which causes leaves and fruits post-harvest rot, has been evaluated. In addition, their action against the phytopathogenic fungi Rhizoctonia solani, Eutypa lata, Monilia laxa, Pyrenochaeta lycopersici, Phoma punicae, Aspergillus niger and Gaumanomyces graminis has been evaluated. All the fungi species used came from a collection of the Benakeio Phytopathological Institute.

The following techniques were used to evaluate the antimicrobial activity of the produced polyphenols against important phytopathogenic fungi species:

• Measurement of suspension zones (well diffusion assay)
• Mycelium spread rate in Petri dishes

in order to determine the minimum effect of different concentrations and combinations of 72 extracts of olive, pomegranate and orange polyphenols.

A segment of the mycelial tissues from the original medium (PDA plates) was placed in test tubes containing a sloping PDA medium and refrigerated at 3 ^οC for longer storage. The experiments and the evaluation of the activity of the olive mill waste polyphenol treatments were performed using sections and fungal spores from this material.

In the second phase of the study, the efficacy of the 11 most effective polyphenol extracts (as obtained from the laboratory experiment) against four of major phytopathogenic fungi species (Verticillium dahliae, Rhizoctonia solani, Pyrenochaeta lycopersici and Botrytis cinerea) was evaluated in vivo (greenhouse conditions). The extracts were applied on 15-day-old tomato plants (3-leaves growth stage) of variety Bella Dona.

In the greenhouse experiment, the height of the tomato plants, the fresh and dry weight of the aboveground and underground parts of the plants, as well as the number of flowers and fruits were evaluated.

The results of the laboratory experiment (in vitro) showed that the olive polyphenol extract, as well as its mixtures with a small percentage of orange or pomegranate extracts significantly reduced (up to 100%) the growth of the phytopathogenic fungi Botrytis cinerea, Verticillium dahliae, Rhizoctonia solani, Eutypa lata, Monilia laxa, Pyrenochaeta lycopersici and Phoma punicae. However, the extracts evaluated did not significantly reduce the growth of the phytopathogenic fungi Fusarium oxysporum, Aspergillus niger and Gaumanomyces graminis.

The results of the greenhouse experiment (in vivo) showed that the extracts were moderately to very effective against the four tested phytopathogenic fungi. In particular, the mixture containing the olive:pomegranate:orange extracts in a ratio of 80:12:8 caused significant reduction of the four fungi infection, with the result that the growth of the infected tomato plants was similar with that of the uninfected control plants.

The above results are very encouraging and show the potential of olive, pomegranate and orange polyphenol extracts to be developed in the future into effective natural pesticides.