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Department of Veterinary Morphophysiology, Via Leonardo da Vinci 44—10095 Grugliasco (To), Italy
Silvano Scannerini
Cinzia M. Bertea
Massimo Maffei
Department of Plant Biology, Viale P. A. Mattioli 25—I-10125 Torino, Italy
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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A hyaline sterile fungus forming epiphyllous mycelial nets was isolated from meristem cultures of Mentha piperita. Histological studies indicated that the culture isolate is able to colonize stems and leaves with no damage to the host plant. In vitro-grown peppermint plants displayed enhanced vegetative growth when infected by the fungus, with mycelium extending from green tissues to growing rootlets. The production of very thin hyphae growing away from host meristems and the asymptomatic nature of the symbiosis were commonly observed in cultures, where the isolate never sporulated. No attribution to a precise morphospecies was therefore possible and the fungal culture was named sterile mycelium PGP-HSF. Through comparison of the 18 S rDNA sequence of the epibiont to those available in literature and in GenBank we were able to determine that the mutualist of peppermint is a member of the Pyrenomycetes, belonging to the subclass Sordariomycetidae.
Key words: Ascomycetes, Dicotiledones, epibiotic, essential oils, Lamiaceae, micropropagation, molecular systematics, plant growth, small subunit ribosomal DNA
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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).
However, plant-fungal interactions are much more than simple physical associations. Analogously to other symbiotic associations like mychorrizae, similar mechanisms of cell recognition and biochemical interaction must be present within leaf endophytes (Peters et al 1998
).
Glandular trichomes represent a highly efficient chemical apparatus developed by many aromatic plants i.e., Lamiaceae, to discourage bacterial and fungal colonization. Considering the role of terpenoids in antipathogen defense and their well-known effects on fungal growth and spore formation (Inouye et al 1998
), systemic endophytism in aromatic plants should deserve more attention.
It is known that host secondary metabolites may inhibit colonization of some species of Colletotrichum, restricting them to quiescent infections (Okane et al 1998
). Relationships of leaf endophytes with terpenoids producing plants were studied in Pinus spp. (Sieber et al 1999
and references therein) and in Sequoia sempervirens, whose needles harbour Pleuroplaconema sp. as the predominant specialized endophyte other than fungal pathogens and saprobes (Espinosa-Garcia and Langenheim 1990
). Terpenoids composition and their differential activity on fungal growth have been shown to be important in redwood-endophyte interactions (Espinosa-Garcia et al 1996
).
Specificity at the tissue level has been confirmed for the endophytic fungi of woody plants (Petrini 1996
), especially those inhabiting tree branches (Kowalski and Kehr 1996
), tree bark and xylem (Stone et al 2000
), aquatic roots (Fisher et al 1991
), needles of conifers (Carroll et al 1977
, Sieber-Canavesi et al 1991
). In these perennial hosts, the high level of endophytic specialization has been interpreted as the result of fungal adaptation to the micro-ecological and metabolic conditions encountered in a given plant tissue or organ (Petrini 1996
).
Within Balansieae, variations in fungus-host relationships, life cycles and physiological capacities are even more evident, going from grass symbionts which are epibiotic with stromata located on stems, florets or leaves to the mutualistic, seedborne and strictly endophytic species of Neotyphodium (Bacon and White 2000
). This condition well correlates with the expected chronology of the origin of these fungi, suggesting a trend toward progressive colonization of the inner tissues and a reduction of destructive effects on hosts (White and Morgan-Jones 1996
, Glenn et al 1996
).
A similar condition has never been confirmed for dicot herbaceous plants, where the stable integration of the endophyte in the host body might have been precluded by the absence of efficient fungal transmission through the host seeds. However, it has been shown recently that the mutualistic endophyte Neotyphodium typhinum (Morgan-Jones & W. Gams) Glenn, Bacon & Hanlin comb. nov. can form a stable external mycelial net on the leaves of the host (Moy et al 2000
). This suggested a possible alternative pathway of fungal dispersal and transmission to hosts, i.e., through epiphyllous-produced conidia.
