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1Title: Microbial diversity of landslide soils assessed by RFLP and SSCP fingerprints.
3Key words: landslide soils/RFLP/SSCP
5Authors: Marco Guida1, Paolo Losanno Cannavacciuolo1, Mara Cesarano2, Marco Borra3, Elio Biffali3,

6Giovanna De Mieri4, Raffaella D’Alessandro4 and Bruna De Felice4§.
81 Department of Biology, University Federico II of Naples, Italy, via Cinthia ed. 7 I-80134,

9Naples, Italy
112 DiSTAR - Department of Earth Sciences, Environment and Resources, University Federico II of Naples,
12Italy, Via Mezzocannone, 8 I-80134, Naples, Italy
133 Zoological Station “Anton Dohrn”, Villa Comunale, 80121 Napoli, Italy
154 DISTABIF- Department of Environmental, Biological and Pharmaceutical Science and Technologies,
16University of Naples II, Via Vivaldi 43, 81100 Caserta, Italy
26§Corresponding author
27Prof. Bruna De Felice, PhD
28Department of Environmental, Biological and Pharmaceutical Science and Technologies,
29University of Naples II,
30Via Vivaldi 43,
3181100 Caserta, Italy
32Tel: ++39-823-274543
33Fax: ++39-823-274571



bacterial and fungal communities in soils triggering landslides in 43Termini-Nerano and Massa Lubrense-Nerano (Naples. some of the identified bacteria. 4 2 .3 37 38 Abstract 39Landslides are a significant component of natural disasters in most countries of the world. were found to favor the 48transformation of clay minerals. 44Fingerprints were generated by single strand conformation polymorphisms (SSCP) and random amplified 45polymorphic DNA (RAPD). These findings suggest a possible relationship between bacterial and fungal 49community colonizing soils and taking place landslides. To address this issue. in both soil types. molecular analysis. Italy) were analysed by genetic profiling techniques. soil transformation. biofilm. Understanding 40these destructive phenomena through the analysis of possible correlation between microbial communities 41and the alteration of the soil responsible for landslides is important in order to reduce their negative 42consequences. forming biofilms and leading to the 47transformation or the formation of minerals. was enriched in species which 46could contribute to degradation process occurring during landslides. The microbial community. 50 51 52 53 54 55 56Keywords: microbial community. Indeed.

SSCP is performed by PCR amplifying a double78stranded DNA fragment that is subsequently denatured to single-stranded DNA and subjected to 79nondenaturing polyacrylamide gel. 75Single-strand conformation polymorphism (SSCP) and random amplified polymorphic DNA (RAPD) 76represent valid alternatives to culturable methods. Culturable methods 72contain significantly less genetic information than the genomes of the total microbiota (Rondon et al. To rise above this problem. The mobility of the single-stranded DNA in the gel is related not only to 80its length but also to its nucleotide sequence (Sunnucks et al. However. 1990. 63Despite the progresses reached in the development of landslide risk assessment in recent years (Xie et al. Huggel et al. 67The complexity of phenomena possibly leading to landslide occurrence is often focused on the 68investigation of macroscopic triggering factors. Baioni 2011. Futagami et al. 2010). a single random oligonucleotide primer 83and small amount of material is needed (Atienzar and Jha 2006). 1998). since it 82requests no prior knowledge about the genome under investigation. 84Up to date. 6 3 . 642003. 71One main purpose in microbial ecology is the understanding of microbial diversity. 2006.5 57 58 Introduction 59 60Landslides are caused by movements of earth secondary to altered stability of the soil and underlying 61materials. Landslides occur as a consequence of several interacting factors. Jamaludin et al. Pardeshi et al. 2000). 81RAPD assay has several advantages when compared to other microbial community analyses. 2009). 2012). evidence of the presence of some kinds of 86bacteria providing the transformation of clay minerals have been reported (Alekseeva et al. 732000). such as man-induced. Torsvik et al. molecular techniques using DNA or RNA extracted directly from the 74soil have been used (Ward et al. only few studies have focused on the correlation between the bacterial community in soils and 85the alteration of the land responsible for landslides. the role of 69microbial community resident in soils in determining soil properties and soil mechanical deterioration has 70been previously supposed (Radina 1973. Lim et al. However. leading to human lives loss and to economical burden both for governments and 66for resident in affected areas. 2013). offering a sensitive strategy to find out microbial 77community from soil (Nair et al. climatic. 2005). such as the ones mentioned above. landslides still remain a major geological hazard in 65several areas of the world. 2002. 62earthquakes or other natural events (Keefer 2002.

