Project Title: "Production of biological control agent (BCA)"

Author: Ana Catarina Borges de Azevedo Costa, University of Minho, Portugal

Host Supervisor: Prof. Daniel Volpe, Universidad de La República, Uruguay

Uruguay faces today a serious issue of tree rothening, mainly in Eucaliptus globolus recent plantations, which represents a major problem in forest zones, especially in the south-southeast part of the country. The industry has been looking for ways to control plant diseases, caused by pathogenic organisms, mainly filamentous fungus, genera Basidiomicetes. These fungus colonize and degrade the wood as much as in alive as in already deceased trees, causing economic losses in all the lumber sector.

Over the past several years, production of Trichoderma harzianum has become a focus of research and industrial development in the search for alternatives to chemical seed, treatments to control soil borne plant pathogens. Trichoderma harzianum is a fungus, from genera Ascomicete, and is a promising biological control agent. To guarantee the effectiveness of the control, large quantities of viable propagules are needed, which in this particularly case are conidiospores.

In this project, we aimed at optimizing the production and drying process of conidiospores produced by Trichoderma harzianum .

The spores were produced by liquid fermentation (working volume of 1.2 L ), using a modified Richard's medium plus tomato source, followed by a filtration, using a press filter, and a drying process To ensure the optimal conditions of viable spores, the effect of the addition of 9 % (v/v) glycerol and medium agitation (500 and 1000 rpm), were tested. During fermentation pH, total spores concentration (spores/mL), viable spores concentration (ufc/mL) and biomass (g/L) were monitored.

The drying process was optimized evaluating its effect in the viability and moisture content in the final product (spores).Spores previously filtered were located in cakes of 1,5 cm heigh into driers and exposed to dry airflows (at five different speeds: 2000, 3000, 5500, 8000 and 9000 mL/min) at ambient temperature (approximately 15 ºC) . The sampling was carried out at five different times (22, 40, 47, 65 and to 72h during the process). Statistic analysis of the data was done using the surface method of reply and contour graph

To verify the effectiveness of the product, a biotest assay was carried by inoculation of Trichoderma harzianum spores and Inocutis (pathogen) in wood pieces of Eucalyptus globulus previously dry . These wood pieces had been incubated at different temperatures (10ºC, T ambient = 15ºC and 32ºC) and moisture contents (44 and 75 %).

The addition of 9 % (v/v) glycerol solution did not improve the productivity (spores/(L.h)) as well as the biomass yield. Spores viability during fermentation (less than 50%) and after drying (less than 20%) was also affected. Fermentations with 9 % (v/v) glycerol added were longer than the one with no glycerol adition. To achieve a spore concentration of 3E8 spores/mL in the bioreactor, 60 hours of operation were necessary in fermentation with glycerol while only take 42 hours without glycerol addition. Concerning the moisture content, it was verified that the addition of glycerol influences this factor. The moisture contents obtained were higher than intended, that is, 5 %.

Agitation during fermentation is another factor that influences productivity, biomass yield and spore viability during fermentation. This is the consequence of the structure and resistance of spores resulting by agitation. Productivities and biomass yields achieve at 500rpm fermentation were lower than using 1000rpm. Also, higher spore viability was obtained during fermentation and drying using lower agitation. The moisture content was not influenced by agitation once differences in moisture values were not found; in both agitations evaluated the moisture content obtained was always lower than 5 %.

Using the surface response for drying process, it was observed that with increasing airflow for a determinate time of dried the viability increases reaching a maximum, and then decrease. The same effect was verified for a determinate airflow when increasing the dry time.

Therefore, it is possible to consider that the optimal conditions to fermentation are a stirring rate of 500rpm and absence of glycerol where viabilities reaches 90 % during fermentation and drying , moisture content lower than 5 %, productivities of 1E10 spores/(L.h) and biomass yields in the order of 7E10.

By the contour graph, it was possible to conclude that the optimal conditions of drying process considering the spore viability, for the ambient temperature considered, occur using airflow of 5125 mL/min and 43 h of drying