Ecology and modes of action of rhizosphere bacteria, antagonistic to phytopathogenic fungi and bacteria

The development of biological control methods against soilborn pathogens is among others based on the searching, evaluating and using of non-pathogenic rhizoshere bacteria. In parallel, the antagonistic action of these bacteria may be exercised against foliar pathogens. This research work deals with the ecology of two rhizosphere bacterial isolates able to control physiological races of Fusarium oxysporum and Vertcillium dahliae, both in vitro and in planta successfully. They are a Paenibacillus sp. (K-165) and a Bacillus sp. (5-127) isolates. The ecology, the duration of survival and the site of not colonisation in soil and into the plant were studied by using the polymerase chain reaction (PCR). Particularly, PCR technique was used to amplify the 16S-23S rRNA interspace region of both strains, by the use of universal primers, and then the nucleotide sequences of the amplified interspace region was determined. Afterwards, the alignment of the strains between other that was already registered in EMBL/GenBank was accomplished and gave as the opportunity to design a set of primer specific for each strain. The PCR technique was applied to chromosomal DNA isolated from natural non-sterile soil inoculated with our strains, using the set of the specific primers. By that technique the detection of as low as 2x10² bacterial cells per grammar of soil was achieved. One of the aims of this work was to study the modes of action of the aforementioned antagonistic bacterium K-165. So, we tried to investigate its capacity to induce resistance against the wiltpathogen Fusarium oxysporum f.sp. raphani (For) and the foliar pathogen Pseudomonas syringae pv tomato DC3000 (Pst). Arabidopsis plants ecotype Col-0 (wild type), mutants etr-1 (ethylene response mutant), npr-1 (non expressed PR-proteins), jar-1 (jasmonate response mutant) and the trangenic NahG (that cannot accumulate salicylic acid) were individually sown in 1 ml pipette tips filled with sterile quartz sand in order to stimulate root elongation, removed after two weeks and placed vertically on a system of rock wool cubes consisting of two spatially separated compartments dividing the root system from the upper part of the plant. Three weeks after inoculation with For, symptoms were already developed in the control plants. Symptom severity expressed as percentage of diseased leaves, reached 56% in control plants 29 days after Fusarium inoculation and at the same time bacterial treatment restricted symptom expression down to 20%. At the end of the experiment (37 days after inoculation) Fusarium wilt symptoms reached almost 90% in control untreated Col-0 plants while in the bacterized ones were only 57%. As far as the etr-1, jar1 mutants and NahG trangenic plants are concerned, the percentage differences of symptom severity between the treated and untreated plants reached the 30%, 36% and 26%, respectively. On the contrary, the analysis of the results from the npr1 Arabidopsis mutant experiment didn't show any significant differentiation between treated and untreated plants with K-165. The npr1l mutants, based on impaired systemic acquired resistance (SAR) expression, reduced SA-induced PR gene expression and enhanced disease susceptibility, indicating the broad involvement of NPR1 regulator protein in plant defense. The absence of induced resistance at the npr1 mutants showed that the induced resistance that caused by the K-165 depends on NPR1 protein. For