Cloning of PCR Amplicons

PCR amplicons were cloned by the pGEM®-T Easy Vector Systems (II) kit (Promega) (Felske and Weller 2004). 5 U of T4 DNA ligase were used to ligate PCR products for 16 h at 4°C into pGEM-T vector at an insert:plasmid ratio of 6:1. The ligation product was added to 50 ml of E. coli JM109 competent cells and the transformation was carried out by heating for 45 s at 42°C, cooling in ice, and incubating at 37°C for 90 min under shaking (750 rpm) in SOC growth medium (20 g Bacto®-tryptone, 5 g Bacto®-yeast extract, 10 ml 1 M NaCl, 10 ml 2 M Mg2+, 10 ml 2 M glucose, per liter).

100 ml of each transformation was plated out on LB agar plates (10 g Bacto®-tryptone, 5 g Bacto®-yeast extract, 5 g NaCl per liter, 1.5% agar) containing ampicillin (100 mg/ml), IPTG (isopropyl-BD-thiogalactopyranoside 0.1 M), X-GAL (5-bromo-4-cloro-3-indolyl-Betagalattoside, 50 mg/ml) (Sambrook et al. 1989), and incubated for 16-24 h at 37°C. The identification of CFU transformed by a PCR product was carried out by the white/blue selection method (Sambrook et al. 1989). The ligation strategy provides that if a nucleotide sequence is inserted into the plasmid, the lacZ operon is no longer expressed so that the synthetic chromo-genic substrate is not processed, giving rise to white colored colonies instead of blue ones (positive clone) (Frackman and Kephart 1999). In addition, a colony PCR was also performed to evaluate the presence of 16S targeting amplicons into transformed cells (Frothingham et al. 1991). White colonies were picked up and resuspended in 50 ml TE buffer (TRIS 10 mM, EDTA 1 mM, pH 7.5), incubated 15 min at 95°C to lyse cells. In order to make bacterial DNA available for PCR, 1 ml of the latter suspension was added with 1 U Taq DNA polymerase (Eurotaq), 8 pmol each of the two primers T7-SP6 (Wang and Wang 1997; Zoetendal et al. 1998), 10 nmol each dNTPs, and 3 mM MgCl2, to reach a final volume of 25 ml.

The amplification conditions were the following:

95 °C for 3 min (denaturing step) 94° C for 10 s 9 46°C for 20 s (25 cycles) 68°C for 60 s

68°C for 10 min (final elongation step)

PCR fragments were checked by electrophoresis on ethidium bromide (0.5 mg/ml) stained 1.5% agarose gel.

In order to avoid repeatedly sequencing of cloned fragments, a screening by amplified ribosomal DNA restriction analysis (ARDRA) was performed (Dunbar et al. 1999). Aliquots of the above described amplified fragments were digested by Hinfl, AluI, Rsal, and TaqI restriction enzymes (Promega) according to the manufacturer' conditions. The size of the digestion patterns was determined by electrophoresis on 2% agarose gel, in which adequate molecular weight markers were also run. PCR cloned amplicons, whose digestion profiles were different for at least one enzyme, were considered different and sequenced separately.

8.2.8 Sequencing and Phylogenetic Investigation

Plasmid DNA was extracted from the CFU that resulted positive to the insertion of 16S rDNA amplicons, as above described, by the QIAprep® Miniprep kit (QIGENE, Inc., Chatsworth, California) according to the manufacturer' instructions and the inserted fragments sequenced by PRIMM.

Nucleotide sequences were compared with those available in the database "Ribosomal Database Project" (RDP 7.0) (Maidak et al. 1997), by the BLAST N system and the SIMILARITY_RANK (RDP) (Maidak et al. 1994). Sequence alignment was performed by the Bioedit and Clustal W programs (Thompson et al. 1994; Hall 1999). Sequence homologies were determined by the neighbor-joining method (Saitou and Nei 1987) and the Jukes and Cantor (1969) algorithm with "bootstrap" values calculated on a 1,000 replicates basis (Felsenstein 1985). Phylogenetic trees were built by the Treecon software (Van de Peer and De Wachter 1997).

8.3 Results and Discussion

In order to evaluate the structure of microbial communities in both bulk and rhizospheric soil, two molecular approaches were used (1) 16S rDNA PCR/DGGE, with an intermediate resolution capacity and (2) cloning/sequencing of 16S rRNA amplicons, that brings to a higher level of information. In particular, we investigated the short-term effects of soil management practices of the MESCOSAGR project, including compost and catalyst treatments, on the diversity of soil bacterial communities.

Compost amendments affected the composition of bacterial communities which were tightly adhered to plant roots. These changes were even more evident when the whole community was investigated by total DNA amplification, instead of examining only the metabolic changes by following RNA extraction/amplification. It is likely that an enhanced nutrients availability due to the soil amendments may have quantitatively and qualitatively affected plant rhizodeposition that has in turn influenced the diversity and activity of the microorganisms colonizing the soil-root interface (Foereid and Yearsley 2004).

