MICROBIAL GENETICS • ORIGINAL PAPER

Microbial diversity of landslide soils assessed by RFLP and SSCPfingerprintsMicrobial fingerprint of landslide soil

Marco Guida & Paolo Losanno Cannavacciuolo &

Mara Cesarano & Marco Borra & Elio Biffali &Raffaella D’Alessandro & Bruna De Felice

Received: 10 November 2013 /Revised: 10 March 2014 /Accepted: 24 March 2014# Institute of Plant Genetics, Polish Academy of Sciences, Poznan 2014

Abstract Landslides are a significant component of naturaldisasters inmost countries around theworld. Understanding thesedestructive phenomena through the analysis of possible correla-tions between microbial communities and the alteration of thesoil responsible for landslides is important in order to reduce theirnegative consequences. To address this issue, bacterial and fungalcommunities in soils triggering landslides in Termini-Nerano andMassa Lubrense-Nerano (Naples, Italy) were analysed by geneticprofiling techniques. Fingerprints were generated by single-strand conformation polymorphisms (SSCP) and random ampli-fied polymorphic DNA (RAPD). The microbial community inboth soil types was enriched in species which could contribute tothe degradation process occurring during landslides, formingbiofilms and leading to the transformation or the formation ofminerals. Indeed, some of the identified bacteria were found tofavour the transformation of clay minerals. These findings

suggest a possible relationship between bacterial and fungalcommunity-colonising soils and the occurrence of landslides.

Keywords Microbial community .Molecular analysis .

Biofilm . Soil transformation . Landslide soils . RFLP . SSCP

Introduction

Landslides are caused by movements of earth secondary toaltered stability of the soil and underlying materials. Theyoccur as a consequence of several interacting factors, such asman-induced, climatic, earthquakes or other natural events(Keefer 2002; Baioni et al. 2011; Huggel et al. 2012).

Despite the progress in the development of landslide riskassessment in recent years (Xie et al. 2003; Jamaludin et al.2006; Pardeshi et al. 2013), landslides still remain a majorgeological hazard in several areas of the world, leading to lossof human lives and economical burden for both governmentsand residents in affected areas.

The complexity of phenomena possibly leading to landslideoccurrence is often focused on the investigation of macroscopictriggering factors, such as those mentioned above. However, therole of the microbial community resident in soils in determiningsoil properties and soil mechanical deterioration has been previ-ously hypothesised (Radina 1973; Futagami et al. 2010).

One of the main purposes of microbial ecology is theunderstanding of microbial diversity. Culturable methods con-tain significantly less genetic information than the genomes ofthe total microbiota (Rondon et al. 2000; De Felice et al. 2009,2010). To solve this problem, molecular techniques usingDNA or RNA extracted directly from the soil have been used(Ward et al. 1990; Torsvik et al. 1998).

Single-strand conformation polymorphism (SSCP) andrandom amplified polymorphic DNA (RAPD) represent valid

Electronic supplementary material The online version of this article(doi:10.1007/s13353-014-0208-y) contains supplementary material,which is available to authorized users.

M. Guida : P. L. CannavacciuoloDepartment of Biology, University Federico II of Naples, Italy, viaCinthia ed. 7, 80134 Naples, Italy

M. CesaranoDiSTAR—Department of Earth Sciences, Environment andResources, University Federico II of Naples, Italy, ViaMezzocannone, 8, 80134 Naples, Italy

M. Borra : E. BiffaliZoological Station “Anton Dohrn”, Villa Comunale, 80121 Naples,Italy

R. D’Alessandro : B. De Felice (*)DISTABIF—Department of Environmental, Biological andPharmaceutical Science and Technologies, University of Naples II,Via Vivaldi 43, 81100 Caserta, Italye-mail: bruna.defelice@unina2.it

J Appl GeneticsDOI 10.1007/s13353-014-0208-y

alternatives to culturable methods, offering a sensitive strategyto ascertain the microbial community from soil (Nair et al.2002; Lim et al. 2005). SSCP is performed by polymerasechain reaction (PCR) amplifying a double-stranded DNAfragment that is subsequently denatured to single-strandedDNA and subjected to non-denaturing polyacrylamide gelelectrophoresis. In fact, the mobility of DNA in the gel de-pends on the secondary DNA strand structure, which is af-fected by the sequence. In fact, the mobility of DNA in the geldepends on the secondary DNA strand structure, which isaffected by the sequence (Sunnucks et al. 2000).

The RAPD assay has several advantages when comparedto other microbial community analyses, since it requires noprior knowledge of the genome under investigation, a singlerandom oligonucleotide primer and only a small amount ofmaterial is needed (Atienzar and Jha 2006).

To date, only a few studies have focused on the correlationbetween the bacterial community in soils and the alteration ofthe land responsible for landslides. However, evidence hasbeen reported on the presence of some kind of bacteria caus-ing the transformation of clay minerals (Alekseeva et al.2009).

The samples analysed in our research were collected fromtwo drillings made in the area of Nerano-Termini and MassaLubrense-Nerano (Naples, Italy).

From the geological point of view, the studied area ischaracterised by the presence of rudist limestones of theUpper Cretaceous sediments, on which they transgress theMiocene limestone consisting of pectin and a clayey sand-stone formation between the ages of Late Cretaceous and earlyOligocene. The materials involved in the landslide of 1963have been ascribed to their training arenaceous clay LowerMiocene (Cotecchia and Melidoro 1966).

In our research, we studied the microorganism communi-ties in the two soils triggering landslides by culture-independent analyses, such as RAPD and SSCP.

Genetic analyses were performed to verify a possible con-nection between soils weathering involved in the landslidesand microorganisms possibly affecting the soil structure alter-ations. For this reason, we used SSCP and RAPD strategies asuseful typing methods to determine the microbial soil com-munity and compare the two different fingerprinting methods.