the Arabidopsis-Pst bioassay, seeds of Col-0 plants were sown and soil mixture was mixed with a 5x10⁷ cfu/g of K-165. Five-week-old plants were challenge inoculated with the virulent pathogen Pst by dipping the leaves in a dense bacterial suspension (2.5x10⁷ cfu/ml). Plants treated with K-165 strain against Pseudomonas syringae pv tomato DC3000 showed a clear differentiation between treated and untreated plants, proving that K-165 also induces resistance against a bacterial pathogen. cDNA fragments of the ISR involved genes PR-1, PR-2, PR-5, Hel, Atvsp, and Tub were amplified with PCR, subcloned and sequenced. Leaf tissues were harvested at different days after inoculation with Pst, for RNA analysis. Gene expression was analysed by RT-PCR. The RT-PCR results showed that these genes are potentiated, leading to enhanced expression after challenge inoculation with Pst and that PR genes played a crucial role in the development of induced resistance. In this case it could be concluded that pathogen-induced systemic resistance is associated with potentiation of PR genes. The results for both of the experiments with the biocontrol bacterium K-165 against the root pathogen Fusarium oxysporum f.sp. raphani (For) and the leaf pathogen Pseudomonas syringae pv tomato (Pst) showed us that the pathogenesis- related proteins are responsible for the induced resistance for the host-pathogen-biocontrol agent system.Η ανάπτυξη μεθόδων βιολογικής καταπολέμησης εδαφογενών παθογόνων, στηρίζεται μεταξύ άλλων στην αναζήτηση, αξιολόγηση και χρησιμοποίηση ανταγωνιστικών βακτηρίων της ριζόσφαιρας. Παράλληλα όμως η ανταγωνιστική δράση αυτών των βακτηρίων δύναται να εξασκηθεί και εναντίον παθογόνων τις φυλλόσφαιρας. Η εργασία αυτή αφορά στην οικολογία δύο στελεχών ριζοσφαιρικών βακτηρίων τα οποία ανταγωνίζονται επιτυχώς in vitro και in planta φυσιολογικές φυλές των μυκήτων Fusarium oxysporum και Vericillium dahliae. Πρόκειται για ένα βακτήριο του γένους Paenibacillus sp. (στέλεχος Κ-165) και ένα Bacillus sp. (στέλεχος 5-127). Η μελέτη της οικολογίας, της διάρκειας επιβίωσης και ο εντοπισμός των βακτηρίων στο έδαφος και στο φυτό, πραγματοποιήθηκε με τη χρήση της τεχνικής της αλυσιδωτής αντίδρασης της πολυμεράσης (PCR). Για το σκοπό αυτό υποκλωνοποιήθηκε η συντηρημένη περιοχή του γονιδιώματος ανάμεσα στο 23S και 16S rRNA (ενδοριβοσωμική περιοχή), με τη χρήση καθολικών εκκινητών. Ακολούθησε προσδιορισμός της νουκλεοτιδικής αλληλουχίας για κάθε ένα στέλεχος και σύγκριση αυτών μεταξύ τους και μεταξύ άλλων καταχωρημένων στο διαδίκτυο ακολουθιών. Η σύγκριση αυτή επέτρεψε την εύρεση μη συντηρημένων και μοναδικών αλληλουχιών για το κάθε στέλεχος, γεγονός που οδήγησε στον σχεδιασμό εξειδικευμένων εκκινητών για κάθε ένα βακτηριακό στέλεχος. Παράλληλα, αναπτύχθηκε η μεθοδολογία για την απομόνωση γενωματικού βακτηριακού DNA από φυσικό έδαφος μολυσμένο με γνωστή συγκέντρωση κυττάρων των βακτηρίων μας. Απεδείχθη ότι η τεχνική PCR είναι δυνατόν να ανιχνεύσει την παρουσία DNA βακτηρίων των οποίων η συγκέντρωση μπορεί να περιοριστεί μέχρι και 2x102 κύτταρα ανά γραμμάριο ξηρού βάρους εδάφους. Στα πλαίσια της μελέτης του τρόπου δράσεως του ανταγωνιστικού ριζοβακτηρίου Κ-165, μελετήθηκε η δυνατότητα επαγωγής διασυστηματικής αντοχής από το στέλεχος αυτό κατά του παθογόνου μύκητα Fusarium oxysporum f.sp. raphani, όπου ως φυτά-πρότυπα χρησιμοποιήθηκαν φυτά Arabidopsis thaliana αγρίου τύπου Col-0, μεταλλαγμένα etr-1 (δεν παράγουν αιθυλένιο), jar-1 (δεν παράγουν το γιασμονακό οξύ), npr-1 (δεν επάγουν την παράγωγη πρωτεϊνών που σχετίζονται με την