Udvikling af et biologisk bejdsemiddel til frø

English Summary

Background and objectives

Background

Seed borne disease of cultivated plants are of major importance world wide. While in general, chemical seed treatment effectively controls seed borne diseases of cereals, certain limitations and environmental disadvantages have been associated with the use of chemicals. This has increased the demand for alternatives to chemical control and coating seeds with antagonistic microorganisms may be such an alternative. Many isolates showing high efficacy under laboratory and/or field conditions have been selected as promising biological control agents (BCAs). However, difficulties with production, formulation and shelf-life or lack of efficacy when applied under field conditions have often complicated and limited the commercialization of such organisms.

Objectives

The aim of this project is to develop a BCA for seed treatment primarily in order to control seed borne diseases of cereals as an alternative to chemical treatment, thereby providing an environmentally safer product. The research focuses on the development of methods for production and formulation which ensure adequate shelf-life and activity as well as high biocontrol efficacy of the BCA under field conditions. The project utilizes a promising isolate of Gliocladium roseum (IK726), selected on the basis of high biocontrol efficacy in the field. The main targets of the investigations are the biocontrol of the seed borne pathogens, Fusarium culmorum and Bipolaris sorokiniana.

Development of G. roseum isolate IK726

Production and formulation

A BCA based on IK726 can only be an alternative to chemicals if active propagules can be mass produced and formulated in a reproducible and cost-effective way. Furthermore, IK726 must be marketed as a dried formulation in order to avoid contamination and to ease handling and delivery. G. roseum produces two types of propagules: hyphae and asexual spores (conidia). Since hyphae do not survive rapid drying, all investigations concerning IK726 use conidia. IK726 produces abundant conidia both in solid (SF) and in liquid fermentation (LF). A yield of 3-8 x 10⁸ cfu/g preparation is repeatedly produced on sphagnum/peat (SF) with a total production time of 16-17 days (drying included). For conidia produced by SF followed by formulation in clay products, yields of 2.1-6 x 10⁹ cfu/g preparation are obtained. By formulating conidia from LF with similar clay products, 2.0-6.5 x 10⁹ cfu/g preparation are obtained with a production period of 9 days. The cfu content per g preparation of IK726 is actually as high or even higher than the content of active propagules in available commercial products based on for example, Trichoderma spp. Several experiments have shown that IK726 conidia produced by SF are more robust with regard to dessication tolerance and shelf-life than those produced by LF and this should be taken into account when deciding upon the production method.

Product shelf-life

An adequate shelf-life is a crucial factor for commercial success of a BCA. A minimum shelf-life of 1 year, with minimal losses in viability, preferably without refrigeration, is therefore recommended. Several viability tests of dried IK726 preparations stored at 4°C to 10°C have revealed that viability at this storage temperature is independent of production method and formulation, since shelf-life of both standard and optimized formulation have been stable for more than 1 year. However, the achievement of stable shelf-life of more than 3-4 months at 20°C is much more difficult. Significantly this problem has only partly been solved for many of the commercial products already on the market. With IK726, however, by optimization of production and formulation methods it is now possible to maintain viability at a high level for about 1 year at 20°C both for sphagnum/peat and for clay formulations. The stabilization of shelf-life at temperature ranging from 4° til 20° has thus improved the feasibility for commercial exploitation of IK726.

Germination of conidia

Although, drying conidia of IK726 results in significant reduction in germination rate and in germination speed compared to freshly harvested conidia, both fresh and dried conidia start to germinate within 4 hours. Compared to most fungi used for biocontrol, the conidia of IK726 germinate relatively quick and this can be a significant factor when control of pathogens that affect plant establishment such as Pythium spp.- which start to germinate 0-4 hours after sowing the seed - is required.

Biological control activity

A high and stable control efficacy of stored BCAs is a significant factor for successful application of such products in practical agriculture. The quality of IK726 preparations has been evaluated in controlled growth chamber experiments with wheat and barley by measuring the biocontrol efficacy against F. culmorum and B. sorokiniana. Control efficacy is related to formulation, storage temperature and storage time of preparations. For storage at 4°C both standard and optimized formulations of sphagnum/peat and clay preparations have shown stable and efficient biocontrol of F. culmorum independent of length of the storage period (from a few weeks and up to one year). For successful storage at 20°C, the method of formulation of IK726 is decisive for retainment of biocontrol efficacy. Thus, with standard formulation of sphagnum/peat or clay preparations, control efficacy is high and stable only for approximately 2 months. By optimized formulation, however, stable and high control efficacy was retained during a test period of 10 months with storage at 20°C. Moreover seed coating with IK726 is compatible with several stickers. This can be a valuable trait allowing the integration of IK726 with existing seed coating technologies.