The TRA and MIN tillage practices in the Torino site did not appear to affect bacterial diversity, as it is shown by the gel and relative cluster analysis obtained for the rhizospheric soil DNA from the Torino experimental site after 1 year of treatment (Fig. 8.2). However, results of the COM treatment resulted to be different from other tillage practices. In fact, samples were pooled in two main groups: one containing all replicates from compost-amended plots (95.7% of similarity index), a second cluster in which almost all other samples had a 95.4% of similarity. This is also figuratively evident by DNA bands (one shown by an arrow) which are present only in compost-amended soils and indicate the changes occurred in the composition of bacterial communities induced by the compost additions.

Rhizospheric DNA, obtained from soils of the experimental site of Napoli, was similarly characterized by DGGE (Fig. 8.3) that shows that the changes in communities structure induced by compost amendments were evident after the second year of treatment, whereas TRA and MIN treatments did not have any

am 9E.8

«

mm

1

i

ii

1

1

:

Fig. 8.2 Gel electrophoresis under denaturing conditions (DGGE) and relative cluster analysis. Rhizospheric DNA (R) extracted from soils under traditional (TRA) and minimal (MIN) tillage and compost (COM) amendment, after 1 and 2 years of treatments for the Torino (TO) site. Four replicates per treatment (A, B, C, D)

TO

R

MIN 1 D

TO

R

TRA 1C

TO

R

TRA 1A

TO

R

TRA 1B

TO

R

MIN 1 B

TO

R

MIN 1 C

TO

R

MIN 1A

TO

R

TRA 1D

TO

R

COM 1A

TO

R

COM 1C

TO

R

COM 1B

TO

R

COM 10

Fig. 8.2 Gel electrophoresis under denaturing conditions (DGGE) and relative cluster analysis. Rhizospheric DNA (R) extracted from soils under traditional (TRA) and minimal (MIN) tillage and compost (COM) amendment, after 1 and 2 years of treatments for the Torino (TO) site. Four replicates per treatment (A, B, C, D)

MA R MIN 1D NARTRA1D NA R COM 1A NA R COM 1 C NA R COM 1 0 NA R COM 1 D NAR MIN IA NA R MIN IB NAR IKA1C NA R MIN 1C NAR TRA1 B NAR TRA1A NAR COM 2C NAR COM 2D NAR COM28 NA R COM 2A NA R MIN 2C NAR TRA2A NAR TRA2B NAR MIN 2D NA R MIN 2A NA R MIN 2B NAR TRA2D NAR TRA2C

Fig. 8.3 Gel electrophoresis under denaturing conditions (DGGE) and relative cluster analysis. Rhizospheric DNA (R) extracted from soils under traditional (TRA) and minimal (MIN) tillage and compost (COM) amendment, after 1 and 2 years of treatments for the Napoli (NA) site. Four replicates per treatment (A, B, C, D)

effect on bacterial diversity. Cluster analysis also indicates that all samples extracted after the first treatment year were separate from those after the second experimentation year. This pool segregation may be attributed to climate and timing variability (annual temperature and rain, period of sampling). Since the analytical focus was on the bacteria colonizing the soil-root interface, the observed changes in diversity might be also due to different plant activities (growth, rhizodeposition, etc.).

Bulk soils from treated plots have been also investigated to evaluate the direct effect of applied treatments without taking into account the contribution of plant root exudates (data no shown). The community structure of soil bacteria were not significantly affected by either TRA and MIN tillage practices or compost amendments for the first year. Only a slight effect was noted after the second experimental year on both the whole communities and the metabolically active bacteria at time of sampling (after harvest).

Fig. 8.3 Gel electrophoresis under denaturing conditions (DGGE) and relative cluster analysis. Rhizospheric DNA (R) extracted from soils under traditional (TRA) and minimal (MIN) tillage and compost (COM) amendment, after 1 and 2 years of treatments for the Napoli (NA) site. Four replicates per treatment (A, B, C, D)

77.8

77.8

PI R NO 1A PI R NO 1B PIR NO 1C PI R CAT 1B PI R CAT 1A PIR CAT 1C

89.1

97 6

Fig. 8.4 Gel electrophoresis under denaturing conditions (DGGE) and relative cluster analysis. Rhizospheric RNA (R) extracted from soils amended with iron-porphyrin (CAT) and non-amended (NOCAT) after 1 year of treatment for the Piacenza (PI) site. Three replicates per treatment (A, B, C)