Methods

Research sites and soil sampling

Soil samples were collected from two areas involved in land-slides, the Nerano-Termini and Massa Lubrense-Nerano(Naples, Italy) climate zones, and from one area outside thelandslides zone, Pompeii (Naples, Italy).

Samples were drilled out from the ground (in situ sam-pling), as undisturbed samples, by using the Mazier sampler.To avoid possible contamination of these soil materials, wecollected the two samples using sterilised steel dies.

Three samples were collected from each site: A1, Nerano-Termini soil taken at a depth of 21.55 m; A2,Massa Lubrense-Nerano soil taken at a depth of 8.10 m; B1, Nerano-Terminisoil taken at a depth of 21.42 m; B2, Massa Lubrense-Neranosoil taken at a depth of 7.97 m; C1, Nerano-Termini soil takenat a depth of 21.55 m; C2, Massa Lubrense-Nerano soil takenat a depth of 7.85 m.

As a control, sample soil was collected from regions out-side of the landslides zone: D1, Pompeii soil taken at a depthof 7.87 m; D2, Pompeii soil taken at a depth of 8.15 m; D3,Pompeii soil taken at a depth of 7.96 m. The studied area ischaracterised by the presence of altered terrains constituted bypyroclastic deposits of the Vesuvius eruption (AD 79), whichconsists of white pumice, followed by grey pumice of pyro-clastic flows. Lava lithics are also present, mainly in the upperpart of the stratigraphic sequence.

Total DNA extraction from bulk soil

Total DNAwas extracted from 0.25 g of soil sample using thePowerSoil® DNA Isolation Kit (MO BIO, Carlsbad, CA,USA). The yield and quality of purified DNA was checkedby agarose gel electrophoresis (0.8 % w/v agarose) and UVvisualisation of the ethidium bromide-stained gels.

RAPD analysis

Genomic profiles of the microbial community were studiedusing RAPD-PCR with ten different 10-mer random primers(Supplementary Table 1). Only one primer, OPU18 (OperonTechnologies, Alameda, CA, USA), provided consistent andreproducible band patterns and, therefore, it was selected forfurther analysis.

Briefly, the RAPD reaction was performed with 20 ng ofDNA soil in a total volume of 25μL containing 2.5μL of 10×enzyme assay buffer, 2 nM of random (10 bp) primer, 100μMeach of dATP, dCTP, dGTP and dTTP, and 2.5U of AmpliTaqDNA Polymerase (Life Technologies, NY, USA). The amplifi-cation was performed in a Perkin Elmer 9600 Thermal Cyclerprogrammed for 45 cycles as follows: first cycle of 1 min at94 °C, 1 min at 38 °C, 1 min at 72 °C, followed by a finalextension cycle of 15 min at 72 °C. PCR products were re-solved on 2.5% agarose gel and stainedwith ethidium bromide.

SSCP analysis of PCR-amplified 16S rRNA and ITS genefragments

SSCP analysis of microbial communities was performed asdescribed by Schwieger and Tebbe (1998). Bacterial 16S

J Appl Genetics

rRNA gene sequences were amplified by PCR using theprimer pair Unibac-II-515f (5′-GTG CCA GCA GCC GC-3′) and Unibac-II-927rP (5′-CCC GTC AAT TYM TTT GAGTT-3′) (Zachow et al. 2008). PCR was started with an initialdenaturation step at 95 °C for 5 min, followed by 32 cycles of95 °C, 20 s; 54 °C, 15 s; 72 °C, 30 s; and elongation at 72 °C,10 min. The PCR was performed by using a total volume of50 μl containing 1U AmpliTaq DNA Polymerase, 1.5 mMMgCl2, 0.2 μM of each primer and 1 μl of template DNA.Fingerprinting of fungal communities by SSCP was carriedout as described by Schwieger and Tebbe (1998). A nestedPCR was applied to obtain genetic fingerprints of fungalcommunities. In a first PCR, the fungus-specific primer pairITS1f/ITS4rP (White et al. 1990) was used, whereas theprimer pair ITS1f/ITS2rP (White et al. 1990) was used forthe second PCR. The first PCR (20 μl) contained of 1UAmpliTaq DNA polymerase, 2.25 mM MgCl2, 0.5 mg/mlBSA, 1.5 % DMSO, 0.2 μM of each primer and 1 μl oftemplate DNA. PCR was started with an initial denaturationstep at 95 ° C for 7 min, followed by 30 cycles of 94 °C, 45 s;56 °C, 2 min; 72 °C, 2 min; and elongation at 72 °C, 10 min.Samples served as templates for the second PCR. For bacterialDNA, the amplicons were separated at 400 Vand 26 °C in 8%acrylamide gels and for fungal DNA 9% acrylamide gels. Theelectrophoretic separation was conducted under non-denaturing conditions using the DCode Universal MutationDetection System (Bio-Rad, Hercules, CA, USA). Silverstaining was used for the routine detection of DNA bands inSSCP gels (Bassam et al. 1991).