παθογένεση, PRs) και διαγονιδιακά NahG (δεν παράγουν σαλικυλικό οξύ). Για την επαγωγή της διασυστηματικής αντοχής τα φυτά αναπτύχθηκαν σε πετροβάμβακα με τέτοιον τρόπο ώστε να διαχωρίζεται το ριζικό σύστημα από το υπέργειο τμήμα, και πραγματοποιήθηκε εφαρμογή του ανταγωνιστικού παράγοντα σε μορφή ταλκ. Πέντε μέρες αργότερα ακολούθησε μόλυνση των φυτών με το μύκητα. Τα ανοσοποιημένα φυτά περιόριζαν την εμφάνιση συμπτωμάτων μέχρι και 50% συγκρινόμενα με το μάρτυρα, 30 μέρες μετά τη μόλυνση, τόσο στα φυτά αγρίου τύπου Col-0, όσο και στα etr-1, jar-1 και NahG. Όσον αφορά στα μεταλλαγμένα npr-1 δεν παρατηρήθηκε ανοσοποίηση στα φυτά που υπέστησαν εφαρμογή του ανταγωνιστικού βακτηρίου Κ-165. Το γεγονός αυτό μας οδηγεί στο συμπέρασμα ότι η επαγωγή ανθεκτικότητας που προκαλείται από το Κ-165 ριζοβακτήριο, συνδέεται με την έκφραση γονιδίων PR. Τα μεταλλαγμένα npr-1 φυτά Arabidopsis έχουν επιλεχθεί για τη μελέτη των γονιδίων της επίκτητης διασυστηματικής αντοχής (SAR), τα οποία εγκλείονται στο μονοπάτι που οδηγεί από το σαλικυλικό οξύ στην ενεργοποίηση των γονιδίων των πρωτεϊνών PR. Τα φυτά αυτά έχουν υποστεί μετάλλαξη στο γονίδιο nrp1, δεν εκφράζουν τις πρωτεΐνες παθογένεσης και δεν εκδηλώνουν το φαινόμενο της SAR. Το npr1 γονίδιο κωδικοποιεί σε μια ρυθμιστική πρωτεΐνη, την NPR1, η οποία ενεργεί στο τελικό μέρος της μεταφοράς του σήματος στην επίκτητη διασυστηματική αντοχή. Τα δεδομένα του πειράματος αυτού αποδεικνύουν ότι η διασυστηματική αντοχή των φυτών που οφείλεται στο Κ-165 εξαρτάται από την παρουσία της πρωτεΐνης NPR1. Για την περαιτέρω μελέτη του τρόπου της επαγωγής αντοχής από το Κ-165 διερευνήθηκε η ικανότητα επαγωγής ανθεκτικότητας σε φυτά Arabidopsis, μετά από εφαρμογή του βακτηρίου Κ-165, εναντίον σημαντικών βακτηριολογικών ασθενειών του φυλλώματος (Pseudomonas syringae pv tomato) με σκοπό τον έλεγχο της ικανότητας επαγωγής του φαινομένου της ανοσοποίησης σε περισσότερα διαφόρου φύσεως παθογόνα και παθογόνων της φυλλικής επιφάνειας των φυτών αυτών. Ακολούθησε μοριακή διερεύνηση της έκφρασης γονιδίων τα οποία εμπλέκονται στο μονοπάτι της επαγόμενης διασυστηματικής αντοχής ( ISR και SAR ) με σκοπό τον πιθανό εντοπισμό του αιτίου της διέγερσης (επαγωγής) των λανθανόντων μηχανισμών αντοχής του φυτού ενάντια στο παθογόνο. Υποκλωνοποιήθηκαν τμήματα των αμυντικών γονιδίων PR-1, PR-2, PR-5, AtVsp, Hel και Pdfl.2, και έγινε ανάλυση της νουκλεοτιδικής αλληλουχίας τους. Για τη μελέτη των επιπέδων έκφρασης των γονιδίων αυτών εφαρμόστηκε η μέθοδος της αλυσιδωτής αντίδρασης της πολυμεράσης, με μήτρα μόρια mRNA (RT-PCR). Για την πραγματοποίηση των αντιδράσεων σχεδιάστηκαν εξειδικευμένοι εκκινητές για κάθε ένα γονίδιο, και ως RNA μήτρα συλλέχθηκε RNA από τη φυλλική επιφάνεια μολυσμένων φυτών Arabidopsis με το παθογόνο βακτήριο τα οποία είχαν υποστεί επέμβαση του ανταγωνιστικού βακτηρίου Κ-165. Τα αποτελέσματα του πειράματος αυτού αποδεικνύουν ότι παρατηρείται διαφοροποίηση στην έκφραση των PR γονιδίων, γεγονός το οποίο οδηγεί στο συμπέρασμα ότι οι πρωτεΐνες που σχετίζονται με την παθογέγεση αποτελούν καθοριστικό παράγοντα για την επαγωγή ανθεκτικότητας στα φυτά με το βακτήριο Κ-165. Τα αποτελέσματα αμφοτέρων των πειραμάτων ανοσοποίησης με το βακτήριο Κ-165, έναντι του μύκητα Fusarium oxysporum f.sp. raphani και έναντι του παθογόνου βακτηρίου της φυλλικής επιφάνειας Pseudomonas syringae pv tomato, οδηγούν στην άποψη ότι οι πρωτεΐνες που σχετίζονται με την παθογένεση (PRs) ευθύνονται για την επαγωγή ανθεκτικότητας στο σύστημα ξενιστή-παθογόνου-βιολογικού παράγοντα, το οποίο μελετήθηκε στην παρούσα διατριβή

