Plant-associated microorganisms: a view from the scope of microbiology.

Microorganisms interact with plants because plants offer a wide diversity of habitats including the phyllosphere (aerial plant part), the rhizosphere (zone of influence of the root system), and the endosphere (internal transport system) [3, 5]. Interactions of epiphytes, rhizophytes or endophytes may be detrimental or beneficial for either the microorganism or the plant and may be classified as neutralism, commensalism, synergism, mutualism, amensalism, competition or parasitism. Plant-associated microorganisms play essential roles in agricultural and food safety, and contribute to the environmental equilibrium. Their study has been classically based on cultivation-dependent methods, which often recover only 0.01–10% of direct counts. However, studies based on molecular analysis have estimated more than 4,000 species per gram of soil. Most of these microorganisms are probably non-cultivatable, such as the plant symbiotic vesicular-arbuscular mycorrhiza (VAM) endomycorrhizae common in many angiosperms and gymnosperms, or are transiently in a viable but noncultivatable state [1]. Microbial composition and population levels in the rhizosphere and phyllosphere are determined mainly by carbon sources released as root exudates and often change with cultivars of the same plant species. The degree of change in microbial composition of the phyllosphere or rhizosphere is associated even with the type of genetic modification in some transgenic plants. Cultivation-dependent methods based on PCR, RFLP, fatty acid profiles (FAME), nutritional (Biolog) and many others have been used largely to characterize specific groups of plant-associated bacteria and fungi. However, conventional cultivation-dependent methods are biased towards the selective enrichment of fastgrowing microorganisms adapted to high substrate concentrations, which can represent a minor fraction of the microbial community. Cultivation-independent PCR-based fingerprinting techniques to study smallsubunit (SSU) rRNA genes (rDNA) in the prokaryote microbial fraction have been developed to study diversity, structural composition and dynamics of microbial communities associated with plants. Amplicons are separated by denaturing gradient gel electrophoresis (DGGE) or temperature gradient gel electrophoresis (TGGE), or submitted to terminal restriction fragment length polymorphism (T-RFLP) analysis. For example, using T-RFLP in a study of corn-associated bacteria, signals related to Cytophaga/Bacteroides/Flavobacterium phylum, Holophaga/Acidobacterium phylum, a-proteobacteria, b-proteobacteria and c-proteobacteria were detected, whereas cultivation-based methods detected mainly c-proteobacteria [4]. Crop production losses due to biotic agents (pests, diseases and weeds) are controlled mainly by spraying crops with a vast amount of synthetic chemical pesticides, which can severely affect planet health due to their non-target effects. New methods of crop protection have been developed based on historical observations in agriculture and forestry of the benefits obtained from naturally occurring microbial communities. Beneficial interactions may result from competition, antagonism and hyperparasitism against plant pathogens, insects and weeds. In most cases, the practice of biological control implicates the introduction of massive amounts of microbial pesticides into the environment. Therefore, specific analysis methods are necessary to study the traceability, residue analysis and environmental impact of a pesticide. However, the search for specific genotypic markers in microbial pesticides is difficult and timeconsuming because it is accomplished by DNA fingerprinting methods to identify strain-specific DNA fragments that can be sequenced and characterized (SCAR), and used to develop real-time PCR (quantitative PCR) analysis. The use of reporter gene systems in plant-associated microorganisms has also provided Int Microbiol (2003) 6: 221–223 DOI 10.1007/s10123-003-0141-0