Extraction, re-amplification and sequencing of DNAfrom silver-stained SSCP profiles

Selected bands of the SSCP community profiles were cut outwith a sterile razor blade, and the single-stranded DNA of

�Fig. 1 Results from random amplified polymorphic DNA (RAPD)fingerprints on landslide soils. a RAPD fingerprints of DNA from soilmicrobial communities. M: DNA Molecular Weight Marker XIV(Roche); A1: Nerano-Termini, depth of 21.55 m; A2: Massa Lubrense-Nerano, depth of 8.10 m; B1: Nerano-Termini, depth of 21.42 m; B2:Massa Lubrense-Nerano, depth of 7.97 m; C1: Nerano-Termini, depth of21.55 m; C2: depth of 7.85 m; NC: negative control. Cloned ampliconswhich allowed the identification of microorganisms in our samples areshown in the figure and are indicated as “a” followed by a number.Amplicons a1 to a11 and a14 to a28 allowed microorganismsidentification in Nerano-Termini soil. Amplicons a2 to a5, a8 and a10to a13 allowed microorganisms identification in Massa Lubrense-Neranosoil (see Table 1). b The phylogenetic tree of bacterial community profilesgenerated from the RAPD analysis was obtained using the neighbour-joining method and the bootstrap consensus tree was inferred from 1,000replicates. The percentage of replicate trees in which the associated taxaclustered together in the bootstrap test are shown next to the branches.The soil of origin is indicated for each clone: T indicates Nerano-Terminisoil andM indicates Massa Lubrense-Nerano soil

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these bands was eluted for 3 h at 37 °C and 500 rpm in 50μl of“crush and soak” buffer (0.5 M ammonium acetate,10 mM Mg2+ acetate, 1 mM EDTA [pH 8.0] and 0.1 %sodium dodecyl sulphate) (Sambrook et al. 1989). The elutedDNAwas precipitated with ethanol and finally resuspended in12 μl of 10 mM Tris–HCl, pH 8.0. Extracted DNA fragmentswere re-amplified by PCR, cloned using the TA Cloning Kit(Invitrogen) and sequenced.

DNA sequence analysis

Sequencing of the amplicons was performed using theBigDye Terminator v3.1 Cycle Sequencing Kit (AppliedBiosystems), with the same primers described previously, inan ABI PRISM 3100 automated DNA sequencer (AppliedBiosystems). The sequences were matched in BLAST(Altschul et al. 1990) and phylogenetic analysis was per-formed using MEGA version 5.0 (Tamura et al. 2011) after

multiple alignments of the data by ClustalW (Thompson et al.1994). Distance matrix and neighbour-joining methods(Saitou and Nei 1987) were applied for tree construction.

The bootstrap consensus tree was inferred from 1,000replicates (Felsenstein 1985) and the evolutionary historywas inferred using the neighbour-joining method. The per-centage of replicate trees in which the associated taxa clus-tered together in the bootstrap test (1,000 replicates) areshown next to the branches.

Results

RAPD fingerprint profiles

In order to analyse the microbial community, fingerprints wereperformed by RAPD analysis using DNA extracted from the

Table 1 Distribution of sequences obtained from bacterial community profiles generated from random amplified polymorphic DNA (RAPD) analysis

Soil RAPD clone Best sequence homology Sequence ID E value Max identity

T a1 Enterobacter cloacae FP929040.1 2e-78 75 %

T, M a2 Escherichia coli O157:H7 AB294212.1 2e-43 97 %

T, M a3 Pseudomonas aeruginosa HQ326208.1 1e-115 99 %

T, M a4 Candidatus Mycoplasma turicensis DQ157153.1 8e-78 97 %

T, M a5 Uncultured Pseudomonas sp. AM084245.1 1e-94 97 %

T a6 Cronobacter turicensis FN543093.2 3e-131 95 %

T a7 Citrobacter koseri ATCC CP000822.1 2e-114 91 %

T, M a8 Erwinia billingiae strain Eb661 FP236843.1 2e-87 91 %

T a9 Serratia marcescens FGI94 CP003942.1 8e-100 89 %

T, M a10 Uncultured alpha Proteobacterium AJ633947.1 7e-34 95 %

T, M a11 Uncultured Gram-positive bacterium AJ633938.1 4e-76 92 %

M a12 Mycobacterium sp. AJ783967.1 1e-82 91 %

M a13 Bacillus sp. AM990469.1 7e-29 97 %

T a14 Leuconostoc mesenteroides GQ456941.1 3e-04 92 %

T a15 Uncultured Aquamonas sp. FM994919.1 6e-44 98 %

T a16 Leptothrix cholodnii SP-6 CP001013.1 2e-130 83 %

T a17 Methylibium petroleiphilum CP000555.1 5e-149 86 %

T a18 Thiomonas sp. FP475956.1 1e-168 89 %

T a19 Thiomonas intermedia K12 CP002021.1 3e-152 86 %

T a20 Delftia sp. CP002735.1 2e-147 86 %

T a21 Polaromonas naphthalenivorans CP000529.1 9e-89 77 %

T a22 Delftia acidovorans CP000884.1 3e-101 79 %

T a23 Alicycliphilus denitrificans BC CP002449.1 2e-96 79 %

T a24 Ramlibacter tataouinensis CP000245.1 2e-78 76 %

T a25 Alicycliphilus denitrificans K601 CP002657.1 2e-98 79 %

T a26 Acidovorax citrulli CP000512.1 8e-115 81 %

T a27 Variovorax paradoxus CP001635.1 5e-105 80 %

T a28 Chromobacterium violaceum AE016825.1 9e-70 74 %

T Nerano-Termini soil; M Massa Lubrense-Nerano soil

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soil samples. The selected RAPD primer produced between10 and 16 fragments, 200–1,500 bp long (Fig. 1a).

Twenty-eight RAPD fragments were excised, cloned andsequenced. Table 1 shows the best sequence homologyBLAST matching obtained from clones of both soil types.Two clone sequences were not attributable to a specific taxo-nomic order (uncultured alpha Proteobacterium and uncul-tured Gram-positive bacterium), while the remaining 26clones were found to belong to 24 different genera.

Figure 1b shows the phylogenetic relationships betweenclones and microorganisms identified with the BLASTanalysis.