The addition of the iron-porphyrin biomimetic catalyst in the CAT treatment to stabilize SOM may have altered its mineralization rate, and thus, nutrients availability to microorganisms. We did not find any change in composition of bacteria communities for the soil in field plots of Torino and Napoli (data not shown). However, slight variations were noted for the experimental site of Piacenza, whose electrophoretic pattern showed that two out of three rhizospheric samples amended with the biomimetic catalyst (CAT) segregated separately from other ones

The effect of the catalyst on the microbial communities of the heavy textured soils from the Piacenza site may be attributed to the longer persistence of the iron-porphyrin on the surface of soil aggregates that for Napoli and Piacenza (see Chaps. 4 and 7). This effect is likely to be of more extent in following treatments years with additional amount of catalyst adsorbed on soil surfaces (see Chap. 3). However, it is noteworthy that the effect was shown in rhizospheric soil samples rather than in bulk soils, thereby suggesting that plant root exudates play a key role for life and diversity of soil microorganisms.

The cloning and sequencing approaches have been used to characterize bacterial populations at a phylogenetic level. In particular, following the DGGE characterization (Fig. 8.2), we also investigated the PCR amplicons relative to rhizospheric DNA for compost-amended (COM) soils (66 clones, marked with a C and progressively numbered) and TRA soils (62 clones, marked with an A and progressively numbered) of the Torino site after the first treatment year. After restriction analysis, that was necessary to avoid the sequencing of the same PCR fragment as described above, the selected clones sequenced were 52 and 41, respectively.

Our results indicate that 90% of sequenced clones showed a strict correlation with the Ribosomal Database Project sequences (Table 8.1). For all clones we report the classification in Table 8.1 only at the level of phylum, because in some cases the information were not enough to identify the level of species.

Table 8.1 Genomic library of clones amplified on rhizospheric DNA extracted from soil (a) amended with compost (COM) and (b) under traditional tillage (TRA) at the experimental site of Torino. Phylogenetic attribution and relative similarities values were by Ribosomal Database Project (RDP)

Clones

Phyla

%Identity

(a)

C1

Proteobacteria

96%

C3

Acidobacteria

99%

C4

Proteobacteria

95%

C5

Actinobacteria

95%

C7

Proteobacteria

99%

C8

Acidobacteria

98%

C10

Uncultured

90%

C11

Uncultured

98%

C14

Acidobacteria

100%

C16

Proteobacteria

99%

C17

Verrucomicrobia

96%

C19

Actinobacteria

99%

C20

Proteobacteria

91%

C21

Actinobacteria

97%

C22

Verrucomicrobia

99%

C24

Proteobacteria

94%

C26

Acidobacteria

99%

C27

Acidobacteria

99%

C29

Proteobacteria

99%

C30

Acidobacteria

96%

C31

Actinobacteria

99%

C32

Acidobacteria

97%

C34

Uncultured

99%

C35

Actinobacteria

100%

C36

Proteobacteria

100%

C37

Actinobacteria

98%

C38

Proteobacteria

99%

C44

Actinobacteria

99%

C45

Firmicutes

99%

C47

Acidobacteria

98%

C48

Actinobacteria

99%

C49

Actinobacteria

97%

C50

Actinobacteria

96%

C52

Actinobacteria

98%

C53

Acidobacteria

100%

C54

Acidobacteria

99%

C56

Actinobacteria

96%

C57

Acidobacteria

98%

C58

Verrucomicrobia

97%

C59

Acidobacteria

99%

C60

Actinobacteria

99%

C62

Uncultured

99%

222

C. Crecchio et al.

Table 8.1 (continued)

Clones

Phyla

%Identity

C63

Proteobacteria

97%

C64

Actinobacteria

97%

C65

Actinobacteria

99%

C67

Gemmatimonades

95%

C68

Proteobacteria

99%

C70

Uncultured

97%

C71

Uncultured

98%

C73

Actinobacteria

99%

C74

Acidobacteria

99%

C75

Verrucomicrobia

96%

(b)

A3

Uncultured

85%

A4

Acidobacteria

99%

A5

Acidobacteria

96%

A7

Actinobacteria

98%

A8

Uncultured

93%

A9

Verrucomicrobia

98%

A12

Uncultured

98%

A22

Actinobacteria

97%

A23

Acidobacteria

98%

A24

Actinobacteria

96%

A26

Proteobacteria

99%

A27

Proteobacteria

100%

A30

Acidobacteria

96%

A33

Actinobacteria

96%

A36

Uncultured

98%

A37

Uncultured

100%

A39

Verrucomicrobia

98%

A40

Proteobacteria

99%

A42

Actinobacteria

100%

A44

Proteobacteria

96%

A46

Acidobacteria

99%

A71

Acidobacteria

97%

A91

Verrucomicrobia

91%

A93

Proteobacteria

97%

A94

Actinobacteria

99%

A96

Proteobacteria

93%

A97

Chloroflexy

96%

A100

Proteobacteria

99%

A105

Proteobacteria

88%

A106

Actinobacteria

99%

A107

Proteobacteria

95%

A109

Acidobacteria

93%

A112

Proteobacteria

98%

A115

Acidobacteria

100%

A122

Proteobacteria

100%

Table 8.1 (continued)