At present no data are available in the literature concerning the isolation of specialized endophytic fungi from herbaceous aromatic plants. However, beneficial interactions similar to those found for woody and grass hosts could be present also in perennial herbaceous species, even in the absence of specialized mechanisms of host infection. This could be possible in the case of strictly vegetative self-propagating species like Mentha piperita L., where the isolation of a sterile fungus possessing plant growth promoting effects prompted us to define its taxonomy and study its biology.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In vitro cultures –
Meristem cultures were established starting from mature peppermint plants. Nodal segments (approximately 3 cm long) of young stems were freed from leaves, leaving the nodal portion of each petiole (explant), and carefully washed under running tap water. Surface sterilization was obtained by immersion for 5 min in 70% ethanol, followed by soaking for 10 min in sodium hypochlorite (1%) plus 0.01% Tween 20 as surfactant and rinsing five times in sterile distilled water. Subsequent procedures were conducted aseptically. The explants were transferred to 12 mL of MS medium (Murashige and Skoog 1962
) supplemented with 2% sucrose, 1 mg/L thiamine, and 1% agar in SigmawareTM culture tubes (25 x 150 mm) provided with cotton plugs. The pH of the medium was adjusted to 5.7 before autoclaving at 121 C for 20 min. All in vitro cultures were carried out in a growth chamber (26/22 C day/night with a photoperiod of 16 h, 45 µmol m-2 s-1).
Culture procedures and fungal isolation –
Three weeks after explanting on MS medium, axillary buds were clearly visible on stem cuttings. Buds were then removed from nodes and transferred separately to new medium for further growth, taking care not to spread on or contaminate aseptic explants and surrounding media with the mycelium. Starting from buds, the fungus was isolated and transferred to 2% malt extract agar (MEA) slants. Colony morphology and growth of the isolate were studied on MEA and meanwhile growth and spore formation were checked on potato dextrose agar (PDA), cornmeal agar (CMA), oat agar (OA) and V8-juice agar media (Christensen et al 1993
). Axillary buds at the time of transfer and 30 d-old axillary bud-derived plants were employed for histological studies. Fungal re-isolation was obtained on MEA, plating each pair of leaves excised from 30 d-old micropropagated plants. A voucher specimen and culture has been deposited as MUT 88.
Histological studies – Both cultured explants and bud-derived micropropagated plants were employed for histological studies. Tissues were fixed in 3% glutaraldehyde 0.1 M phosphate buffer pH 7, washed in the same buffer and post-fixed in OsO4 for 1 h, dehydrated in a ethanol graded series. After dehydration, samples to be employed for scanning electron microscopy (SEM) were transferred to acetone 100 and then dried in a Emitech K850 critical point dryer, coated with gold, and viewed at 20 kV with a stereoscan 120 Cambridge Instruments scanning electron microscope. For conventional microscopy, tissues were embedded in LR White resin and sectioned at 0.5 and 2 µm thick. Sections were stained with toluidine blue.
DNA isolation, PCR amplification and sequencing –
Total genomic DNA was extracted by rapidly growing mycelium subcultured on M102 liquid medium (Bacon 1988
) without the addition of chloramphenicol. Seven d-old mycelium was collected by centrifugation, washed with distilled water and ground in liquid nitrogen. Extractions were made starting from 0.4–0.6 g (wet weight) of ground mycelium, using the procedure of Lee and Taylor (1990)
, as modified by Glenn and colleagues (Glenn et al 1996
). Four complementary segments corresponding to most of the 18 S rDNA gene were amplified using primers NS1, NS2, NS3, NS4, NS5, NS6, NS7, and NS8 (White et al 1990
). For PCR amplification, working reaction mixtures consisted of equal volumes of DNA samples and a master mix made of all the other necessary components, at the following concentrations: 10 mM tris HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 µM of each of the four deoxyribonucleotide triphosphates, 0.5 µM of each of the eight primers, 2.5 units of Taq polymerase and 5–10 ng of template DNA in 100 µL reaction volume. Amplification was accomplished using a Perkin Elmer GeneAmp System 2400 thermal cycler. Thermal cycling was run at the following conditions: one initial cycle of denaturation at 95 C for 3 min, annealing at 55 C for 30 s, and extension at 72 C for 1 min. This cycle was followed by 35 cycles with denaturation at 95 C for 30 s, annealing at 55 C for 30 s, and extension at 72 C for 1 min. A final cycle was run with an extension of 72 C for 10 min. Annealing temperature was raised to 58 C for primers NS3 and NS4. Amplification yielded enough DNA for automated sequencing, after purification with a Sephaglas BandPrep DNA purification kit (Amersham Pharmacia Biotech). Purified samples were sequenced by Genome Express (Grenoble, France) using an AbiPrism automated sequencer (model 373, version 3.3.1). Primers were used for sequencing both strands of 18S rDNA.
