FAIR-CT95-0722 Production, Processing and Practical Application of Natural Antifungal Crop Protectants

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Biotechnology : FAIR Area 1.2 - Green Chemicals and Polymers Chain : Integrated Crop Protection & Biological Control : Paints/Coatings/Plastics

	Contract No:	FAIR-CT95-0722
	Date Prepared:	July 2001, September 1999, July 1998, April 1998
	Source:	Final Report Progress Report Summary Year 3 Progress Report Summary Year 2 Progress Report Summary Year 1

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Final Report

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Progress Report Summary Year 3

Summary

Objectives Spoilage and plant pathogenic fungi are responsible for some 20% loss of the potential global plant production for food and non-food use. The very large amounts of chemical crop protectants used to control these losses are detrimental to environmental and human health. This project has been initiated to develop and implement non-chemical crop protection using natural antifungal agents (green chemicals): antagonistic microorganisms or antifungal metabolites from plant or microbial origin. These natural crop protectants will be designed for use with food crops or non-food products vulnerable to fungal deterioration.

Activities The research work to be conducted in this 4-year project has been subdivided in four consecutive phases:

• Selection of green chemicals and evaluation of their in vitro activity (months 1-6)
• Further selection of green chemicals and evaluation of their in situ activity (months 7-16)
• Optimisation of the application of the green chemicals (months 17-30)
• Scaling-up of the applications (months 31-48)

Progress In the reporting period, research has been focused on production, formulation, and in situ efficacy of the green chemicals selected for the third phase. These trials included applications against economically important diseases occurring during cultivation and storage of crops. Also, in vitro cytotoxicity of volatile plant-derived green chemicals in human cells has been studied using several assays and model systems especially designed for such, compounds.

Results

As far as plant extracts, essential oils and volatiles are concerned the main points are as follows.

• S-carvone completely suppressed development of Penicillium at the basis of Dutch iris bulbs but the dosages required for this effect also brought about a discoloration of the shoots,, indicating phytotoxicity. Indeed such bulbs showed a lower emergence and a lower percentage of flowers than those treated with lower doses. Repetitive administration of carvone at lower dosages did not suppress fungal development.

• Application of clove extract in a sucrose coating on wheat grains inactivated all fungal propagules present on the grains but also completely inhibited in vitro germination. In contrast, no negative effect on germination ability was observed when grains were sowed in soil. In order to optimise the application of clove extract in a coating on wheat grains, several coating materials were evaluated for adhesive properties, additional antifungal action, and protection of wheat grains against the phytotoxic action of clove extract. Chitosan and sorbitol did not appear more suitable than sucrose that was used thus far, and paraffin and gelatine did not protect the grains against the clove extract. A pH-controlled coating did not offer additional control of fungal infestation.

Other applications of clove extract that were examined included gas-phase application and surface disinfecting in watery solutions. In a 16-week storage trial, gas-phase application of clove extract resulted in a considerable reduction of fungal infestation. In vitro germination of the grains was dose- and time-dependent. Complete disinfection of grains was achieved with watery solutions of clove extract. Antifungal action was time and dose dependent and inversely related to in vitro germination ability.

The highlights of the work on antagonistic microorganisms were as follows:

• Cultivation of Bacillus sp. NIC by submerged fermentation yielded 2 x 10⁹ spores-ml^-1. The fluidised bed drying technique was a suitable approach for the formulation of spores on glucose as carrier. In such formulations viability of spores did not decrease over a 90 days storage at room temperature. Bacillus sp. NIC was particularly effective against oomycetes. When applied in coating on cucumber seeds, the organism increased the emergence rate in soil infested with Pythium ultimum. A reduction of the damage of late blight of potato (caused by Phytophthora infestans) was achieved by foliar application.

• Laboratory scale fermenter cultivations of Pseudomonas marginalis in optimised media conditions resulted in high yields (0.2 - I x 10¹¹ cells.ml^-1) and a high antifungal activity in the culture broth. The appropriate cultivation medium and the proper cultivation strategy led to rapid and vigorous growth, which resulted in high antifungal activity. P. marginalis retained acceptable viability for application in liquid formulations, although viability decreased 2 log cycles during the first 10, weeks of storage at 4 ºC. On the other hand a further decrease was not observed in the subsequent 16 weeks. The right cultivation time was crucial for production of preparations with a long shelf life. P. marginalis survived very well in dry products, which were produced by solid state fermentation, and stored 4ºC. At 28 ºC, viability decreased about two log cycles in one month. In small-scale in situ tests, P. marginalis had a good control efficacy against a moderate Pythium damping off infection in rockwool substrate. The effect of liquid formulations of P. marginalis was almost as good as that of chemical fungicide Previcur®

• Solid state cultivation of Fusarium aquaeductuum was scaled up to the level of 15 kg per batch. Using media with spent distiller solids and malt extract as substrates the yield of conidia was again increased to 2 x 10⁸ cfu.g^-1 products after one week of cultivation. The organism survived very well in dry solid state products. After six months storage at 4 ºC, the viability of F. aquaeductuum did not change, while at 28 ºC the viability decreased slightly. Spray drying for formulation on acetylised starch was not suitable for F. aquaeductuum. Only half of the conidia survived the spray drying and already, after 20 days storage no viable conidia could be detected. Product formulation of F. aquaeductuum will be optimised for application against Penicillium on flower bulbs, against Didymella bryoniae on cucumber (soil treatment) and against Rhizoctonia solani on cabbage, tomato, and cucumber nursery plants.