The analysis of the microorganisms found in each soilsample has allowed the identification of differentorders.Burkholderiales (46 %), Enterobacteriales (27 %) andPseudomonadales (8 %) were the main orders identified inNerano-Termini soil, while Enterobacteriales (22 %) andPseudomonadales (22 %) were mainly represented in MassaLubrense-Nerano soil (samples A2, B2 and C2). Moreover,microorganisms belonging to Mycoplasmatales, also if less

represented, were found in both soils (samples A1, A2, B1,B2, C1, C2).

Bacteria belonging to Lactobacillales and Neisseriales or-ders were specifically found in Nerano-Termini soils (samplesA1, B1 and C1), while Actinomycetales and Bacillales werefound only in Massa Lubrense-Nerano soil.

In detail, seven species belonging to the Enterobacterialesorder [Enterobacter cloacae (clone a1), Escherichia coliO157:H7 (clone a2), Cronobacter turicensis (clone a6),Citrobacter koseri ATCC (clone a7), Erwinia billingiae strainEb661 (clone a8), Serratia marcescens FGI94 (clone a9) anduncultured Aquamonas sp. (clone a15)]. In Nerano-Terminisamples, Leuconostoc mesenteroides (clone a14), unculturedalpha Proteobacterium (clone a10), uncultured Gram-positivebacterium (clone a11) and 12 microorganisms belonging toBurkholderiales [Leptothrix cholodnii SP-6 (clone a16),Methylibium petroleiphilum, Thiomonas sp. (clone a17),Thiomonas intermedia K12 (clone a19), Delftia sp. (clonea20), Polaromonas naphthalenivorans (clone a21), Delftiaacidovorans (clone a22), Alicycliphilus denitrificans BC

Fig. 2 Results from RAPDfingerprints on control soil.RAPD fingerprints of DNA fromcontrol soil microbialcommunities.M: DNAMolecularWeight Marker XIV (Roche);D1:Pompeii, depth of 7.87 m; D2:Pompeii, depth of 8.15 m; D3:Pompeii, depth of 7.96 m. Clonedamplicons which allowed theidentification of microorganismsin our samples are shown in thefigure and are indicated as “a”followed by a number (from a29to a46) (see Table 2)

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(clone a23), Ramlibacter tataouinensis (clone a24),Alicycliphilus denitrificans K601 (clone a25), Acidovoraxcitrulli (clone a26), Variovorax paradox (clone a27)(Table 1)] have also been identified. No specific speciesbelonging to the Enterobacteriales order was found inMassa Lubrense-Nerano soil and Pseudomonadales [uncul-tured Pseudomonas sp. (clone a5) and Pseudomonasaeruginosa (clone a3)] bacteria were found in both soils.Moreover, both the uncultured Gram-positive bacterium(clone a11) and the uncultured alpha Proteobacterium (clonea10) were present in the two soil types.

Figure 2 shows the microbial community fingerprints per-formed by RAPD analysis using DNA extracted from the soilsamples collected from areas outside of the landslides zone ascontrols (Pompeii, Campania, Italy). Table 2 shows the bestsequence homology BLAST matching obtained from the con-trol soil clones, revealing no significant overlapping withclones obtained from the landslide soils. Eighteen microor-ganisms were identified and five were not attributable to aspecific order [uncultured Actinobacteria (clone a 29), uncul-tured alpha Proteobacterium (clone a30), unclassified betaProteobacterium (clone a31), unclassified gammaProteobacterium (clone a32), uncultured Verrucomicrobia(clone a39)], while the others were found to belong to eightdifferent orders.

The main bacteria orders identified in Pompeii control soilwere Bacillales (28 %) and Flavobacteriales (11 %).Specifically, Bacillus subtilis (clone a36), Bacillus cereus(clone a37), Bacillus brevis (clone a42), Bacillus lentus (clone

a43) and Bacillus laterosporus (clone a46) species belong tothe Bacillales order andChryseobacterium sp. (clone a34) andFlavobacterium sp. (clone a35) were found to belong toFlavobacteriales. Other microorganisms identified wereChthoniobacter flavus (clone a33, Chthoniobacterales order),Rhizobium sp. (clone a38, Rhizobiales order), Acinetobactersp. (clone a40, Pseudomonadales order), Aureobacterium sp.(c lone a41, Act inomycetales order) , uncul turedAcetobacteraceae (clone a44, Rhodospirillales order) anduncultured Rickettsiella (clone a45, Legionellales order).

SSCP profiles of bacterial communities

To establish the diversity of microbial isolates from the land-slide soils, fingerprints were also performed by SSCP analysisof 16S rRNA genes (Fig. 3a).

In order to assign single-stranded PCR products generatedto specific microorganisms, each band was extracted frompolyacrylamide gels, subjected to PCR with the same primersas in the first amplification and then cloned and sequenced.

Eleven SSCP fragments were excised, cloned and se-quenced. Table 3 shows the results obtained from the se-quence analysis of both soil types and the correspondingphylogenetic tree is shown in Fig. 3b. The nucleotide se-quences retrieved from the SSCP profile showed similaritiesto 16S rRNA gene sequences deposited in public databases,with a maximum identity ranging from 92 to 99 %.