Phylogenetic analysis –
Reference sequences included in this study other than that of the peppermint isolate were obtained from Glenn and colleagues (1996)
and from GenBank database (Table I
). 18 S rDNA sequences were aligned using Clustal W (Thompson et al 1994
). The alignments have been deposited in Tree Base (accession number S668). Parsimony analyses were carried out from the alignments using PAUP v. 3.1.1 (Swofford 1993
) on a Macintosh iMac ver. 1.00 computer. Alignment gaps were treated as missing data. Heuristic searches were performed with general, stepwise addition (random sequence addition) and branch swapping (tree bisection-reconnection) algorithms, using collapsing zero length branches and saving all minimal length trees (MULPARS) as options in effect. Ten replications of each heuristic search were performed. To measure the relative support of the resulting clades, bootstrap values (Efron 1982
, Felsenstein 1985
) were calculated using PAUP v. 3.1.1. Bootstrapping was performed with 250 replications. Saccharomyces cerevisiae E. C. Hansen, Taphrina deformans (Berk.) Tul., and Candida tropicalis (Castellani) Berkhout were used as outgroup taxa, based on previous publications (Spatafora and Blackwell 1993
, Glenn et al 1996
) (Table I
); Hypocrea schweinitzii (Fr.) Sacc., Hypomyces poliporinus Peck, and Microascus trigonosporus C. W. Emmons & B. O. Dodge were used as outgroups at a narrow taxonomic scale. The 18 S rDNA sequence of the peppermint isolate was submitted to GenBank (AF292054) for availability to the scientific community. Alignments are available from TreeBase under Study Accession Number S668.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), and 4 C in dark or after treatment with UV lights at 25 C was ineffective for the development of spore formation.
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). The fungus was detectable only in green tissues excised from the first two nodes, starting from the stem base. Mycelial stroma were present in the parenchymatic cells of mature leaves, especially at the first signs of senescence (Fig. 6
). Instead, mycelium was more easily detected in SEM preparations of meristems, stems and leaves after brief incubation on MEA medium. As shown in Fig. 7
, after 10 d of incubation on MEA, mycelium proliferated from excised plant tissues. In 30-d-old plants, the fungus was reisolated from each pair of leaves, going from the base up to the apical shoot. SEM observations of the infected tissues revealed the presence of superficial fungal hyphae, appressed closely to the host surface. They were solitary and branched on both the upper and lower face of 2-d MEA incubated leaves (Fig. 8
), or arranged in mats of numerous hyphae on tissues after prolonged time of culture (Fig. 9
). Mats of anastomosed hyphae, with short branches apically enlarged and tightly appressed to the cuticle surface were frequently observed (data not shown).
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The first ssrDNA data set used in this study consisted of 27 taxa chosen based upon their position in past and contemporary classification systems and possession of particular morphological and ecological features (see ref. list in Table I ). The phylogenetic analysis of all taxa with approximately 1200 bp yielded five equally parsimonious trees of 563 steps, with consistency index (CI) excluding uninformative characters, retention (RI) and rescaled (RC) indices of 0.606, 0.746, and 0.501, respectively. The strict consensus of the five trees is 571 steps in length with a CI of 0.596, a RI of 0.735 and a RC of 0.486 and is presented in Fig. 10 along with bootstrap values greater than 50%.
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In subclade B the Clavicipitales comprising Balansia henningsiana (A. Møller) Diehl, Neotyphodium coenophialum, Epichloë amarillans J. F. White as typical grass endophytes and Atkinsonella hypoxylon (Peck) Diehl, a grass epibiont (Glenn et al 1996
), formed a monophyletic group (bootstrap 58%), which together with the genus Claviceps and Cordyceps is a sister group to the clade containing the Hypocreales. The remaining taxa of the Hypocreales are Nectria cinnabarina (Tode) Fr., already indicated by Spatafora and Blackwell (1993)
as non monophyletic, and Acremonium chrysogenum (Thirum. & Sukapure) W. Gams. The latter has been referred by Glenn and colleagues (1996)
as one of the Acremonium species which, together with the Acremonium -like anamorph of Emericellopsis and of Nectria vilior Starbäck, have affiliations to the Hypocreales. The clade with Microascus and the genus Ceratocystis of the Microascales is a sister group to the taxa sampled for the Hypocreales and Clavicipitales. Monascus purpureus and Eurotium rubrum of the Eurotiales are sister taxa positioned between the derived pyrenomycetes and the outgroup.