94In our research. the studied area is characterized by the presence of rudist limestones of 90the Upper Cretaceous sediments. Italy). and 99comparing the two different fingerprinting methods. on which they transgress the Miocene limestone consisting of pecten and 91a clayey sandstone formation between the ages of Late Cretaceous and early Oligocene. For this reason we used 98SSCP and RAPD strategy as useful typing methods to determine microbial soil community. 96Genetic analyses were performed to verify a possible connection between soils weathering involved in the 97landslides and microorganisms possibly affecting the soil structure alterations. 89From the geological point of view. The materials 92involved in the landslide of 1963 have been ascribed to their training arenaceous clay Lower Miocene 93(Cotecchia and Melidoro 1966).7 87The samples analyzed in our research were collected from two drillings made in the area of Nerano-Termini 88and Massa Lubrense-Nerano (Naples. 8 4 . such as RAPD and SSCP. we studied microorganism communities in the two soils triggering landslides by culture- 95independent analyses.

Nerano-Termini soil taken to a 112depth of 21. and 2.5% agarose gel and stained with ethidium bromide. 106Samples were drilled out from the ground (in situ sampling). 10 5 . A2.USA). CA). 1min at 38◦C. To avoid a possible contamination of these soil materials. PCR products 129were resolved on 2. 1 min at 72◦C. 2nM of random (10 bp) primer.9 100 101 Methods 102 103Research sites and soil sampling 104Soil samples were collected from two areas involved in landslides. 113 114 115Total DNA extraction from bulk soil 116Total DNA was extracted from 0.85m.8% 118w/v agarose) and UV visualization of the ethidium bromide stained gels.55m. Italy) climate zones.5 125μL of 10x enzyme assay buffer. dGTP.5U of AmpliTaq DNA polymerase (Life Technologies. C1.42m. 100 μM each of dATP. NY. as undisturbed samples. we collected the two samples using 108sterilized steel dies. 119 120RAPD analysis 121Genomic profiles of microbial community were studied using RAPD-PCR with 10 different 10-mer 122random primers (data not shown). taken to a depth of 7.10m. The yield and quality of purified DNA was checked by agarose gel electrophoresis (0. The amplification was 127performed in a Perkin-Elmer 9600 thermocycler programmed for 45 cycles as follows: 1st cycle of 1min at 12894◦C. Nerano-Termini soil taken to a depth of 11121. CA). 109Three samples were collected from each site: A1. Only one primer OPU18 (Operon Technologies. Nerano-Termini and Massa Lubrense- 105Nerano (Naples. Alameda.55m. dCTP.97m. by using the Mazier 107sampler. the RAPD reaction was performed with 20 ng DNA soil in a total volume of 25 μL containing 2.25 g of soil sample using the PowerSoil® DNA Isolation Kit (MO BIO. B2. C2 Massa Lubrense-Nerano soil. 117Carlsbad. Nerano-Termini soil taken to a depth of 21. Massa Lubrense-Nerano soil taken to a depth of 7. 124Briefly. 110Massa Lubrense Nerano soil taken to a depth of 8. B1. provided 123consistent and reproducible band patterns and therefore it was selected for further analysis. followed by a final extension cycle of 15min at 72◦C. and 126dTTP.

