Mikrobiologiske bekæmpelsesmidler og toksiske metabolitter

Summary

Background

Many of the microorganisms used as biological control agents (BCAs) are producers of toxic secondary metabolites in vitro. However, information concerning production of such toxins under natural conditions in vivo is sparse. The occurrence of toxins in or on plant products will, to a high degree, depend on the method of delivery of the BCA and on the ecology of the potentially toxin producing organism. It is widely held, that microorganisms only produce metabolites when they are active. Therefore focus in this report will be on the ability of the organism to spread, survive and establish in soil, in the rhizosphere and phyllosphere and on postharvest products. At the moment 18 BCAs are marketed in Denmark on dispensation until official EU approval has been obtained. This report primarily deals with microorganisms used for biological control of plant pathogens with specisal focus on the organism Trichoderma harzianum.

Objectives.

The main objective of this report is to elucidate the environmental conditions under which metabolites produced by microorganisms may pose a human health risk in agricultural and horticultural crops in relation to the application of BCAs. In order to achieve this end, the spread, survival and establishment of microorganisms in crops will be related to the method of application and ecological parameters in the soil and rhizosphere, phyllosphere and post harvest environments. The natural occurrence of populations of toxin producing microorganisms will be examined partly to exemplify how ecological parameters affect toxin production by these organisms and partly to evaluate whether there are greater health risks associated with ingestion of toxins from BCAs in compared to ingestion of toxins from the indigenous microbiota.

Natural occurring microbiota

Microorganisms can be detected on all fresh plant products such as fruits, vegetables and cereals. The composition of the microbiota on such products depend on the cropping conditions, the type of product and the storage conditions. Amongst the fungi the genera Cladosporium, Alternaria, Epicoccum, Aspergillus, Penicillium and Fusarium are highly common. Amongst the yeasts especially Cryptococcus spp., Sporobolomyces spp. and Aureobasidium pullulans occur and within the bacteria Pseudomonas, Xanthomonas, Chromobacterium and Klebsiella are the dominating genera

Ecological habitats

Soil, rhizosphere, phyllosphere and post harvest products are the main ecological habitats that BCAs may occupy either following direct application or by secondary spread. These habitats offer very different opportunities for microbial growth and establishment. Soil and rhizosphere are characterized by an indigenous microflora, rich in species diversity and amount of biomass, in which an intensive competition takes place for nutrient rich substrates, such as plant debris and root exudates. The phyllosphere is a very dynamic habitat with drastic and frequent fluctuations in temperature, humidity and radiation and where nutrients are often very limited. Therefore the microorganisms in the phyllosphere have to be highly adapted in order to survive and establish in this habitat. The postharvest environment is more defined and stable. Fruit and vegetables are mainly stored at a high relative humidity and a low temperature in order to maintain the quality of the product and to reduce development of postharvest pathogens. Cereals should be stored with a low water content (ca. 13%). At such low water availability microbial growth is very limited.

Biological control

In this report microbial control of plant pathogens is defined as: " The artificial introduction of antagonistic microorganisms into the environment to control plant pathogens". Most of the organisms used for biological control have been isolated from natural environments such as the soil, rhizosphere, phyllosphere, fruits and seeds. The mechanisms involved in biological control are generally classified into four groups: antibiosis, competition, mycoparasitism and induced resistance. However in practice these mechanisms often are not clearly separable, as antagonists often have multiple modes of action.

Mode of application

The method used for application of BCAs depends on the life-cycle of the pathogen, its pathogenesis and this, in turn, determines at which stage of plant development application should take place.

Soil and seedborne plant pathogens

Soilborne plant pathogens are controlled by incorporating, drenching or spraying the BCA on growth medium or by seed coating. In general, it has proven to be difficult to establish a sufficiently high and active population of a BCA when it is introduced into a complex ecosystem with a high microbial buffering capacity, such as soil. The normally observed decline in an introduced population varies according to environment and species, but bacteria and fungi may survive from a few weeks and up to several years, mainly as inactive spores (fungistasis). Direct application of BCAs to greenhouse crops, when grown in smaller volumes of e.g soilless potting mixture seems more economically feasible than application to soil on a field scale. Furthermore soilless or artificial growth media probably possess a lower microbial buffering capacity at crop start, which offers good opportunities for the establishment of an early introduced BCA.