Five clone sequences were not attributable to a specifictaxonomic order [three sequences related to uncultured

Table 2 Distribution of sequences obtained from bacterial community profiles generated from RAPD analysis on control samples

Soil RAPD clone Best sequence homology Sequence ID E value Max identity

C a29 Uncultured Actinobacteria AY922236.1 5e-70 96 %

C a30 Uncultured alpha Proteobacterium EF208568.1 7e-31 90 %

C a31 Unclassified beta Proteobacterium AB194098.1 9e-60 79 %

C a32 Unclassified gamma Proteobacterium GU187295 6e-41 95 %

C a33 Chthoniobacter flavus AM905294.1 3e-23 96 %

C a34 Chryseobacterium sp. KF751764.1 2e-45 98 %

C a35 Flavobacterium sp. M22863.1 2e-83 94 %

C a36 Bacillus subtilis AF011544.1 5e-23 79 %

C a37 Bacillus cereus AB020313.1 2e-44 80 %

C a38 Rhizobium sp. U56239.1 3e-62 94 %

C a39 Uncultured Verrucomicrobia AB826713.1 1e-73 96 %

C a40 Acinetobacter sp. AF132598.1 3e-34 92 %

C a41 Aureobacterium sp. Y15325.1 6e-33 98 %

C a42 Bacillus brevis D10696.1 2e-43 94 %

C a43 Bacillus lentus AB028644.1 2e-41 91 %

C a44 Uncultured Acetobacteraceae HM112259.1 8e-51 82 %

C a45 Uncultured Rickettsiella HF912419.1 1e-53 92 %

C a46 Bacillus laterosporus BD223215.1 2e-35 97 %

C control soil (Pompeii, Italy)

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bacteria (clones b4, b9 and b10], one sequence was related touncultured archaeon (clone b11) and one sequence was relatedto uncultured alpha Proteobacterium (clone b8), while theremaining six clones were found to belong to five differentorders.

It is interesting that, similarly to the RAPD analysis, mostsequences were related to bacteria belonging to the

Pseudomonadales order [18 % in Nerano-Termini (samplesA1, B1 and C1) and 33 % inMassa Lubrense-Nerano (samplesA2, B2 and C2)] . Act inomycetales (c lone b1) ,Enterobacteriales (clone b5), Mycoplasmatales (clone b6),P s e u d om o n a d a l e s ( c l o n e s b 2 a n d b 7 ) a n dAlphaproteobacteria (clone b8) microorganisms were commonto the two soil types studied. Instead, uncultured bacteria

Fig. 3 Results from single-strandconformation polymorphism(SSCP) analysis of the bacterialcommunity on landslide soils. aSSCP analyses of polymerasechain reaction (PCR)-amplified16S rRNA genes of soil samples.A1: Nerano-Termini, depth of21.55 m; A2: Massa Lubrense-Nerano, depth of 8.10 m; B1:Nerano-Termini, depth of21.42 m; B2: Massa Lubrense-Nerano, depth of 7.97 m; C1:Nerano-Termini, depth of21.55 m; C2: depth of 7.85 m.Cloned amplicons which allowedthe identification ofmicroorganisms in our samplesare shown in the figure and areindicated as “b” followed by anumber. All showed ampliconsallowed microorganismsidentification in Nerano-Terminisoil. Amplicons b1, b2 and b5 tob8 allowed microorganismsidentification in Massa Lubrense-Nerano soil (see Table 3). bPhylogenetic tree of 16S rRNASSCP clones was obtained usingthe neighbour-joiningmethod andthe bootstrap consensus tree wasinferred from 1,000 replicates.The percentage of replicate treesin which the associated taxaclustered together in the bootstraptest are shown next to thebranches. The soil of origin isindicated for each clone: Tindicates Nerano-Termini soil andM indicates Massa Lubrense-Nerano soil

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(clones b4, b9 and b10), uncultured archeon (clone b11) andDesulfuromonadales [uncultured Geobacter sp. (clone b3)]were specifically found in the Nerano-Termini soil sample.However, the profiles generated with SSCP analysis showeda reduced number of identified bacteria when compared toRAPD analysis.

Fingerprints from the control soil were performed also bySSCP analysis of 16S rRNA genes (Fig. 4). SSCP fragmentswere excised, cloned and sequenced. Table 4 shows the resultsobtained from sequence analysis of the control soil, whichrevealed no match with clones from landslide soils.

Eleven bacterial microorganisms have been identified withthis analysis. Two microorganisms were not attributable toany order [uncultured beta Proteobacterium (clone b16) anduncultured Verrucomicrobia bacterium (clone b18)], whileother microorganisms were found to belong to six differentorders. Similarly to the RAPD analysis on Pompeii controlsoil, SSCP revealed that the most often represented orderswere Bacillales (27 %) and Flavobacteriales (18 %). Theformer includes Bacillus cereus (clone b15), Bacillus lentus(clone b19) and Bacillus brevis (clone b21), while the latter arerepresented by Flavobacterium sp. (clone b13) andChryseobacterium sp. (clone b14).

Besides this, other identified microorganisms wereMycobacterium sp. (clone b12, Actinomycetales order),Rhizobium sp. (clone b17, Rhizobiales order), unculturedAcetobacteraceae bacterium (clone b20, Rhodospirillales or-der) and unclassifiedMyxococcales (clone b22,Myxococcalesorder).

Fingerprinting of fungal communities by SSCP

In order to characterise fungal communities, two different soiltypes were analysed by using SSCP analysis. SSCP patternswere obtained with a nested PCR using the fungi-specificprimer pair ITS. Amplicons were separated on acrylamide

gels for fungal DNA. In total, 18 different bands, as shownin Fig. 5a, were isolated from SSCP profiles.

