Evolutionary basis and ecological role of toxic microbial secondary metabolites

J. theor. Biol. (1982) 97,325-332

Evolutionary Basis and Ecological Role of Toxic Microbial Secondary Metabolites E. B. LILLEHOJ Southern Regional Research Center, Science and Education Administration, U.S. Department of Agriculture, New Orleans, LA 70179, U.S.A. (Received 20 January 1981, and in final form 11 March 1982) This presentationdevelopsa theory of the evolutionary origin and ecological implications of toxic microbial secondary metabolites. The theory is

based on a model system that outlines cause-effect associations between pertinent biotypes in the aflatoxin contamination of developing maize kernels.The modelsuggeststhat the aflatoxin-producing fungi are natural

digestive tract inhabitants of a number of insect species that feed on developing kernels. During feeding, the insect larvae introduce fungal

propagules and provide infection sites on damaged kernels. The fungal associationwith insectsexhibits extraordinary variability, ranging from symbiotic to pathogenic. Elaboration of aflatoxin by the fungusfacilitates the pathogenic process in host insects. The theory contends that genetic information for secondarymicrobial metabolitesevolved during ecosystem disequilibria. During periods of ecologicalstability, mechanismsevolved for repression of toxic secondary metabolite biosynthesis. The theory broadly suggeststhat contemporary agricultural activities presents the

requisite milieu for production or toxic microbial secondary metabolites. 1. Introduction In the microbial context, use of the term, “secondary metabolite” has been restricted to substances that are not essential for growth of the producing organism (Aharonowitz & Demain, 1980; Bu’Lock, 1965). In spite of an extraordinary, multifaceted literature, the fundamental basis of the role of microbial secondary metabolites in nature remains somewhat ambiguous. A preliminary working hypothesis has been developed that defines environmental aspects of secondary metabolites (Lillehoj, 1980); the current presentation provides additional, pertinent information. 2. Ecology and Evolution

of Microbial

Secondary Metabolites

The question of the advantage to a producing microbe of secondary metabolism has aroused spirited interest (Demain, 1974). Conventional 325

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wisdom has assumed that the ability of a microbial species to elaborate antibiotic substances provides competitive benefit (Janzen, 1977). However, Waksman (1948) expressed skepticism about the premise since antibiotics could not be readily isolated from natural substrates that contained the producing organisms. Further studies demonstrated that adding nutrients to soils elicited production of the secondary metabolites (Gottlieb, 1976); this observation suggested that nutrient limitations restricted natural accumulation of the metabolites. Although early studies did not provide definitive evidence to support or refute the microbial benefit proposal, subsequent studies have produced two compelling points that support the advantage concept: (1) characterization of hundreds of secondary metabolites with complex structures (Turner, 1971) and their specificity in chemical structure-biological response interactions (Corcoran & Hahn, 1975; Detroy, Lillehoj & Ciegler, 1971) and (2) linkage of specific genetic components of the producing microbes to the biosynthesis of secondary metabolites (Hopwood & Merrick, 1977). Understanding the evolutionary aspects of microbial secondary metabolites requires evaluation of the interaction between species in an ecological system and the potential role of the metabolites in mediating a chemical signalling function. The advantages in elaboration of biologically active substances in a competive interaction are intuitively obvious. However, presence of antagonistic metabolites would often be incompatible with symbiotic interactions; these activities are characterized by mutually beneficial associations that are relatively stable in the absence of major climatic or other environmental changes (Lynch & Poole, 1979). Ecological systems can be characterized from a nutritional standpoint by their carbon assimilation efficiencies (Odum & Odum, 1976). Member species in an ecosystem are either producers or consumers of photosynthate. In a specific enviroment, evolutionary processes have provided for efficient capture of radiation and distribution of the photosynthate; in a balanced interaction, the competition for available carbon is intense. Abrupt environmental changes can upset the equilibrium and provide an opportunity for colonization by species that are adapted to the new milieu (Baker, 1965; Dobzhansky, 1970; Lewontin, 1965). Microbial activity in the interaction between species in an ecosystem has been broadly grouped into two categories: (1) biotrophy, deriving nutrients from living material, and (2) necrotrophy, deriving nutrients from non-living material (Thrower, 1966). Many of the fungi that produce mycotoxins are considered necrotrophs but they can also function as biotrophs (parasites) under certain conditions (Detroy et al., 1971). In fastidious types of bio-

