EDITORIAL

Plant-associated microorganisms: a view from the scopeof microbiology

Published online: 21 August 2003� Springer-Verlag and SEM 2003

Microorganisms interact with plants because plants offera wide diversity of habitats including the phyllosphere(aerial plant part), the rhizosphere (zone of influence ofthe root system), and the endosphere (internal transportsystem) [3, 5]. Interactions of epiphytes, rhizophytes orendophytes may be detrimental or beneficial for eitherthe microorganism or the plant and may be classified asneutralism, commensalism, synergism, mutualism,amensalism, competition or parasitism.

Plant-associated microorganisms play essential rolesin agricultural and food safety, and contribute to theenvironmental equilibrium. Their study has been classi-cally based on cultivation-dependent methods, whichoften recover only 0.01–10% of direct counts. However,studies based on molecular analysis have estimated morethan 4,000 species per gram of soil. Most of thesemicroorganisms are probably non-cultivatable, such asthe plant symbiotic vesicular-arbuscular mycorrhiza(VAM) endomycorrhizae common in many angiospermsand gymnosperms, or are transiently in a viable but non-cultivatable state [1]. Microbial composition and popu-lation levels in the rhizosphere and phyllosphere aredetermined mainly by carbon sources released as rootexudates and often change with cultivars of the sameplant species. The degree of change in microbial com-position of the phyllosphere or rhizosphere is associatedeven with the type of genetic modification in sometransgenic plants.

Cultivation-dependent methods based on PCR,RFLP, fatty acid profiles (FAME), nutritional (Biolog)and many others have been used largely to characterizespecific groups of plant-associated bacteria and fungi.However, conventional cultivation-dependent methodsare biased towards the selective enrichment of fast-

growing microorganisms adapted to high substrateconcentrations, which can represent a minor fraction ofthe microbial community. Cultivation-independentPCR-based fingerprinting techniques to study small-subunit (SSU) rRNA genes (rDNA) in the prokaryotemicrobial fraction have been developed to study diver-sity, structural composition and dynamics of microbialcommunities associated with plants. Amplicons areseparated by denaturing gradient gel electrophoresis(DGGE) or temperature gradient gel electrophoresis(TGGE), or submitted to terminal restriction fragmentlength polymorphism (T-RFLP) analysis. For example,using T-RFLP in a study of corn-associated bacteria,signals related to Cytophaga/Bacteroides/Flavobacteriumphylum, Holophaga/Acidobacterium phylum, a-proteo-bacteria, b-proteobacteria and c-proteobacteria weredetected, whereas cultivation-based methods detectedmainly c-proteobacteria [4].

Crop production losses due to biotic agents (pests,diseases and weeds) are controlled mainly by sprayingcrops with a vast amount of synthetic chemical pesti-cides, which can severely affect planet health due to theirnon-target effects. New methods of crop protection havebeen developed based on historical observations inagriculture and forestry of the benefits obtained fromnaturally occurring microbial communities. Beneficialinteractions may result from competition, antagonismand hyperparasitism against plant pathogens, insectsand weeds. In most cases, the practice of biologicalcontrol implicates the introduction of massive amountsof microbial pesticides into the environment. Therefore,specific analysis methods are necessary to study thetraceability, residue analysis and environmental impactof a pesticide. However, the search for specific genotypicmarkers in microbial pesticides is difficult and time-consuming because it is accomplished by DNA finger-printing methods to identify strain-specific DNAfragments that can be sequenced and characterized(SCAR), and used to develop real-time PCR (quantita-tive PCR) analysis. The use of reporter gene systems inplant-associated microorganisms has also provided

Int Microbiol (2003) 6: 221–223DOI 10.1007/s10123-003-0141-0

Emilio Montesinos

E. MontesinosInstitute of Food and Agricultural Technology-CIDSAV,University of Girona, 17071 Girona, SpainE-mail: emonte@intea.udg.esTel.: +34-972-418476Fax: +34-972-418399

powerful tools for evaluating in situ gene expression,colonization, mechanism of action, and monitoring ofrelease. The most successful methods are based on thechromogenic gusA (a b-glucuronidase from Escherichiacoli), luminescent lux/luc (luciferases from Vibrio fischerior firefly lantern) or fluorescent gfp (green fluorescentproteins from jellyfish).

Since the introduction of genetically modified (GM)crops in 1996, their impact on associated microbiota,and horizontal transfer of antibiotic selectable markershave both been a cause of concern for large-scalecommercial introduction. World areas of GM cropshave increased at a sustained rate, with the principalcrops being soybean, corn, cotton and canola. Mostcommercial transgenic plants contain selectablemarkers under bacterial promoter control such asantibiotic-resistance genes, the most common being thebla gene, which codes for a TEM b-lactamase (ampi-cillin resistance), and the nptII gene, which codes for aneopentenyltransferase (kanamycin resistance). Theacquisition of genes coding for antibiotic resistancefrom transgenic plants by plant-associated bacteria,and their subsequent transfer from these bacteria tohuman or animal bacterial pathogens, has been amajor concern in the large-scale use of transgeniccrops. The process would require a sequence of coor-dinated and complex events, and the occurrence ofsuch a transfer under natural conditions would be rare,but there is no reason to assume that the phenomenonwould not take place, even at a reduced probability, innature. Gene transfer from transgenic plants to bac-teria has been observed only under laboratory condi-tions, when certain barriers were absent, in complexmodel systems consisting of two bacterial species andtransgenic tobacco with high transgene copy numberand a high degree of sequence homology [2]. However,very few field studies have addressed this issue. Thehigh proportion of ampicillin-resistant bacteria occur-ring naturally in agricultural fields fertilized often withmanure, may put into perspective the potential impactof the antibiotic selectable marker acquisition by corn-associated microbiota.