Dominant bands were excised from SSCP gels and re-amplified by PCR, cloned and sequenced. Bioinformatic anal-ysis of sequences cloned from SSCP bands showed the pres-ence of fungal DNA in our samples. The identity of single-stranded PCR products isolated are shown in Table 5. On thebasis of BLASTn results, six clones could not be attributed to

Table 3 Phylogenetic distribution of the 16S rRNA single-strand conformation polymorphism (SSCP) clones

Soil SSCP clone Best sequence homology Sequence ID E value Max identity

T, M b1 Mycobacterium sp. AJ783967.1 5e-27 92 %

T, M b2 Pseudomonas aeruginosa HQ326208.1 0.0 98 %

T b3 Uncultured Geobacter sp. AM712156.1 2e-172 96 %

T b4 Uncultured bacterium HE574367.1 2e-173 96 %

T, M b5 Escherichia coli O157:H7 AB294212.1 4e-24 96 %

T, M b6 Candidatus Mycoplasma DQ157153.1 5e-23 95 %

T, M b7 Uncultured Pseudomonas sp. AM084245.1 5e-23 95 %

T, M b8 Uncultured alpha Proteobacterium AJ633947.1 5e-23 95 %

T b9 Uncultured bacterium FN826015.1 0.0 99 %

T b10 Uncultured bacterium HE574388.1 2e-177 97 %

T b11 Uncultured archaeon FM242736.1 5e-173 96 %

T Nerano-Termini soil; M Massa Lubrense-Nerano soil

Fig. 4 Results from SSCP analysis of the bacterial community on controlsoil. SSCP analysis of PCR-amplified 16S rRNA genes of control soilsamples. D1: Pompeii, depth of 7.87 m; D2: Pompeii, depth of 8.15 m;D3: Pompeii, depth of 7.96 m. Cloned amplicons which allowed theidentification of microorganisms in our samples are shown in the figureand are indicated as “b” followed by a number (from b12 to b22) (seeTable 4)

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any taxonomic order [two sequences were associated to un-cultured marine fungi (clones c1 and c3), one to unculturedBasidiomycota (clone c6), two to uncultured fungus (clonesc7 and c16) and one to Sordariomycetes sp. (clone c14)]. Theremaining 12 clones were found to be related to sequencesfrom fungi belonging to three orders (Malasseziales,Eurotiales and Hypocreales). No overlap between the fungiorders found for the two soil samples was shown after cloneanalysis.

In detail, in Nerano-Termini soil (samples A1, B1 and C1),Malasseziales fungi were mainly represented (43 %, clones c2

and c4), but also Eurotiales (clone c5), unculturedBasidiomycota (clone c6) and uncultured fungi (clones c1,c3 and c7) were identified. In Massa Lubrense-Nerano soil(samples A2, B2 and C2), Sordariomycetes (clone c14) anduncultured fungi (clone c14) were identified, andHypocrealesrepresented the most frequent order (82%) (clones c8, c9, c10,c11, c12, c13, c15, c17 and c18).

In order to characterise the fungal communities from con-trol soil, SSCP analysis was performed using the fungi-specific primer pair ITS (Fig. 6). Table 6 shows the distribu-tion of sequences obtained from the fungal community pro-files generated by SSCP analysis on control samples, whichrevealed nomatch with clones from landslide soils. Ten fungalmicroorganisms were found in Pompeii control soil. Twomicroorganisms were not attributable to any order [unculturedfungus (clone c27) and uncultured Ascomycota (clone c28)].

The remaining eight microorganisms were found to belongto five different orders; the most frequent order wasCryptococcus (30 %) [including Cryptococcus phenolicus(clone c22), Cryptococcus aerius (clone c23), Cryptococcusalbidus (clone c24)], followed by Hypocreales order (20 %)[including Fusarium oxysporum (clone c19) and Trichodermaviride (clone c20)].

Other identified orders were Capnodiales (clone c21),Sporidiales (clone c25) and Sporidiobolales (clone c26).

Discussion

Landslides are one of the most severe natural disturbances,characterised by the rapid movements of earth or other solidmaterial. They can cause extensive damage to life, havingimportant landscape- and ecosystem-wide effects on nutrientavailability. Studies on landslides are often focused on land-slide distribution, nutrient availability and plant successions(Guariguata and Larsen 1990; Myster and Walker 1997).

Table 4 Distribution of the 16S rRNA SSCP clones on control samples

Soil SSCP clone Best sequence homology Sequence ID E value Max identity

C b12 Mycobacterium sp. JQ316216.1 3e-28 95 %

C b13 Flavobacterium sp. AM779887.1 2e-60 84 %

C b14 Chryseobacterium sp. DQ673674.1 1e-67 90 %

C b15 Bacillus cereus FJ982661.1 3e-84 77 %

C b16 Uncultured beta Proteobacterium AB058673.1 8e-60 85 %

C b17 Rhizobium sp. L39920.1 3e-26 97 %

C b18 Uncultured Verrucomicrobia HM750069.1 2e-63 94 %

C b19 Bacillus lentus AB021189.1 4e-22 93 %

C b20 Uncultured Acetobacteraceae JQ291060.1 6e-20 89 %

C b21 Bacillus brevis AJ831420.1 1e-70 98 %

C b22 Unclassified Myxococcales AJ233950.1 3e-77 97 %

C control soil (Pompeii, Italy)

Fig. 5 Results from SSCP analysis of fungal community on landslidesoils. a SSCP fingerprint patterns of fungal communities obtained fromsoil samples. A1: Nerano-Termini, depth of 21.55 m; A2: MassaLubrense-Nerano, depth of 8.10 m; B1: Nerano-Termini, depth of21.42 m; B2: Massa Lubrense-Nerano, depth of 7.97 m; C1: Nerano-Termini, depth of 21.55m;C2: depth of 7.85 m. Cloned amplicons whichallowed the identification of microorganisms in our samples are shown inthe figure and are indicated as “c” followed by a number. Amplicons c1 toc7 allowed fungi identification in Nerano-Termini soil. Amplicons c8 toc18 allowed fungi identification in Massa Lubrense-Nerano soil (seeTable 5). b Phylogenetic tree of fungal community profiles generatedby SSCP analysis was obtained using the neighbour-joining method andthe bootstrap consensus tree was inferred from 1,000 replicates. Thepercentage of replicate trees in which the associated taxa clustered to-gether in the bootstrap test are shown next to the branches. The soil oforigin is indicated for each clone: T indicates Nerano-Termini soil andMindicates Massa Lubrense-Nerano soil

J Appl Genetics

Previously, it has been reported that the processes of thetransformation of clay minerals such as intensification ofremoval of exchange bases and dissolution of silicates andiron oxides occurred in the presence of the alkaliphiliccyanobacterial community (Alekseeva et al. 2009).However, there has been little study on the determination ofthe relationships between microbial community compositionand the alteration of the land responsible for landslides.