• Gliocladium catenulatum was found to be a broad-spectrum antagonist. Under commercial cultivation conditions, G. catenulatum reduced the damage of black stem rot of cucumber (caused by Didymella bryoniae) by 75-85%. Timing of the application is crucial. The first application has to be within 7 days after planting, followed by a second treatment one month later. On a smaller scale G. catenulatum effectively controlled silver scurf (caused by Helminthosporium solani) and Rhizoctonia canker diseases of potato. The control efficacy equalled or was even higher than that of the chemical fungicides Amistar® (containing azoxystrobin as active ingredient) and Fungazil® (containing imazalil as active ingredient). Solid state cultivation of Gliocladium catenulatum J1446 was scaled up to the level of 15 kg per batch. Using media with spent distiller's solids and malt extract as substrates the yield of conidia was again increased to 9 x 10⁷ cfu.g^-1 after two weeks of cultivation. The organism survived very well in dry solid state products. During a six-month storage, viability decreased from 1 x 10⁸ cfu.g^-1 to 6 x 10⁷ cfu.g^-1 at 4 ºC and to 2 x 10⁷ cfu.g^-1 at 28 ºC.

The main results from the toxicology studies were as follows:

• In the course of the project, a database has been compiled containing toxicological data on the plant-derived green chemicals selected for the project. The data include local effects on mammals, sensitisation and intolerance, general systemic effects (both acute and subacute), reproductive toxicity, carcinogenicity, genotoxicity, and in vitro toxicity. The database is continuously updated and extended. Currently, the database comprises 7 herbs, 9 plant volatiles, and 1 essential oil.

• Using short-term mammalian in vitro assays, cytotoxicity, of the plant volatiles S- carvone, carvacrol, decanal, trans-cinnamaldehyde, and thymol were evaluated. On the basis of IC₅₀ values ranging from 0.3 mM to 0.7 mM, these compounds are classified as mildly to moderately toxic. The adverse effects of carvone, carvacrol and thymol on the viability of human cells were reversible, those of decanal not.

• A model system has been developed to evaluate the in vitro cytotoxicity of plant volatiles in the gas phase. In this system plant volatiles evaporate from the source and diffuse via the head space to human cells. On the basis of neutral red uptake and total protein content of cells after exposure, the relative cytotoxicity of the plant volatiles was found to decrease in the following order: thymol > carvacrol, carvone > caraway oil > decanal.

Future actions For the final year of the project, five green chemicals have been selected for further research. These include the ethanolic extract of clove, the bacteria Bacillus sp. NIC and Pseudomonas marginalis, and the fungi Fusarium aquaeductuum and Gliocladium catenultum J1446. Research will be focused on scaling up the production and application of the selected green chemicals in formulated preparations. Also these preparations will be evaluated for human toxicity, persistence and biodegradability.

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Progress Report Summary Year 2

Objectives
Spoilage and plant pathogenic fungi are responsible for some 20% loss of the potential global plant production for food and non-food use. The very large amounts of chemical crop protectants used to control these losses is detrimental for the environment and human health. This project has been initiated to develop and implement non-chemical crop protection using natural anti-fungal agents (green chemicals): antagonistic micro-organisms or anti-fungal metabolites from plant or microbial origin. These natural crop protectants will be designed for use with food crops or non-food products vulnerable to fungal deterioration.

Description of work

The research work to be conducted in this four year project has been subdivided in four consecutive phases:

• Selection of green chemicals and evaluation of their in vitro activity (months 1-6)
• Further screening and selection of green chemicals and evaluation of their in situ activity (months 7-16)
• Optimisation of the application of the green chemicals (months 17-30)
• Scaling-up of the applications (months 31-48).

State of progress

This year research has been focused on the in situ performance of the green chemicals, on their toxicity to non-target organisms, and on cultivation of the antagonists. Except for one, all of the milestones of phase two have been accomplished. Only development and production of formulations for plant volatiles had to be postponed to the next phase. Evaluation of the human toxicity of plant-derived green chemicals which was originally scheduled in phase 1, has taken much more time than foreseen and consequently this task has been prolonged.