Impact of Elemental Sulfur on the Rhizospheric Bacteria of Durum Wheat Crop Cultivated on a Calcareous Soil

Previous experiments have shown that the application of fertilizer granules containing elemental sulfur (S0) as an ingredient (FBS0) in durum wheat crops produced a higher yield than that produced by conventional ones (F), provided that the soils of the experimental fields (F vs. FBS0) were of comparable quality and with the Olsen P content of the field’s soil above 8 mg kg−1. In this experiment the FBS0 treatment took place in soil with Olsen P at 7.8 mg kg−1, compared with the F treatment’s soil with Olsen P of 16.8 mg kg−1, aiming at reducing the imbalance in soil quality. To assess and evaluate the effect of FBS0 on the dynamics of the rhizospheric bacteria in relation to F, rhizospheric soil at various developmental stages of the crops was collected. The agronomic profile of the rhizospheric cultivable bacteria was characterized and monitored, in connection with the dynamics of phosphorus, iron, organic sulfur, and organic nitrogen, in both the rhizosoil and the aerial part of the plant during development. Both crops were characterized by a comparable dry mass accumulation per plant throughout development, while the yield of the FBS0 crop was 3.4% less compared to the F crop’s one. The FBS0 crop’s aerial part showed a transient higher P and Fe concentration, while its organic N and S concentrations followed the pattern of the F crop. The incorporation of S0 into the conventional fertilizer increased the percentage of arylsulfatase (ARS)-producing bacteria in the total bacterial population, suggesting an enhanced release of sulfate from the soil’s organic S pool, which the plant could readily utilize. The proportion of identified ARS-producing bacteria possessing these traits exhibited a maximum value before and after topdressing. Phylogenetic analysis of the 68 isolated ARS-producing bacterial strains revealed that the majority of the isolates belonged to the Pseudomonas genus. A large fraction also possessed phosphate solubilization, and/or siderophore production, and/or ureolytic traits, thus improving the crop’s P, Fe, S, and N balance. The aforementioned findings imply that the used FBS0 substantially improved the quality of the rhizosoil at the available phosphorus limiting level by modulating the abundance of the bacterial communities in the rhizosphere and effectively enhancing the microbially mediated nutrient mobilization towards improved plant nutritional dynamics

<i>Calendula officinalis</i>—A Great Source of Plant Growth Promoting Endophytic Bacteria (PGPEB) and Biological Control Agents (BCA)

The application of beneficial bacteria may present an alternative approach to chemical plant protection and fertilization products as they enhance growth and resistance to biotic and abiotic stresses. Plant growth-promoting bacteria are found in the rhizosphere, epiphytically or endophytically (Plant Growth Promoting Endophytic Bacteria, PGPEB). In the present study, 36 out of 119 isolated endophytic bacterial strains from roots, leaves and flowers of the pharmaceutical plant Calendula officinalis were further identified and classified into Bacillus, Pseudomonas, Pantoea, Stenotrophomonas and Rhizobium genera. Selected endophytes were evaluated depending on positive reaction to different plant growth promoting (PGP) traits, motility, survival rate and inhibition of phytopathogenic fungi in vitro and ex vivo (tomato fruit). Bacteria were further assessed for their plant growth effect on Arabidopsis thaliana seedlings and on seed bio-primed tomato plantlets, in vitro. Our results indicated that many bacterial endophytes increased seed germination, promoted plant growth and changed root structure by increasing lateral root density and length and root hair formation. The most promising antagonistic PGPEB strains (Cal.r.29, Cal.l.30, Cal.f.4, Cal.l.11, Cal.f.2.1, Cal.r.19 and Cal.r.11) are indicated as effective biological control agents (BCA) against Botrytis cinerea on detached tomato fruits. Results underlie the utility of beneficial endophytic bacteria for sustainable and efficient crop production and disease control