The PCR was performed by using a total volume of 50 µl containing 1U 138AmpliTaq DNA polymerase. 1989). followed by 32 cycles of 95°C. 2008).25 mM MgCl2. followed by 30 cycles of 94°C. and the single- 153stranded DNA of these bands was eluted for 3 h at 37°C and 500 rpm in 50 μl “crush and soak” buffer (0. 54°C.5% DMSO. 151Extraction. and 137elongation at 72°C. 1990) was used for the second PCR. The electrophoretic 148separation was conducted under non-denaturing conditions using the DCode Universal Mutation Detection 149System (Bio-Rad. 15 s. In a first PCR the fungus-specific primer pair ITS1f/ITS4rP (White et al. 1.11 130 131SSCP analysis of PCR-amplified 16S rRNA and ITS gene fragments 132Single Strand Conformational Polymorphism Analysis (SSCP) of microbial communities was performed as 133described by Schwieger and Tebbe (Schwieger and Tebbe 1998). and elongation at 72°C. 0.2 µM of each primer and 1 µl of template DNA. pH 8. The first PCR (20 143µl) contained of 1 U AmpliTaq DNA polymerase. Extracted DNA fragments were re-amplified by PCR. 2 min. cloned using TA Cloning 157kit (Invitrogen) and sequenced.5 mM MgCl2. Bacterial 16S rRNA gene sequences were 134amplified by PCR using the primer pair Unibac-II-515f (5′-GTG CCA GCA GCC GC-3′) and Unibac-II135927rP (5′-CCC GTC AAT TYM TTT GAG TT-3′) (Zachow et al. 56°C. Hercules. CA). 1 mM EDTA [pH 8. 146Samples served as templates for the second PCR. 10 min. 0.0. 1991). Silver staining was used for the routine detection of DNA bands in SSCP 150gels (Bassam et al.0] and 0.1% sodium dodecyl sulfate) 155(Sambrook et al.5 mg/ml BSA. PCR was started with an initial 136denaturation step at 95°C for 5min. 1990) was used. the amplicons were separated at 400 147V and 26°C in 8 % acrylamide gels and for fungal DNA 9% acrylamide gels.2 µM 144of each primer and 1 µl of template DNA. 1.5 154M ammonium acetate. PCR was started with an initial denaturation step at 95° C for 1457min. 139Fingerprinting of fungal communities by SSCP was carried out as described by Schwieger and Tebbe 140(Schwieger and Tebbe 1998). 72°C. 2. For bacterial DNA. The eluted DNA was precipitated with ethanol and finally resuspended in 12μl of 15610 mM Tris–HCl. 30 s. 142whereas the primer pair ITS1f/ITS2rP (White et al. 45 s. 158 159DNA sequence analysis 12 6 . re-amplification and sequencing of DNA from silver-stained SSCP profile 152Selected bands of the SSCP community profiles were cut out with a sterile razor blade. 72°C. 0. 10 min. 2 min. 10 mM Mg2+-acetate. A nested PCR was applied to obtain genetic fingerprints of fungal 141communities. 20 s.

1994).1 Cycle Sequencing Kit 161(Applied Biosystems).13 160Sequencing of the amplicons was performed using the BigDye Terminator v3. The sequences were matched in BLAST (Altschul et al. in an ABI 3100 automatic DNA sequencer 162(Applied Biosystems). Distance matrix and neighbour-joining methods (Saitou and Nei 1987) 165were applied for tree construction. 167The evolutionary history was inferred using the Neighbor-Joining method and the bootstrap consensus tree 168inferred from 1000 replicates.0 (Tamura et al. 166The bootstrap consensus tree was inferred from 1000 replicates (Felsenstein 1985). with the same primers described before. 1990) and phylogenetic 163analysis was performed using MEGA version 5. The percentage of replicate trees in which the associated taxa clustered 169together in the bootstrap test (1000 replicates) are shown next to the branches. 2011) after multiple alignment of data by 164ClustalW (Thompson et al. 14 7 .

