Development of formulation technology for biofungicides for greenhouse tomatoes, cucumber and pepper crops

Project Code: BPI07-040

Project Lead

Russell Hynes - Agriculture and Agri-Food Canada

Objective

To characterize the biocontrol organism Streptomyces sp. Di-944, complete development of new microencapsulating technology/formulation, and conduct efficacy trials to test new formulations and demonstrate the value of the product

Summary of Results

Background

Disease control is a challenge in field and greenhouse tomato. To comply with market demands, Canadian growers need pest management options that include effective biocontrol products against root, stem, and fruit diseases that affect the crop quality and yield. This project is aimed at developing formulations and application technologies of the common soil bacterium, Streptomyces sp Di-944 (hereafter referred to as Di-944). It produces an antifungal metabolite that affects various vegetable diseases, among them Fusarium wilt, Fusarium crown and root rot (FORL), and Rhizoctonia damping-off, in tomato. The project involved the development and evaluation of microencapsulation technology for Di-944 as a formulation for delivery of the active ingredient.

Approaches

Characterization studies of the partial 16S ribosomal ribonucleic acid (rRNA) gene sequences of the biocontrol micro-organism revealed a species-specific signature for Streptomyces sp Di-944 following analysis using GeneBank. This sequence data was used to confirm identity of the micro-organism through the course of the project.

Formulation studies were initiated by selecting the ingredients that allow for Di-944 growth while maintaining its performance as a biopesticide. Using a novel, complex coacervation protocol, microencapsulation of Di-944 was developed as the preferred formulation for its delivery to the rhizosphere of tomato plants.

Experiments were conducted to select (i) compatible microencapsulation ingredients; (ii) oil carrier for Di-944 propagules; and (iii) pH level for optimal coacervation.

The conditions for the preparation of the formulation were determined through screening experiments that evaluated parameters including paddle stirring speed, duration, and reaction time.

A solid state fermentation protocol for Rhizoctonia solani AG4 was developed by using heat-sterilized barley and wheat grains, with growth carried out in sterile bags, which permitted gas exchange and promoted fungal propagation.

Damping-off of tomato caused by R. solani AG4 was calibrated through a series of dose-response experiments to allow for predictable disease levels in soil-peat growth mixtures. The ability of the product to protect tomato seedlings by inhibiting growth of R. solani AG4 was assessed through in-vitro experiments. Shelf-life studies were also conducted to determine survival rate of Di-944 in the formulated product.

Results

Di-944 was highly effective in reducing R. solani AG4 mycelial growth in laboratory assays. Up to 100% suppression was achieved in agar plate assays. Levels of inhibition by the formulated product were similar to that of Di-944 on its own.

Tomato roots inoculated with formulated Di-944 suppressed the growth of R. solani AG4 in laboratory trials, resulting in inhibition of damping-off disease. The formulated Di-944 was four times more effective than the non-formulated product, possibly as a result of the propagules being concentrated in the microcapsules and the adhesion of the formulation to the roots of the tomato plants.

Shelf-life studies indicated that Di-944 has high survivability – three months as a minimum – in complex coacervate, as measured by viable propagules in the formulation. When stored at 22 degrees Celsius, however, the texture of the product changed and became thick over time, likely due to association of smaller coacervate particles into larger ones. This may reduce the adherence of the formulation to plant roots.

Conclusions

An ubiquitous soil microorganism, Streptomyces sp. Di-944 is neither a human nor a plant pathogen. It can be readily grown under laboratory conditions in an inexpensive medium. The organism produces effective anti-microbial agents and has good survival characteristics. As such, it is estimated that Di-944 has potential for commercial development due to its safety and efficacy. Additional studies on efficacy of the formulated biofungicide under laboratory and commercial greenhouse conditions, as well as testing of longer shelf-life, are required to clarify its potential for commercialization.

Further experiments to examine growth and spore production in order to establish the conditions for production of the more resistant spores and a conceivably longer shelf-life product would be beneficial. Experiments will also be required to address the delivery method of the biofungicide, e.g. by root dipping, or through plant growth medium (i.e. inoculating soil or peat moss). And finally, future studies could also examine the potential biofungicidal activity of Di-944 against other root rot pathogens, such as Fusarium spp, Pythium spp., and other isolates of R. solani.

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