Genomic and Metabolomic Insights into Secondary Metabolites of the Novel Bacillus halotolerans Hil4, an Endophyte with Promising Antagonistic Activity against Gray Mold and Plant Growth Promoting Potential

The endophytic bacterial strain Hil4 was isolated from leaves of the medicinal plant Hypericum hircinum. It exhibited antifungal activity against Botrytis cinerea and a plethora of plant growth promoting traits in vitro. Whole genome sequencing revealed that it belongs to Bacillus halotolerans and possesses numerous secondary metabolite biosynthetic gene clusters and genes involved in plant growth promotion, colonization, and plant defense elicitation. The Mojavensin cluster was present in the genome, making this strain novel among plant-associated B. halotolerans strains. Extracts of secreted agar-diffusible compounds from single culture secretome extracts and dual cultures with B. cinerea were bioactive and had the same antifungal pattern on TLC plates after bioautography. UHPLC-HRMS analysis of the single culture secretome extract putatively annotated the consecutively produced antimicrobial substances and ISR elicitors. The isolate also proved efficient in minimizing the severity of gray mold post-harvest disease on table grape berries, as well as cherry tomatoes. Finally, it positively influenced the growth of Arabidopsis thaliana Col-0 and Solanum lycopersicum var. Chondrokatsari Messinias after seed biopriming in vitro. Overall, these results indicate that the B. halotolerans strain Hil4 is a promising novel plant growth promoting and biocontrol agent, and can be used in future research for the development of biostimulants and/or biological control agents

Compatible Consortium of Endophytic <i>Bacillus halotolerans</i> Strains Cal.l.30 and Cal.f.4 Promotes Plant Growth and Induces Systemic Resistance against <i>Botrytis cinerea</i>

Evaluating microbial-based alternatives to conventional fungicides and biofertilizers enables us to gain a deeper understanding of the biocontrol and plant growth-promoting activities. Two genetically distinct Bacillus halotolerans strains (Cal.l.30, Cal.f.4) were evaluated for the levels of their compatibility. They were applied individually or in combination under in vitro and greenhouse conditions, using seed bio-priming and soil drenching as inoculum delivery systems, for their plant growth-promoting effect. Our data indicate that application of Cal.l.30 and Cal.f.4 as single strains and as a mixture significantly enhanced growth parameters of Arabidopsis and tomato plants. We investigated whether seed and an additional soil treatment with these strains could induce the expression of defense-related genes in leaves of young tomato seedling plants. These treatments mediated a long lasting, bacterial-mediated, systemic-induced resistance as evidenced by the high levels of expression of RP3, ACO1 and ERF1 genes in the leaves of young tomato seedlings. Furthermore, we presented data showing that seed and soil treatment with B. halotolerans strains resulted in an effective inhibition of Botrytis cinerea attack and development on tomato leaves. Our findings highlighted the potential of B. halotolerans strains as they combine both direct antifungal activity against plant pathogens and the ability to prime plant innate immunity and enhance plant growth

The Nitrogen-Fixation Island Insertion Site Is Conserved in Diazotrophic <i>Pseudomonas stutzeri</i> and <i>Pseudomonas</i> sp. Isolated from Distal and Close Geographical Regions