The structural and functional analysis of microbialgenomes and the proteins encoded by genes ofimportant plant-associated microbes will provide in-sights into several aspects of these molecular interac-tions and will be crucial for the development of moreefficient control measures, and accurate diagnostic andmonitoring methods. More than 10,000 species offungi, bacteria and viruses are estimated to cause plantdiseases, several of which are of major economicimportance. The automation of sequencing technolo-gies and advancements in bioinformatics provide toolsleading to better knowledge not only of the genome ofplant pathogens or microorganisms beneficial to plantsbut also of ways of incorporating genes from microbesinto plants as microbial-derived resistance. The ge-nomes of 733 prokaryotic and eukaryotic organismshave been completely sequenced or are ongoing (146

organisms have been completely sequenced and 587 areongoing), and those of 944 viruses have been com-pleted. However, of the completed genomes, only 9 areplant-associated bacteria and 44 plant viruses. Of theongoing 587 sequences, only 32 (14 prokaryotes and 18eukaryotes) pertain to bacteria or fungi associated withplants. Of the 1,677 genomes sequenced or ongoing,only 85 correspond to plant-associated organisms.Most plant-associated microorganisms whose genomeshave already been sequenced are bacteria, includingAgrobacterium tumefaciens, Pseudomonas syringae pv.tomato, Xanthomonas campestris pv. campestris,Ralstonia solanacearum, Xanthomonas axonopodis pv.citri and Xylella fastidiosa (several strains), Bradyrhiz-obium japonicum, Sinorhizobium meliloti, and Meso-rhizobium loti. Ongoing genome analyses includebacteria such as Clavibacter michiganensis subsp.sepedonicus, Azotobacter, Bacillus thuringiensis, Pseu-domonas fluorescens, Rhizobium leguminosarum, andfungal pathogens including Aspergillus spp., Botryoti-nia fuckeliana, Fusarium spp., Mycospherella gramini-cola, Ustilago maydis, and Phytophthora spp. (http://ergo.integratedgenomics.com/GOLD/).

The easy commercial exchange of agriculturalproducts (fresh products and plant material) hascaused the highest spread of plant pathogens anddiseases ever observed. Quarantine systems to preventthe introduction of new plant pathogens in protectedareas, and eradication measures to avoid epidemicspread are the first protection barriers. However, manypathogens remain latent in the plant material and invery low numbers, and methods with high sensitivity,specificity and reliability are required. New methods ofplant-pathogen analysis have evolved parallel to theadvancement of methods of analysis in human andveterinary microbiology. Automation and electronicdata management are essential for the increase inproductivity and efficiency of routine analysis for thedetection of plant pathogenic bacteria and viruses. Thespecificity of detection by serological techniques hasimproved greatly with the use of specific monoclonaland recombinant antibodies, enrichment-ELISA pro-tocols, and on-site-testing using tissue print-ELISAand lateral flow devices. Molecular techniques basedon hybridization or amplification and especially PCRhave been refined by co-operational PCR, multiplexPCR, and multiplex-nested RT-PCR. In many cases,the need for quantification and field compatibility ofthe technique has led to new devices for portable rapidcycling real-time PCR [6]. However, new methods witha high potential for determining many pathogenssimultaneously including in-situ fluorescence hybrid-ization and DNA microarrays are under development.

These are only a few examples of the fact thatmicrobiology, through its different projections and spe-cifically via the approaches of molecular microbiologyand microbial ecology, plays an essential role in pro-viding answers to the main questions related to planthealth.

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References

1. Colwell RR, Grimes DJ (2000) Nonculturable microorganismsin the environment. American Society for Microbiology,Washington, D.C.

2. Kay E, Vogel TM, Bertolla F, Nalin R, Simonet P (2002) In situtransfer of antibiotic resistance genes from transgenic (trans-plastomic) tobacco plants to bacteria. Appl Environ Microbiol68:3345–3351

3. Lindow SE, Hecht-Poinar EI, Elliot VJ (eds) (2002) Phyllo-sphere microbiology. American Phytopathological Society, St.Paul, Minn.

4. Lukow T, Dunfield PF, Liesack W (2000) Use of the T-RFLPtechnique to assess spatial and temporal changes in the bacterialcommunity structure within an agricultural soil planted withtransgenic and non-transgenic potato plants. FEMS MicrobiolEcol 32:241–247

5. Lynch JM (1990) The rhizosphere. Wiley, New York6. Weller SA, Elphinstone JG, Smith NC, Boonham N, Stead DE

(2000) Detection of Ralstonia solanacearum strains with aquantitative multiplex, real-time, fluorogenic PCR (TaqMan)assay. Appl Environ Microbiol 66:2853–2858

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