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trophy between microbes and higher organisms such as those observed in plant pathogen-host plant relationships, the microbes have coevolved with their hosts and they may produce toxins as part of the pathogenic process. Although microbes have adapted to evolutionary pressures based on nutrient quality and access, categorical descriptions are hazardous since they ignore the inherent variability of the dominant factor, the environment. Lewis (1973) summarizes the flexibility, “Indeed, it is probable that environmentally induced shifts from biotrophy to necrotrophy convert mutualistic symbiosis into parasitic ones”. It appears that both biotrophic and necrotrophic microbes utilize metabolites as interspecific chemical signals. Coevolution between microbes and insects has also occurred. Symbiotic relationships between intestinal microbes and host insects are very common with balanced, integrated systems allowing microbes to break down dietary constituents (e.g. cellulose) to units that the host can digest and to supply required amino acids, vitamins and other growth factors (Wilson, 196.5; Lynch & Poole, 1979). The microbe-insect symbiosis probably also reflects a stable interaction that has evolved with periods of parasitism during ecosystem disequilibrium and symbiosis in the relatively stable interactions (Harper, 1977; Lynch & Poole, 1979). 3. Evolution

and Agoecosystems

In the practice of agriculture, humans have introduced an ingredient of disequilibrium into stable ecological communities. Generally, man’s activities tend to homogenize the associated flora and fauna. The extent of the disequilibrium is closely linked to the scope and intensity of agricultural practices. During the past 50 years, the full impact of the industrial revolution and attendant technologies on agriculture has been realized including: (1) mechanization of farm implements, (2) broad utilization of industrially derived fertilizers and pesticides, (3) wide use of high-producing crop varieties drived from plant breeding programs, and (4) continuous cropping of limited plant species with restricted variation in interspecific genetic constituents. These factors, acting in concert, impose a marked degree of homogeneity in agroecosystems, that produces a dilemma; the more effort humans put into intensive agricultural practices the higher the cost becomes in terms of plant disease and pest population increases. Harper (1977) has summarized the situation, “Pest epidemics can often be ascribed to some activity of man that has brought a floristically diverse ecosystem closer to a monoculture”.

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4. Aflatoxin

Contamination

of Developing

Maize Kernels:

Model

The presence of mutagenic, teratogenic, and carcinogenic substances in agricultural commodities (Detroy et al., 1971) provides an opportunity for development of a model that relates the role of a secondary microbial metabolite group (aflatoxin) in the context of ecology, evolution, and agriculture to specific information from developing maize kernels. The two fungi that produce the toxin, Aspergillus flavus and Aspergillus parasiticus, are broadly distributed (Detroy et al., 1971) and they have been widely detected in agricultural commodities during recent years. Categorical information on the nutrient requirements for toxin synthesis has been acquired with defined media; these investigations have identified two critical factors that contribute to high yields: (1) elevated levels of available carbon relative to nitrogen, and (2) requistite levels of certain trace elements (Detroy et al., 1971). Integration of a number of ecological components in the nutritional context of available carbon is proposed in Fig. 1. In the undisturbed state Ecologxol equllibrlum

Green

plants

X

Photosynthote dfstributed mteroctmg

ropldly among species

No secondary

metabohtes

Photosynthote in plants and

occumulotes insects

Ecological disequilibrium

Green

plants

I

Secondary

FIG.

metabolites

1. Association between secondary metabolite production and ecosystem interactions.

of ecological equilibrium a balance exists between interacting plants, insects, and microbes; the photosynthate would be distributed rapidly and the competition for carbon would prohibit synthesis of secondary metabolites. In conditions of ecological disequilibrium, such as agroecosystems, in which a single plant species predominates, the attendant number of species of insects and microbes would be reduced (Batra, 1982). Since one of the principal goals of agriculture is the production of carbon compounds, crop development results in accumulation of significant levels of carbon that can be assimilated by microbes; under these conditions, secondary meta-