.. 191Uncultured Gram-positive bacterium. fingerprints were performed by RAPD analysis using DNA 173extracted from the soil samples. also if less represented. Thiomonas intermedia K12. 181The analysis of the microorganisms found in each soil sample has allowed the identification of different 182orders. 189In details.. Acidovorax citrulli. Table 1 shows the best sequence homology 176BLAST matching obtained from both soil types clones.500 bp long (Fig. Delftia acidovorans. Moreover. Delftia sp. while Enterobacteriales (22%) and Pseudomonadales (22%) were 185mainly represented in Massa Lubrense-Nerano soil. Alicycliphilus denitrificans BC. Variovorax paradox) have been 196identified only in Nerano-Termini samples (Table 1). 17528 RAPD fragments were excised.1A). cloned and sequenced. 179Fig. The selected RAPD primer produced a number comprised between from 17410 to 16 fragments. Mycobacterium sp. Ramlibacter 195tataouinensis. 178while the remaining 26 clones were found to belong to 24 different genera. Erwinia billingiae strain Eb661. and Pseudomonas aeruginosa) bacteria were found in both 16 8 . Citrobacter koseri 190ATCC. Alicycliphilus denitrificans K601. 187Bacteria belonging to Lactobacillales and Neisseriales orders were specifically found in Nerano-Termini 188soils. 1B shows the phylogenetic relationships between clones and microorganisms identified with BLAST 180analysis. 200–1. 183Burkholderiales (46%). microorganisms belonging to 186Mycoplasmatales. were found in both soils. 192Uncultured Aquamonas sp. Serratia marcescens FGI94. while Actinomycetales and Bacillales were found only in Massa Lubrense-Nerano soil. Methylibium petroleiphilum. Two clone sequences were not attributable to a 177specific taxonomic order (uncultured alpha Proteobacterium and uncultured Gram-positive bacterium). Leuconostoc mesenteroides. Enterobacteriales (27%) and Pseudomonadales (8%) were the main orders 184identified in Nerano-Termini soil.15 170 Results 171RAPD Fingerprint Profiles 172In order to analyze the microbial community. Uncultured alpha Proteobacterium. ten species belonging to Enterobacteriales order (Cronobacter turicensis. Thiomonas sp. 194Polaromonas naphthalenivorans.. Bacillus sp. 197No specific species belonging to Enterobacteriales order was found in Massa Lubrense-Nerano soil and 198Pseudomonadales (uncultured Pseudomonas sp.) and twelve microorganisms belonging to Burkholderiales (Leptothrix 193cholodnii SP-6.

fingerprints were performed also 204by SSCP analysis of 16S rRNA genes (Fig. 18 different bands. Table 2 shows results obtained from sequence 209analysis obtained from both soil types and the corresponding phylogenetic tree is showed in Fig. most sequences were related to bacteria belonging to 216the Pseudomonadales order (18% in Nerano-Termini and 33% in Massa Lubrense-Nerano). as well as the RAPD analysis. both the uncultured Gram-positive bacterium and the uncultured alpha Proteobacterium 200were present in the two soil types.2A). cloned and sequenced. 226Amplicons were separated on acrylamide gels for fungal DNA. the profiles generated with SSCP analysis showed a reduced number of identified bacteria when 221compared to RAPD analysis. uncultured archea and 219Desulfuromonadales (uncultured Geobacter sp. The 210nucleotide sequences retrieved from the SSCP profile showed similarities to 16S rRNA gene sequences 211deposited in public databases with a maximum identity ranging from 92 to 99%. while the remaining 6 clones were found to belong to 5 different genera. 215It is interesting that. 205In order to assign single-stranded PCR products generated to specific microorganisms.17 199soils. subjected to PCR with the same primers as in the first amplification 207and then cloned and sequenced. SSCP patterns were obtained with a nested PCR using the fungi-specific primer pair ITS. 201 202SSCP profiles of bacterial communities 203To establish the diversity of microbial isolates from the two studied soils. 212Five clone sequences were not attributable to a specific taxonomic order (three sequences related to 213uncultured bacteria. each band was 206extracted from polyacrylamide gels. 217Actinomycetales.) were specifically found in Nerano-Termini soil sample. were isolated from SSCP profiles. 222 223Fingerprinting of fungal communities by SSCP 224In order to characterize fungal communities. Instead. showed in Fig. one sequence related to uncultured archaeon and one sequence related to uncultured 214alpha Proteobacterium). Moreover. 220However. In total. Enterobacteriales. uncultured bacteria. 208Eleven SSCP fragments were excised. two different soil types were analysed employing SSCP 225analysis. Mycoplasmatales and Alphaproteobacteria microorganisms were 218common to the two soil types studied. 2273. 18 9 . 2B.