Dose-response relationship

Dose-response curves for the relationship between dosage of germinable conidia of IK726 per seed and the percentage control of F. culmorum and B. sorokiniana have been established. For conidia stored at 4°C a strong linear relationship (r=0.73) exists between dosage (cfu/seed) and control efficacy against F. culmorum and it is estimated that about 5 x 10³ cfu/seed is sufficient for effective control (>80%). For freshly harvested conidia a similarly strong relationship exists (r=0.95) and the effective dose is of the same magnitude. This proves that control efficacy is reproducible for both type of conidia and that stored conidia are as effective as freshly harvested conidia when tested in green house experiments. For control of B. sorokiniana a dosage of approximately 1 x 10⁴ cfu/seed of both fresh and stored conidia of IK726 gives effective control (>80%). A dosage of ³10⁴ cfu/seed should, therefore, be sufficient to ensure effective protection against both pathogens. Possible direct effects of IK726 on plant establishment have also been examined. Varying the doses from 10¹ to 4 x 10⁴ cfu/seed had no significant effect on plant emergence or plant dry weight. Thus, it is possible to increase the dosage of IK726 on seeds, for example in order to control other pathogens, without any negative influence on plant establishment.

Shelf-life and activity of conidia stored on seed

Commercialization of biological seed treatment will be a more attractive alternative to chemicals if seed coated with IK726 can be stored. For cereal seed, in the Nordic countries, this means storage temperatures of about 15-20°C. In practice, seed of spring barley is maximumly stored for 8 months and seed of winter wheat is rarely stored for more than 3 months. Viability on seeds has been tested for about six months. In this period shelf-life has been stable on seeds stored at 20°C. Moreover the biocontrol efficacy against Bipolaris foot-rot has been high (>80%) and stable during a test period of 7½ months for coated seed stored at this temperature.

Field experiments

It is often seen that potentially useful BCAs are unable to ensure stable and effective biocontrol under natural growing conditions. During this project six field experiments have been conducted. The results from all these trials demonstrated that coating seed of wheat and barley with IK726 significantly controls disease caused by seedborne inoculum of F. culmorum. IK726 is active against the pathogen at soil temperatures that are typical for cereal cultivation in Denmark, since average temperatures varied between 6.2°C and 12 °C in the periods from sowing to plant emergence. Growth chamber experiments has further revealed that IK726 also controls F.culmorum effectively at temperatures from 10°C to 20°C. In addition the results from these field trials confirm that stored conidia are as effective in controlling F. culmorum as are freshly harvested conidia. In conclusion, the efficacy data from the field experiments demonstrate that seed coating with IK726 should be a realistic alternative to chemical control, especially for control of seed borne infections caused by F. culmorum.

Potential of IK726 against other pathogens

Preliminary experiments have revealed that IK726 also controls other plant pathogens. Screening experiments with control of common bunt (Tilletia caries) on wheat have shown that IK726 seed treatment can reduce the frequency of bunt both in the field and in growth chamber experiments. Coating seeds of sugar beet with IK726 has also significantly reduced damping-off caused by soil borne inoculum of P. ultimum in growth chamber experiments. Preliminary investigations with reisolation of G. roseum from barley and wheat roots indicate that IK726 is rhizosphere competent (able to colonize the root from the seed) on these crop plants. This further indicates the potential of IK726 to prevent attack from soil borne pathogens since rhizosphere competence is regarded as a significant factor for high control efficacy against such pathogens.

Future development of IK726

It is expected that a significant part of the project dealing with producetion and formulation can be patented. This will improve the prospects for commercialization of IK726. Several agreements of collaboration and projects should help ensure the future development of IK726. An agreement on collaboration and options has been signed with the seed company, Dæhnfeldt a/s, in order develop and use IK726 for coating seeds of vegetables and flowers. A three year project was started in 1998 in order to develop biocontrol for practical application to control seed and soil borne pathogens mainly in cereals ad it is planned that the efficacy of IK726 against other seed borne diseases will be tested in field trials. In connection with the above, collaboration with Cillus a/s on biological seed treatment of cereals has just started. Moreover, Danisco Seed is at the moment testing IK726 for coating seed of sugar beet. A more formalized collaboration is expected during 1998/99 with this company.

Conclusion

The results of the project "Development of a biological control agent for seed treatment" underline the good possibilities for commercial use of G. roseum (IK726) for seed treatment in order to control seed borne diseases. IK726 can be produced cost-effectively and reproducibly. Formulated preparations have stable shelf-lives of about 1 year at storage temperatures of up to 20°C. The control efficacy of stored IK726 preparations is high and reproducible in green house tests. Furthermore, high and stable biocontrol efficacy has been demonstrated in a series of field trials under varying environmental conditions. In addition, IK726 can be stored on seeds for more than half a year without significant loss of control efficacy. An investigation on the novelty value made by Plougmann, Vingtoft & Partners established that it probably will be possible to protect considerable parts of the technology developed in the project with patents. Furthermore, the project points to new areas where IK726 can be utilized for disease control.