Seed coating

Seed coating mainly protects the seed against seedborne pathogens. However if the antagonist is rhizosphere competent, i.e. able to colonize the developing root after seed application, then also the plant may be protected against soilborne pathogens

Phyllosphere pathogens

Plant pathogens in the phyllosphere are mainly controlled by spray application of BCAs. The survival of BCAs is generally limited to a few days or weeks due to poor climatic adaption and poor ability to compete for nutrients in the phyllosphere. Therefore repeated treatment with the BCA are often needed. Successful biocontrol in the phyllosphere generally depends on the capacity of the BCA to exploit sparse sources of nutrients such as necrotic tissue, pollen, honey dew, etc. or freshly made wounds, not yet colonized by the natural microbiota and of the availability of these resources.

Postharvest pathogens

Post harvest pathogens are controlled by spraying or dipping. Most fruits and vegetables are stored at low temperature, often near 0°C. However, low temperature storage is one of the main limitations to efficient biological postharvest disease control, since many of the known antagonists have no or very low activity at such temperatures.

Toxin production by the natural microbiota

Fungi belonging to Aspergillus, Penicillium, Fusarium, and Alternaria are often detected on harvested products and they are strongly involved in spoilage of crops both in the field and during transport and storage. Many of these species can also produce the toxic secondary metabolites (mycotoxins) that are frequently detected in plant products used for human and animal consumption, such as cereals. Mycotoxins may be produced during the growth of the crop or during storage of the harvested products. Both fungal growth and mycotoxin production are dependent on environmental factors such as substrate, temperature, water activity, microbial interactions etc, with the limits for mycotoxin production usually being more narrow than those for growth of the organism. The risk of fungal growth and thereby potential mycotoxin formation in harvest products increases, if products are handled incorrectly and/or stored under conditions which accelerate the natural deterioration and senescence processes (especially in fruits and vegetables) or with too high a water content (primarily cereals).

Production of secondary metabolites by BCA's

Many BCAs can produce secondary metabolites in vitro and in many cases these are thought to be involved in biocontrol (antibiosis). However, it should be noted that in vitro production of metabolites in many cases is not correlated with biocontrol efficacy in vivo. Furthermore, there are only a few reports of in situ production of secondary metabolites in relation to biocontrol of plant pathogens. Reported cases mainly concern Pseudomonas fluorescens, P. aureofaciens and Trichoderma virens (former Gliocladium virens). These studies indicate that availability and amount of nutrient (exudates, organic material) is essential for metabolite production and for the amount produced. Since the knowledge of in situ metabolite production by BCAs is incomplete and since isolates within species can vary in production of metabolites, general conclusions concerning metabolite productivity cannot be drawn. Thus, presently, potential risks must be evaluated in the light of the natural occurrence and ecology of the BCA in question and on the basis of knowledge of potential in vitro metabolite production of the specific organism in question.

Trichoderma harzianum

Isolates of T. harzianum are the active ingredient in two BCA products marketed in DK and in several products registered in the USA. The species T. harzianum differs from other Trichoderma species in respect to in vitro production of metabolites and requirements for growth. T. harzianum is a typical soilborne saprophytic fungus which occurs naturally in all soil types (10² to 10⁴ cfu/ g soil). Dependent on the isolate, the fungus is active between 5° and 36°C and at a minimum water activity (a_w) of 0.91. Rhizosphere competence does not seem to be a natural trait of T. harzianum. T. harzianum can occur naturally in the phyllosphere and on postharvest products as contamination. Conidia applied to aerial plant organs generally survive poorly and a rapid decrease in the population density to below 1% is often observed after 2 to 3 weeks. Furthermore, conidia of T. harzianum are unable to germinate on intact living plant tissue without an exogenous nutrient supply. Metabolites produced by T. harzianum in vitro have not been detected in situ either in soil, the rhizosphere or on postharvest products

General considerations concerning risk assessment

Much of the information needed for risk assessment of BCAs is available while it is mainly general in nature. For each individual case more specific ducumentation on e.g. ecology and metabolite production by the BCA may be required. Methods to obtain this kind of data are available, but the need of such data should be carefully considered, since such methods generally are costly. Aspects which might be considered for more detailed study are dealt with in chapter 7.

Although a postharvest product may be contaminated with BCAs, none of the organisms used at the moment are known to cause postharvest problems in terms of their growth and sporulation on plant products. Therefore in most cases, the risk of metabolite production on harvested products can be considered minimal.