<div><p>The presence of nitrogen fixers within the genus <i>Pseudomonas</i> has been established and so far most isolated strains are phylogenetically affiliated to <i>Pseudomonas stutzeri</i>. A gene ortholog neighborhood analysis of the nitrogen fixation island (NFI) in four diazotrophic <i>P. stutzeri</i> strains and <i>Pseudomonas azotifigens</i> revealed that all are flanked by genes coding for cobalamin synthase (<i>cobS</i>) and glutathione peroxidise (<i>gshP</i>). The putative NFIs lack all the features characterizing a mobilizable genomic island. Nevertheless, bioinformatic analysis <i>P. stutzeri</i> DSM 4166 NFI demonstrated the presence of short inverted and/or direct repeats within both flanking regions. The other <i>P. stutzeri</i> strains carry only one set of repeats. The genetic diversity of eleven diazotrophic <i>Pseudomonas</i> isolates was also investigated. Multilocus sequence typing grouped nine isolates along with <i>P. stutzeri</i> and two isolates are grouped in a separate clade. A Rep-PCR fingerprinting analysis grouped the eleven isolates into four distinct genotypes. We also provided evidence that the putative NFI in our diazotrophic <i>Pseudomonas</i> isolates is flanked by <i>cobS</i> and <i>gshP</i> genes. Furthermore, we demonstrated that the putative NFI of <i>Pseudomonas</i> sp. Gr65 is flanked by inverted repeats identical to those found in <i>P. stutzeri</i> DSM 4166 and while the other <i>P. stutzeri</i> isolates harbor the repeats located in the intergenic region between <i>cobS</i> and glutaredoxin genes as in the case of <i>P. stutzeri</i> A1501. Taken together these data suggest that all putative NFIs of diazotrophic <i>Pseudomonas</i> isolates are anchored in an intergenic region between <i>cobS</i> and <i>gshP</i> genes and their flanking regions are designated by distinct repeats patterns. Moreover, the presence of almost identical NFIs in diazotrophic <i>Pseudomonas</i> strains isolated from distal geographical locations around the world suggested that this horizontal gene transfer event may have taken place early in the evolution.</p></div

16S rRNA phylogenetic tree.

<p>Neighbor-Joining phylogenetic tree of 16S rRNA gene constructed using the partial nucleotide sequence from the 11 <i>P. stutzeri</i> isolates and related sequences obtained from NCBI <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105837#pone.0105837-Anzai1" target="_blank">[40]</a>. Numbers shown at nodes indicate bootstrap values (percentage of 1000 replicates). The trees were constructed by the neighbour-joining method using MEGA v.5. Reference strains are highlighted in bold. The bar scale indicates the rates of substitution per nucleotide position. Sequence accession numbers are given in parentheses. ^T = type strain.</p

Rep-PCR genomic fingerprinting of <i>P. stutzeri</i> strains.

<p>Rep-PCR genomic fingerprinting of <i>P. stutzeri</i> A1501, <i>P. stutzeri</i> DSM4611 and 11 isolates (Gr16, Gr17, Gr18, Gr19, Gr20, Gr50, Gr45, Gr46, Gr57, Gr65). M: DNA ladder λ DNA HindIII and φX174 DNA HaeIII.</p

Schematic representation and comparison of the Nitrogen Fixation Islands and flanking genes of diazotrophic <i>P. stutzeri</i> strains A1501, DSM4166, B1SMN1, KOS6 and <i>P. azotifigens</i> DSM 17556.

<p>The nitrogen fixation island of <i>P. stutzeri</i> KOS6 was assembled downloading from the Integrated Microbial Genomes (IMG) (<a href="https://img.jgi.doe.gov/cgi-bin/w/main.cgi" target="_blank">https://img.jgi.doe.gov/cgi-bin/w/main.cgi</a>) three contigs (AMCZ01000041, AMCZ01000045 and AMCZ000005) indicated by brackets. The nitrogen fixation island of <i>P. stutzeri</i> strains B1SMN1 and <i>P. azotifigens</i> DSM 17556 were found in one contig. The colored of arrows are indicating different functional genes as described by IMG.</p

Inverted and/or direct repeats identified in the IRLeft and IRRight regions flanking the nitrogen fixation island of <i>P. stutzeri</i> strains and <i>Pseudomonas</i> sp. Gr65.

<p>The repeats DR1, DR2 and DR3 (boxed) present in the IRLeft and IRRight regions flanking the nitrogen fixation island of <i>P. stutzeri</i> A1501 and DSM4166 (A), <i>P. stutzeri</i> NF13 (B) and <i>Pseudomonas</i> sp. Gr65 (D). The repeats located in the intergenic region between PST_1322- PST_1323 (designated as 1501Μ) and PSTAA_1354- PSTAA_1355 (designated as 4166Μ) (C). The coordinates displayed on the left and the right side of the sequences indicate the position of the sequences in genome of <i>P. stutzeri</i> A1501 or DSM4166 (A and C). The coordinates displayed for <i>Pseudomonas</i> sp. Gr65 were based on the nucleotide sequences of IRLeft and IRRight found in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105837#pone.0105837.s002" target="_blank">files S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105837#pone.0105837.s003" target="_blank">S2</a>.</p