In order to analyze the Mentha isolate positioning at a narrow taxonomic scale, taxa of subclade A were implemented with more species sampled within Diaporthales, Ophiostomatales and Sordariales (Sordariomycetidae). A second subset of 25 selected taxa was run and bootstrap values calculated. Hypomyces and Hypocrea among the Hypocreales and Microascus, belonging to the subclade previously referred as B, were included as outgroups. Figure 11 shows the maximum parsimony strict consensus phylogram of this subset, which yielded twelve equally parsimonious trees of 395 steps, with CI, RI, and RC indices of 0.763, 0.813, and 0.620 respectively. The large clade previously indicated as subclade A is now supported by a high bootstrap confidence level of 97%. Based on this new analysis, members of the Diaporthales and the species of genus Ophiostoma were confirmed as sister groups with a bootstrap value of 100% and 75% respectively. The Mentha isolate positioned again as a sister taxon of Diaporthales and of the genus Ophiostoma, with a bootstrap value for this clade of 78%. A separate clade that was comprised of taxa sampled from Sordariales, received a bootstrap value of 100%. When Acremonium alabamense Morgan-Jones of the Sordariales was excluded from the analysis, the bootstrap values of this grouping remained unvaried (100%), however the exclusion reduced the bootstrap value of the sister group comprising the Mentha isolate to 70% (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Cladistic analysis rejected this belief, placing the Mentha isolate in a subclade comprising the perithecial ascomycetes Ophiostomatales and Diaporthales, Sordariales and Xylariales. This cladistic grouping received a bootstrap value of 97% after increasing the number of species in the molecular analysis, thus completely satisfying the results reported by Spatafora and Blackwell (1993, 1994)
. These authors analyzing ssrDNA data showed that the taxa sampled for the Clavicipitales formed a derived monophyletic group of filamentous fungi that is a sister group to the Hypocreales. These results excluded any possible derivation of clavicipitaceous fungi from the order Xylariales (Spatafora and Blackwell 1994
) and supported the establishment by Kreisel (1969)
of a single order including the families Hypocreaceae and Clavicipitaceae. Accordingly to our results, intraordinal relationships within the orders Xylariales, Diaporthales and Sordariales and within the genus Ophiostoma received maximum cladistic support (93%, 100%, 100%, and 100% respectively), nevertheless none of the supraordinal relationships in the subclade A received bootstrap confidence level high enough to draw conclusions on the Mentha isolate attribution. To reach a better resolved phylogeny more data from other parts of the fungal genome are needed (Cabral et al 1999
), therefore ITS regions sequencing of the isolate is in progress.
Our data excluded possible affiliation of the Mentha isolate to the clavicipitalean grass symbionts and their Acremonium anamorphs. However, several morphologically distinct teleomorphic taxa have Acremonium -like anamorphs, especially within the family Hypocreaceae. As more recently showed by Glenn and colleagues (Glenn et al 1996
) Acremonium is a cosmopolitan and heterogeneous taxon, whose simple morphology may have been derived several times in the evolution of fungi (Glenn et al 1996
). Among the Acremonium species that we have examined, A. alabamense (Sordariales) is the only species that positioned in the subclade A lacking other Acremonium or Acremonium-like representatives and comprising the Mentha isolate.
With respect to the situation found in the Clavicipitaceae, the development of beneficial endophytisms has been regarded as depending on the improvement of nutritional host-fungus relationships and concurrent reduction of destructive effects on the host (White and Morgan-Jones 1996
). Ecological characters, such as host affiliation and nutritional mode, are therefore much too complicated to be useful at a taxonomic level, but can be viewed in a phylogenetic context. Since the Mentha isolate and the grass mutualists of the Clavicipitaceae are not related, an independent evolutionary event leading to the colonization of Mentha probably occurred.
Based on the 18 S rDNA sequence analysis, Glenn and colleagues have placed the anamorphs of Epichloë; and related mutualistic clavicipitaceous fungi into the new genus Neotyphodium, which now comprises fungi with unique morphology and ecology with respect to endophytism in the Poaceae (Glenn et al 1996
).
The isolate PGP-HSF inhabits the epibiotic niche of peppermint meristems, but it has been observed to grow endophytically also. The occurrence of an epibiotic stage in the life cycles of some endophytic Epichloë; and Neotyphodium species has been documented and justified by the incorporation of some epiphytic hyphae in the spaces between developing leaves of meristems (White et al 1996
). The localization of mycelium in plant meristems insures the close association of the fungus with the host tissues undergoing cell division and differentiation and the maintenance of a continuous symbiotic association as host develops. The continuous maintenance of the symbiotic relationship requires that the fungus derive energy sources for growth from the host plant. In clavicipitaceous epibiotic fungi, substrate utilization consists in the availability of carbon rings coming from the waxy cuticle covering grass plants and from other exuded compounds as well as lipids, amino acids, and vitamins. The main energy-yielding compounds are simple sugars, that in the case of endophytic mycelia are derived from the apoplasm through the intercellular fungal hyphae (White and Morgan-Jones 1996
). In Mentha isolates, superficial fungal hyphae with tip enlargements tightly adherent to the host surface have been observed. Mechanisms for the selective and efficient transfer of carbohydrates to the fungus could be present and be similar to those present in the haustoria of powdery mildews (Bushnell and Gay 1978
, Bacon and White 2000
).