Achievements

Plant extracts, essential oils and volatiles:

• An in vitro assay has been developed to evaluate the fungicidal activity of plant volatiles in the gas phase towards conidia. It was shown that at a fixed dose the decimal reduction time varied as much as from 8 days (S-carvone) to 1 hour (decanal)
• S-carvone completely suppressed development of Penicillium hirsutum at the basal plate of Dutch iris bulbs during low temperature storage. This treatment as such did not have any negative effect on flower development
• A gas chromatographic method has been developed and optimised to quantify the concentration of the principle fungicidal components in ethanolic plant extracts
• When applied in a coating on wheat grains, clove extract completely inhibited development of moulds during storage. This effect was solely due to the eugenol in the coating. In general, doses required to suppress mould growth also inhibited germination. In order to protect the grains better against the phytotoxic activity of the green chemicals, wax-based coating were developed.

Antagonistic micro-organisms:

• Cultivation of Bacillus sp by submerged fermentation yielded 1-4 x 10⁹ cells and spores per ml. Application of low doses of this organism reduced damage by late blight on potatoes (Phytophthora infestans) and downy mildew on spinach (Peronospora farinosa) to a large extent
• Cell count in the range 10¹¹ per ml was achieved in submerged fermentation of Pseudomonas marginalis. The organism suppressed damping-off (Pythium sp.) on cucumber and has also a very promising potential for application against storage fungi on seeds and grains.
• By solid state fermentation of Fusarium aquaeductuum on wheat bran, a preparation containing 1 x 10⁸ conidia per g was obtained. When applied as in coating on cucumber seeds, the organism suppressed development of Pythium ultimum on cucumber seedlings. Also this organism effectively reduced grey mould (Botrytis cinerea) on several crops
• Gliocladium catenulatum was found to be a broad spectrum antagonist. It suppressed grey mould (Botrytis cinerea) on sweet pepper leaves and stems, Gerbera petals, tomato stems, and lettuce, and damping-off (Pythium sp.) and black stem rot (Didymella brioniae) on cucumber. Against Helminthosporium solani, the fungus that causes silver scurf on potato during storage, treatment with Gliocladium catenulatum was even more effective than several commercially available synthetic fungicides.

Toxicology:

• The database containing toxicological data on the plant-derived green chemicals selected for the project has been extended and updated. Currently, the database comprises 7 herbs, 8 plant volatiles, and l essential oil
• The in vitro toxicity and mutagenicity plant volatiles has been evaluated in microbial and mammalian short-term assay in order to identify any possible effect at the cellular level and, if any, the degree of this effect.

Future activities

Now that green chemicals have been identified with good in situ anti-fungal activity against several economically important field and storage fungi, formulations will be developed and the application of the formulated green chemicals on selected crops will be further optimised. In addition, the formulated preparations will be evaluated for human toxicity, migration into products, persistence and biodegradability, and shelf life. Finally, after a careful re-evaluation of the selected combinations of formulated green chemicals and crops, the production, formulation, and application will be scaled up.

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Progress Report Summary Year 1

Objectives

The project aims to develop and implement non-chemical crop protection using natural antifungal agents (green chemicals), i.e. antagonistic microorganisms or antifungal metabolites from plant or microbial origin. These natural crop protectants will be designed for use with food crops or non-food products vulnerable to fungal deterioration.

Progress Year1

During the first year of the project, a range of green chemicals were screened for their fungicidal spectrum in standardized bioassays against many different target fungi. Additionally, indicative toxicology treats of the green chemicals were assessed. The following conclusions can be drawn from the progress made in year 1:

Effect of green chemicals derived from plants

• Thymol and carvacrol were strong inhibitors of fungal germination and growth, in liquid as well as via the gas phase. Carvone and cinnamaldehyde inhibited germination best when administered via the gas phase.
• In situ tests proved the potential of cinnamaldehyde as a surface disinfectant for tomatoes, controlling visible fungal growth.

Effect of green chemicals of microbial origin

• Supernatant of L. plantarum was very inhibitory towards all a range of target fungi in vitro. Cell free extracts of Staphylococcus strains inhibited Fusarium-strains, but not A. niger and B. cinerea.G. catenulatum, G. roseum and Bacillus sp. spore-cultures significant inhibited almost all target fungi.

• Among the antagonists tested on crop-tissues, four showed activity against cucumber black stem rot caused by D. bryoniae. Foliar diseases caused by D. bryoniae and B. cinerea (grey mold), could be reduced by Gliocladium strains, F. aquaeductuum and Bacillus sp.. Against potato silver scurf, Gliocladium strains and Bacillus sp. were promising antagonists.

Toxicological database

A start was made to compile a data-base with toxicological information on plant derived green chemicals. In addition, the toxicological position of plant extracts and microbial derived compounds was evaluated in a number of short-term end-point tests, the Ames mutagenicity test and the Photobacter biological activity test.

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