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bolites can be synthesized. Regarding aflatoxin occurrence, the premise contends that the carbon availability in developing maize kernels would provide ideal conditions for toxin production. The presence of aflatoxin in developing maize kernels has prompted investigations of the fungal colonization process (Lillehoj & Hesseltine, 1977). Three fundamental observations have emerged from the studies: (1) insects can serve as vectors of propagules of the toxin-producing fungi, (2) insect-mediated damage of kernels appears to provide access for fungal development, and (3) limited fungal species competition on developing kernels seems associated with the presence of the toxin-producing microbes (Lillehoj & Hesseltine, 1977; Widstrom, 1979). Isolates of the two aflatoxin-producing species have been identified as inhabitants of insect digestive tracts (Sinha, 1971; Srinath, Ragunathan & Majumadar, 1973); these Aspergillus species have also been characterized as pathogens in a number of insects and mites (Steinhaus & Marsh, 1962). Investigation of insects collected from developing kernels throughout the corn-growing regions of the U.S.A. demonstrated a general distribution of associated toxin-producing fungi (Fennel1 etal., 1977). A significant number of the A. fluvus and A. parasiticus isolates from the surface-sterilized insects occurred as monoculture outgrowths. The phenomenon of singlespecies presence in insects has been attributed to the ability of the predominant microbe to elaborate an exotoxin that blocks development of the competing species (Yendol & Hamlen, 1973). Aflatoxin could be the substance that restricts competition since the toxin does possess antibiotic properties (Detroy et al., 1971). In addition to induction of a biological response in a wide array of microbial and animal species, aflatoxin also exhibits toxicity in insect species (Beard & Walton, 1971; Lalor, Chinnici & Llewellyn, 1976; Llewellyn & Chinnici, 1978; Matsumura & Knight, 1967). Identification of the toxin in dead insect larvae that had been infected with A. fluvus suggests that the fungal metabolite could be the compound responsible for the mortality (Murakoshi et al., 1977; Ohtomo el al., 1975). The dual function of microbial secondary metabolites as antibiotics and insecticides has been observed in other microbial compounds; dose rate appears to be the decisive factor between control of the indigenous microbes in the insect and lethality in the host (Ciegler, 1975; Claydon, Grove & Pople, 1977; Reiss, 1975; Richards & Brooks, 1958). A proposed interaction between an A. flaws endosymbiont and insect host is presented in Fig. 2. Three stages of nutrition are characterized: balance, limited imbalance, and extensive imbalance. The proposal suggests that in nutrient balance a symbiotic relationship exists between the microbes

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E. B. LILLEHOJ Insect

host

A f/avus Endosymbiont No oflotoxin

Nutrient balance Symbiosis

A. fhwus

Nutrient

imbalance

Low oflatw.in

A fhwus Pathogen High aflatoxln

FIG.

2. Interrelationships

Nutrient imbalance (extensive) Death

between

A. frauus

and insect hosts.

and host. During periods of limited nutrient imbalance the fungus produces low levels of aflatoxin that imposes an antibiotic effect on other internal microbes and in situations of elevated nutrient imbalance, the fungus assumes a pathogenic character that is mediated by the insecticidal properties of aflatoxin. The proposal is supported by laboratory studies that have demonstrated a shift from symbiotic microbe-insect association to pathogenic relationships mediated by nutritional modifications (Richards & Brooks, 1958). The proposed model identifies an evolutionary relationship between aflatoxin-producing fungi and insects that infest developing maize kernels. In this context, the observed toxin contamination of maize represents an accidental sequela of prior species interactions. However, the condition is not mediated entirely by chance since the kernels of a monocultured crop exhibit unique ecological characteristics with high levels of nutrients and a limited complement of competitive microbes. The opportunistic development of the toxin-producing fungi in the relatively pristine kernel environment presents an example of introduced species exploitation in the destabilized agroecosystem. 5. Evolutionary

Origin and Ecological Metabolites:

Role of Toxic Microbial Theory

Secondary

The model system of aflatoxin contaminatian of preharvest kernels serves as a reference point for extrapolation to a general theory of the evolutionary and ecological implications of toxic microbial secondary metabolites. The theory proposes that production of toxic microbial secondary metabolites provides an advantage to the producing organisms only under certain environmental conditions. The capacity for synthesis of the metabolites evolved during periods of ecological disequilibrium when competitive

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activities are a distinct advantage. However, during periods of greater symbiosis between interacting species that are associated with stable ecosystems, mechanisms for repression of the toxin biosynthesis were evolved; under these conditions toxin production would not be advantageous. The determining influences in the environment that provide the switching mechanism between toxin production and repression are the quantity and quality of the nutrients available to the pertinent microbes. Contemporary agroecosystems are characterized by intensive cropping activity that tends to homogenize the associated flora and fauna, and pertinent nutrient patterns. The theory anticipates a parallel development of increased cropping of limited plant species with enhanced aggressiveness in the attendant biota and associated contamination of commodities by toxic secondary microbial

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