237uncultured Basidiomycota and uncultured fungi were identified. cloned and sequenced. On the basis of 231BLASTn results. in Nerano-Termini soil Malasseziales fungi were mainly represented (43%). Eurotiales and Hypocreales). The identity of single-stranded PCR products isolated are shown in Table 3. but also Eurotiales. The remaining 12 clones were found to be related to sequences from fungi belonging 234to three orders (Malasseziales. six clones could not be attributed to any taxonomic order (two sequences were associated 232to uncultured marine fungi. two to uncultured fungus and one to 233Sordariomycetes sp. 238Sordariomycetes and uncultured fungi were identified. 229Bioinformatic analysis of sequences cloned from SSCP bands showed he presence of fungal DNA in our 230samples. In Massa Lubrense-Nerano.). No overlap between the fungi orders found for 235the two soil samples was showed after clone analysis. 20 10 .19 228Dominant bands were excised from SSCP gels and re-amplified by PCR. and Hypocreales represented the most frequent 239order (82%). one to uncultured Basidiomycota. 236In details.

litter. based on DNA analysis. such 253as RAPD and SSCP. that are heavily influenced by the substratum. it has been suggested that the 22 11 . Moreover. or soil particles. Both analysis provided evidence of a prevalence of 262Pseudomonadales and the presence of Actinomycetales and Enterobacteriales in studied soils. transformation. little has been 249studied to determine the relationships between microbial community composition and the alteration of the 250land responsible for landslides. often with unique 265structural phenomena. and often alter the surfaces as a result of this interaction. and are characterized by distinct physical and chemical gradients that affect 266microbial metabolic processes. Studies on landslides are often focused on landslide 245distribution. it has been reported that the processes of transformation of clay minerals such as 247intensification of removal of exchange bases and dissolution of silicates and iron oxides occurred in the 248presence of the alkaliphilic cyanobacterial community (Alekseeva et al. Myster and Walker 2461997). or even the formation of minerals. Lactobacillales and Neisseriales bacteria were specifically found in Nerano-Termini soils. A biofilm can 264be broadly defined as an organised system of microbial cells associated with surfaces. 259while Actinomycetales and Bacillales were found only in Massa Lubrense-Nerano soil.2009). Bacteria attach to surfaces. However. Previously. nutrient availability and plant successions (Guariguata and Larsen 1990. 258Burkholderiales. 260The comparison of RAPD results with bacterial SSCP results showed that the first method allows the 261identification of a larger population of microorganisms. They can cause extensive damage to life having important landscape and 244ecosystem-wide effects on nutrient availability. 257RAPD analysis allowed also the identification of differences in microbial communities in the two soils. 267form biofilms on them. leading to the destruction 268(dissolution). 251In the present work we analyzed the bacterial and fungal communities in soils triggering landslides in 252Nerano-Termini and Massa Lubrense-Nerano by culture-independent method. 254RAPD fingerprinting results and phylogenetic analysis showed that Nerano-Termini and Massa Lubrense- 255Nerano soils were characterized by a prevalence of microorganisms belonging to Enterobacteriales and 256Pseudomonadales orders. 263It is known that most soil bacteria are organized in biofilms on roots.21 240 241 Discussion 242Landslides are one of the most severe natural disturbances characterized by the rapid movements of the 243earths or other solid material.