In the present work, we analysed the bacterial andfungal communities in soils triggering landslides in

Nerano-Termini and Massa Lubrense-Nerano, comparedto a control area outside the landslides zones, byculture-independent method, based on DNA analysis,such as RAPD and SSCP.

RAPD fingerprinting results and phylogenetic analysisshowed that Nerano-Termini and Massa Lubrense-Neranosoils were characterised by a prevalence of microorganismsbelonging to the Enterobacteriales and Pseudomonadalesorders.

RAPD analysis also allowed the identification of differ-ences in the microbial communities in the two soils.Burkholderiales, Lactobacillales and Neisseriales bacteriawere specifically found in Nerano-Termini soils, whileActinomycetales and Bacillales were found only in MassaLubrense-Nerano soil.

The comparison of RAPD results with bacterial SSCPresults showed that the first method allows the identificationof a larger population of microorganisms. Both analyses pro-vided evidence of a prevalence of Pseudomonadales and thepresence of Actinomycetales and Enterobacteriales in thestudied soils.

Phylogenetic tree construction allowed inferring a possibleevolutive relationship among microorganisms identifiedthrough RAPD and SSCP molecular analyses. However,some of the inferred relationships of the obtained trees arepoorly supported in the bootstrap analysis, and this might bedue to the high divergence of the analysed sequences.

It is known that most soil bacteria are organised in biofilmson roots, litter or soil particles. A biofilm can be broadly

Table 5 Distribution of sequences obtained from fungal community profiles generated by SSCP analysis

Soil SSCP clone Best sequence homology Sequence ID E value Max identity

T c1 Uncultured marine fungus JX269710.1 0.0 99 %

T c2 Uncultured Malassezia EU915312.1 0.0 99 %

T c3 Uncultured marine fungus JX269956.1 0.0 99 %

T c4 Malassezia restricta HQ710822.1 0.0 99 %

T c5 Uncultured Trichocomaceae EU085028.1 0.0 98 %

T c6 Uncultured Basidiomycota HQ433158.1 0.0 98 %

T c7 Uncultured fungus AM260792.1 0.0 98 %

M c8 Fusarium solani JX282606.1 4e-153 97 %

M c9 Fusarium cf. solani JN235271.1 2e-136 98 %

M c10 Nectria haematococca DQ535186.1 2e-137 98 %

M c11 Fusarium solani AB775569.1 3e-135 98 %

M c12 Hypocreales sp. HQ207063.1 4e-134 98 %

M c13 Nectria ipomoeae JQ411380.1 2e-136 98 %

M c14 Sordariomycetes sp. JQ761017.1 2e-152 98 %

M c15 Nectria sp. KC007318.1 5e-44 97 %

M c16 Uncultured fungus KC191755.1 2e-47 99 %

M c17 Fusarium falciforme JX982560.1 2e-80 99 %

M c18 Fusarium solani HE974456.1 2e-47 99 %

T Nerano-Termini soil; M Massa Lubrense-Nerano soil

Fig. 6 Results from SSCP analysis of the fungal community on controlsoil. SSCP fingerprint patterns of fungal communities obtained for con-trol soil samples. D1: Pompeii, depth of 7.87 m; D2: Pompeii, depth of8.15 m; D3: Pompeii, depth of 7.96 m. Cloned amplicons which allowedthe identification of microorganisms in our samples are shown in thefigure and are indicated as “c” followed by a number (from c19 to c28)(see Table 6)

J Appl Genetics

defined as an organised system of microbial cells associatedwith surfaces, often with unique structural phenomena, andare characterised by distinct physical and chemical gradientsthat affect microbial metabolic processes that are heavilyinfluenced by the substratum. Bacteria attach to surfaces, formbiofilms on them and often alter the surfaces as a result of thisinteraction, leading to the destruction (dissolution), transfor-mation or even the formation of minerals. Moreover, it hasbeen suggested that the biofilm microenvironment may stim-ulate bacterial production of specific extracellular enzymesinvolved in the degradation of organic material (Jass et al.2002).

Interestingly, here, the orders and genera mainly identifiedin landslide soils, but not in control soil, Enterobacteriales,Burkholderiales and Pseudomonadales , are well-characterised, biofilm-associated microorganism groups.

Among the Enterobacteriales, E. coli O157 has been dem-onstrated to survive in cold water for up to 12 weeks in mud,sludge and other sediments, including those occurring natu-rally in lakes, seas and along riverbeds (Hall-Stoodley andStoodley 2005). Molecular typing analysis has determinedthat the bacterium is capable of surviving for months, andperhaps years, heavily embedded within biofilm matrices,persisting outside a host organism in animal faeces or faecallyderived material, on inorganic substrata (such as wood ormetal) and in both treated and untreated waters (Cooperet al. 2007).

Besides, the presence of species of Burkholderiales,Burkholderia sp. specifically, as members of anode biofilmcommunities surfaces has also been reported (Chung andOkabe 2009).