Physiological changes paralleled by morphological adaptations of the autotrophic host have been described for some endophytic associations (Bacon and White 2000
). In Myriogenospora, plant host changes in the epidermal cell size and shape suggests the activity of growth regulatory substances which are either produced by the fungus and secreted into the host or that are produced by the host in response to the fungal symbiont (Bacon and White 2000
). We did not observe cytological modifications in tissues of Mentha piperita induced by the fungal host. Electron microscopy analyses of leaf and stem tissues are needed to better ascertain the endophytic development into the leaves and to examine the plant-fungus cell interface.
The growth stimulation observed in Mentha piperita in the presence of the fungus, consisted of a global plant response. The vegetative growth enhancement showed by many grass species in the presence of their fungal symbionts (Hill 1994
), has been principally attributed to increased plant fitness. The latter depends on biotic and abiotic stress tolerance as well as drought, soil acidity, and mineral stress resistance, acquired by endophyte-infected grasses (Belesky and Malinowski 2000
). In axenic conditions, as for peppermint, plant growth stimulation due to improved plant fitness may be excluded. On the contrary, different biotic and abiotic factors such as mineral nutrition improvement, changes in the gas atmosphere of the culture vessel, or plant growth promoting substances of fungal origin may be involved in determining the observed plant host response. Enhanced vegetative growth of infected plants has been also documented in the host of Atkinsonella hypoxylon (Clay 1984
) and interpreted as the result of the diversion of energy from inflorescence formation to vegetative processes or alternatively due to growth regulator effects induced by the epibiotic fungus (White and Morgan-Jones 1996
). The observed peppermint root growth stimulation is probably another consequence of fungal growth in contact with this organ. The mycelium spread rapidly from the apex to the culture medium, thus reaching growing rootlets. In this regard, the ability to synthetize and release hormonal substances by the fungal isolate is highly probable and should be assessed. Investigation of the ability of PGP-HSF fungal isolate to promote peppermint growth after in pot and in vitro inoculation is underway.
In the case of grasses, fungal development, either epibiotic or endophytic, is mainly determined by chemical interactions with the host, relying on a diverse array of fungus-produced enzymes, mycotoxins, alkaloids, and perhaps other compounds possessing hormonal activity (Porter 1994
). The true defensive role of these fungal metabolites has been recently debated and put in discussion, owing to a possible different ecological role played by these alkaloids in the evolution of endophyte-infected grasses (Richardson 2000
). The Mentha isolate has been described inhabiting peppermint leaf surfaces. The individual hyphae of the epiphyllous mycelium were observed to run sparsely within peltate and capitate glandular hairs of both upper and lower leaf surfaces, with no apparent signs of suffering. Plant-fungus chemical interactions are highly probable in this situation, especially considering the culture system employed. Terpenoids due to their volatilization from the plant, go to enrich the plant headspace, thus reaching the epiphytic mycelium at the interface of leaf surfaces. If we consider the role of terpenoids as plant defense compounds because of their effects on fungal growth and reproduction (Inouye et al 1998
), the isolation of a fungus inhabiting peppermint leaves poses the question whether these secondary metabolites can contribute to the observed symptomless plant-fungus interaction as a consequence of pathogenicity attenuation. Endophytes clearly have the potential to colonize many diverse ecological and biochemical niches within their plant host, yet there are few detailed studies on mutualistic endophytes inhabiting dycotyledonous herbaceous hosts. The great bulk of scientific reports concern taxa sampled for Xylariales inhabiting leaving trees (Petrini and Petrini 1985
, Petrini and Fisher 1990
, Brunner and Petrini 1992
, Bills 1996
, Stone et al 2000
), while the number of fungi isolated from herbaceous plants is growing (O'Dell and Trappe 1992
, Pereira and Azevedo 1993
, Fisher et al 1995
, Marais et al 1998
). These taxa are believed to have evolved first as opportunistic pathogens, having acquired only later in their evolution a total asyptomatic behavior.
Owing to the colonization of a unique plant niche and the plant growth response observed, the fungal isolate of Mentha piperita represents a novel finding among beneficial plant-fungus symbiosis in non-graminaceous plants. The strictly vegetative self-propagating habit of this species together with the selective pressure exerted by peppermint terpenoids on leaf fungal community might have favored the establishment and perpetuation of this systemic colonization.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted for publication May 24, 2001.
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LITERATURE CITED |
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