actively swimming by means of its 283flagellum. Ramlibacter tataouinensis Strain TTB310 is able to 298use only acetate. such as reduced inorganic sulphur compounds. as a unicellular organism. sludge and other sediments. Among the identified species belonging to the genus Pseudomonas. 281Additionally. Several organic or inorganic electron donors. coli O157 has been demonstrated to survive in cold water for up to 274twelve weeks in mud. Mn. 272Burkholderiales and Pseudomonadales.. sheath forming. filamentous bacteria. beside 287bacteria and fungi can be absorbed on the surface of clay minerals and the products of their metabolism 288interact with the minerals (Sokolova et al. and in both treated and untreated waters (Cooper et al. specifically. DL-lactate or propionate of 24 12 . pyruvate. seas and 275along riverbeds (Hall-Stoodley and Stoodley 2005). 2002). Burkholderia sp. Pseudomonas aeruginosa is 284ubiquitous in soil and water has forming an antibiotic-resistant biofilm (Drenkard and Ausubel 2002). Molecular typing analyses has determined that the 276bacterium is capable of surviving for months.and manganese-rich water (Siering and Ghiorse 2911996). iron. beta-hydroxybutyrate. could 292be used in carbon dioxide fixation during anoxygenic photosynthetic growth of Thiomonas sp. the typical Pseudomonas bacterium might be found in biofilm in nature.g Fe. 2005). able to oxidize Mn2+ and Fe2+ 290and is usually found in oligotrophic. E. attached to some 282surface or substrate. as members of anode 280biofilm communities surfaces has also been reported (Chung and Okabe 2009). the presence of species of Burkholderiales. which has 293showed particular carbon and energy metabolic capacities (Frigaard and Dahl 2009). including those occurring naturally in lakes. are well-characterised biofilm-associated microorganism groups. on inorganic 278substrata (such as wood or metal). Delftia acidovorans is known to degrade a number of organic compounds such as 2-(4296sulfophenyl)butyrate (SPB) and may be useful for the degradation of linear alkylbenzenesulfonate (LAS) 297surfactant in wastewater treatment (Schulz et al.23 269biofilm microenvironment may stimulate bacterial production of specific extracellular enzymes involved in 270degradation of organic material (Jass et al. the orders and genera mainly identified in soil types in this study. and perhaps years. 273Among the Enterobacteriales. 294Also other bacteria found in our soil samples have been previously found to be able to metabolize soil- 295related compounds. heavily embedded within biofilm 277matrices. etc. 271Interestingly.) makes minerals extremely sensitive to environmental conditions (Stucki 1988). slowly running. gamma-hydroxybutyrate. 2000). or in a planktonic form. 2007). Enterobacteriales. 279Beside. 285Previously. persisting outside a host organism in animal faeces or faecally-derived material. Leptothrix cholodnii is an aerobic. Among the bacterial species identified in Nerano-Termini 289soil. it has been reported that frequent occurrence in soil structure of the ions with variable valence 286(e.