Additionally, the typical Pseudomonas bacterium might befound in biofilm in nature, attached to some surface or sub-strate, or in a planktonic form, as a unicellular organisms,actively swimming by means of its flagellum. Among theidentified species belonging to the genus Pseudomonas,Pseudomonas aeruginosa is ubiquitous in soil and water,

forming an antibiotic-resistant biofilm (Drenkard andAusubel 2002).

Previously, it has been reported that frequent occurrence insoil structure of the ions with variable valence (e.g. Fe, Mnetc.) makes minerals extremely sensitive to environmentalconditions (Stucki 1988); bacteria and fungi can be absorbedon the surface of clay minerals and the products of theirmetabolism interact with the minerals (Sokolova et al.2005). Among the bacterial species identified in Nerano-Termini soil, Leptothrix cholodnii is an aerobic, sheath-forming, filamentous bacteria, able to oxidise Mn2+ andFe2+, and is usually found in oligotrophic, slowly running,iron- and manganese-rich water (Siering and Ghiorse 1996).Several organic or inorganic electron donors, such as reducedinorganic sulphur compounds, could be used in carbon diox-ide fixation during anoxygenic photosynthetic growth ofThiomonas sp., which has showed particular carbon and en-ergy metabolic capacities (Frigaard and Dahl 2009).

Also, other bacteria found only in soil samples from land-slides, but not in control soil, have been previously found to beable to metabolise soil-related compounds. Delftiaacidovorans is known to degrade a number of organic com-pounds, such as 2-(4-sulfophenyl)butyrate (SPB), and may beuseful for the degradation of linear alkylbenzenesulfonate(LAS) surfactant in wastewater treatment (Schulz et al.2000). Ramlibacter tataouinensis strain TTB310 is able touse only acetate, pyruvate, beta-hydroxybutyrate, gamma-hydroxybutyrate, DL-lactate or propionate of tested carbonsources, and it is able to reduce nitrate to nitrite; moreover, abeta-glucosidase activity has been reported (Heulin et al.2003; Gommeaux et al. 2005).

The reported data suggest a role of biofilm-forming bacte-ria in the chemical composition of the soil. The association ofgenetic analysis to identify microorganisms present in soilsamples with compositional analysis would be a valid ap-proach to increase current knowledge about features leadingto landslides.

Table 6 Distribution of sequences obtained from fungal community profiles generated by SSCP analysis on control samples

Soil SSCP clone Best sequence homology Sequence ID E value Max identity

C c19 Fusarium oxysporum AY669123.1 3e-44 98 %

C c20 Trichoderma viride AF140045.1 2e-60 94 %

C c21 Cladosporium cladosporioides FJ797611.1 4e-86 79 %

C c22 Cryptococcus phenolicus AF444351.1 9e-36 92 %

C c23 Cryptococcus aerius AF444376.1 5e-40 93 %

C c24 Cryptococcus albidus KC295598 3e-34 98 %

C c25 Rhodotorula glutinis AF387777 1e-46 95 %

C c26 Sporobolomyces salmonicolor AF335955.1 2e-52 91 %

C c27 Uncultured fungus HM545722.1 4e-34 97 %

C c28 Uncultured Ascomycota AY273330.2 2e-57 97 %

C control soil (Pompeii, Italy)

J Appl Genetics

Other than bacteria, our genetic analyses revealed the pres-ence of fungi in soil samples. Our results regarding the fungalcommunity showed differences among species composition intwo landslide soil types compared to control soil. Some ofthem are known to be involved in biochemical processespossibly related to soil properties. In Nerano-Termini soil,we identified uncultured Trichocomaceae. Trichocomaceousare saprophytic fungi whose role in soil aggregation remainsuncertain. However, recently, it has been reported thatTrichocomaceous fungi are able to create soil aggregates, mostprobably as a result of hyphal enmeshment. Once formed, theaggregates are not stable, likely due to the loss of hyphal wallintegrity during autolysis, leading to the disintegration ofmacroaggregates into microaggregates or reversion to theoriginal size distribution (Daynes et al. 2012).

It is interesting that, in Massa Lubrense-Nerano soil, weidentified saprophytic fungi, Fusarium andNectria. This find-ing supports a possible role of fungal community in determin-ing the physical properties of soils.

Our research, other than providing evidence of microbialcommunity differences between two landslide-subjected soils,compared to control soil, allows the comparison of two mo-lecular analyses in finding bacterial and fungal species. Ourresults showed that RAPD analysis allowed more detailedresults about the bacterial community present in the studiedsoil samples, while SSCP analysis with specific primers forfungi identification resulted in a more detailed profile of thefungal community.

Moreover, the identification of still uncultured microbialspecies underlies the limitation of standard culture-based tech-niques, highlighting the relevance of molecular studies toanalyse microbial communities from complex samples, suchas soil. Previous studies support the importance to identifyuncultured microbial species in soil samples, as they can havespecific metabolic capabilities not expressed by cultured spe-cies. For example, antibiotic resistance genes knowledge(Riesenfeld et al. 2004) and enzyme discovery (Kim et al.2008) can be extended by the discovery of uncultured micro-organisms, allowing research for compounds which inhibitresistance mechanisms.

In conclusion, the present study showed that thebacterial and fungal communities found in Nerano-Termini and Massa Lubrense-Nerano soils are populatedby species which could contribute to degradation pro-cesses leading to soil properties changes possibly in-volved in the occurrence of landslides in the studiedsites. Furthermore, the results of this study can beconsidered as an initial support for evaluating the rela-tionship among bacterial and fungal communitycolonising soils and the occurrence of landslides.However, further studies are necessary in order to con-firm the direct involvement of bacterial and fungalspecies to determine soil properties.

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