likely due to the loss of hyphal wall integrity during autolysis. 320Moreover.25 299tested carbon sources and it is able to reduce nitrate to nitrite. 2008) can be extended by the discovery of uncultured microorganisms. our genetic analyses revealed the presence of fungi in soil samples. 327In conclusion. Previous studies support the importance to identify uncultured 323microbial species in soil samples. while SSCP analysis with specific primers for fungi 319identification resulted in a more detailed profile of the fungal community. highlighting the relevance of molecular studies to analyze microbial communities 322from complex samples. 314This finding supports a possible role of fungal community in determining physical properties of soils. most probably as a result of hyphal enmeshment. 310Once formed. 2012). Gommeaux et al. 2004). 304Other than bacteria. moreover. the present study showed that bacterial and fungal communities found in Nerano-Termini 328and Massa Lubrense-Nerano soils were enriched in species which could contribute to degradation processes 26 13 . it has been reported that 309Trichocomaceous fungi are able to create soil aggregates. The 302association of genetic analysis to identify microorganism present in soil samples with compositional 303analysis would be a valid approach to increase current knowledge about features leading to landslides. recently. Our results about 305the fungal community showed differences among species composition in two soil types. 2003. and enzyme discovery 325(Kim et al. as they can have specific metabolic capabilities not expressed by cultured 324species. 311leading to disintegration of macroaggregates into microaggregates or reversion to the original size 312distribution (Daynes et al. However. In 307Nerano-Termini soil we identified uncultured Trichocomaceae. 315Our research. antibiotic resistance genes knowledge (Riesenfeld et al. allowing the research of 326compounds which inhibit resistance mechanisms. 301Reported data suggest a role of biofilm-forming bacteria in chemical composition of the soil. other than provide evidence of microbial community differences between two landslide- 316subjected soils. 2005). 317Our results showed that RAPD analysis allowed to obtain more detailed results about the bacterial 318community present in the studied soil samples. allows the comparison of two molecular analyses in finding bacterial and fungal species. the aggregates are not stable. such as the soil. For example. the identification of still uncultured microbial species underlies the limitation of standard 321culture-based techniques. 306Some of them are known to be involved in biochemical processes possibly related to soil properties. 313It is interesting that in Massa Lubrense Nerano soil we identified saprophytic fungi. a beta-glucosidase activity has 300been reported (Heulin et al. Fusarium and Nectria. Trichocomaceous are saprophytic fungi 308whose role in soil aggregation remains uncertain.

333 28 14 . However. Furthermore. further studies 332are necessary to confirm the direct involvement of bacterial and fungal species to determine soil properties.27 329leading to soil properties change possibly involved in landslides occurring in the studied sites. 330the results of this study can be considered as an initial support for evaluating the relationship among 331bacterial and fungal community colonizing soils and the occurring of landslides.

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The percentage of replicate trees in which the 478associated taxa clustered together in the bootstrap test are shown next to the branches. 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 38 19 . 474A SSCP analyses of PCR-amplified 16S rRNA genes of soil samples. 1 Results from RAPD fingerprints. lane 4: sample B2. lane 3: sample B1. lane 6: sample C2. lane 5: sample C1. and an additional fragment at 4652. lane 6: sample C2. 483B Phylogenetic tree of fungal community profiles generated by SSCP analysis was obtained using the 484Neighbor-Joining method and the bootstrap consensus tree inferred from 1000 replicates. lane 2: sample A2. 471 472 473Fig. M: the DNA ladder consisting of 15 blunt 464fragments ranging in length from 100 to 1. lane 6: sample C2. 2 Results from SSCP analysis of bacterial community. at 100-bp increments. lane 2: sample 475A2. Lane 1: sample A1. lane 3: sample B1. Lane 1: sample A1.500 bp. 463A RAPD fingerprints of DNA from soil microbial communities. The percentage of 485replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the 486branches. 467B Phylogenetic tree of bacterial community profiles generated from RAPD analysis was obtained using the 468Neighbor-Joining method and the bootstrap consensus tree inferred from 1000 replicates. lane 5: sample C1. 476B Phylogenetic tree of 16S rRNA SSCP clones was obtained using the Neighbor-Joining method and the 477bootstrap consensus tree inferred from 1000 replicates.072 bp (Roche). lane 3: sample B1. lane 2: 482sample A2. The percentage of 469replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the 470branches. lane 4: sample B2. lane 4: sample B2. 479 480Fig. Lane 1: sample A1. lane 7: negative control. lane 5: 466sample C1.37 459 460Figure Legends 461 462Fig. 3 Results from SSCP analysis of fungal community 481A SSCP fingerprint patterns of fungal communities obtained of soil samples.