Clones

Phyla

%Identity

A123

Actinobacteria

95%

A124

Acidobacteria

96%

A126

Acidobacteria

98%

A127

Proteobacteria

91%

A128

Uncultured

95%

A130

Verrucomicrobia

98%

Fig. 8.5 Cluster analysis and phylogenetic tree. Cloned and sequenced amplicons of rhizospheric DNA extracted from soil amended with compost (COM) for 1 year in the Torino site

Actinobactena

Verrucomicrobia

. Acidobacteria

Proteobacteria y Acidobacteria

Fig. 8.5 Cluster analysis and phylogenetic tree. Cloned and sequenced amplicons of rhizospheric DNA extracted from soil amended with compost (COM) for 1 year in the Torino site

Actinobactena

The cluster analysis of the determined sequences (Figs. 8.5 and 8.6) shows the presence of four major groups corresponding to the Proteobacteria, Acidobacteria, Actinobacteria and Verrucomicrobia phyla. In particular, the library relative to the compost (COM) treatment indicates that Proteobacteria constituted about 23% (basically consisting of the g-Proteobacteria subgroup), Acidobacteria 21.2%, Actinobacteria 34.6%, Verrucomicrobia 7.7%, Firmicutes 1.9%, Gemmatimonadetes 1.9%, and 9.7% of other bacteria of unknown phylogenetic classification (Table 8.2). In the library relative to soils under traditional tillage (TRA), Proteobacteria were 29.3% (mainly the g-Proteobacteria subgroup), while Acidobacteria were 24.4%,

Fig. 8.6 Cluster analysis and phylogenetic tree. Cloned and sequenced amplicons of rhizospheric DNA extracted from soil under traditional tillage (TRA) for 1 year in the Torino site

Fig. 8.6 Cluster analysis and phylogenetic tree. Cloned and sequenced amplicons of rhizospheric DNA extracted from soil under traditional tillage (TRA) for 1 year in the Torino site

Table 8.2 Number of clones and their percentage (%) found in the two amplicons libraries for compost-amended (COM) soils (52 clones) and for traditionally tilled (TRA) soils (41 clones)

Phylum_COM_TRA

Table 8.2 Number of clones and their percentage (%) found in the two amplicons libraries for compost-amended (COM) soils (52 clones) and for traditionally tilled (TRA) soils (41 clones)

Phylum_COM_TRA

Gemmatimonadetes 1 (1.9%) 0

Actinobacteria 19.5%, Verrucomicrobia 9.8%, Chloroflexi 2.4%, and 14.6% were bacteria of unknown phylogenetic classification.

In both libraries the prevalent group was that of the g Proteobacteria subphylum, that is a bacterial group responsible for the mineralization of soil carbon and possesses the specific capacity for decomposition of fresh organic matter (Fontaine et al. 2003). Their abundance in the rhizosphere at the soil root interface is reasonable because plant rhizodeposition should account for an enhanced availability of microbial growth substrates.

The information provided by the two libraries suggests that the Actinobacteria is the only group that changes by a significant extent and increasing mostly in compost-amended soils. This variation is to be accounted for the capacity of bacteria belonging to this phylum to degrade complex substrates such as the mature compost used in the amendments. The relative abundance of Actinobacteria in comparison to the total composition of the phyla in the whole sequenced communities agrees with the information provided by the DGGE profiles (Fig. 8.2). In fact, the profiles showed bands which were clearly present only in COM replicates and well indicated that compost-amended soils segregated separately from other soils.

Although the low number of sequenced clones reported here does not give a comprehensive view of the whole bacterial communities, our results do suggest that the application of different molecular approaches contribute to reach an advanced characterization of structure and diversity of soil bacteria and appraisal of their variation as a consequence of specific soil management practices.

Molecular approaches are undoubtedly a powerful tool to valuate microbial structure and diversity in different environments. Among different approaches, cloning and sequencing of whole metagenomes are still quite expensive and show some important drawbacks to be overcome. Very recently, pyrosequencing, a tool used firstly for the determination of the human genome, has been applied for soil metagenome investigation. It is based on the possibility to detect pyrophosphate released during DNA synthesis that is used to produce enzymatically ATP by ATP arylsulphatase; ATP is a suitable substrate for the luciferin-luciferase system that generates bioluminescence directly correlated to the amount of nucleotides incorporated during the PCR synthesis of template DNA (Ronaghi 2001). By running hundreds of thousands of sequences at the same time, each one detectable by a bar code system, and by the support of post-run softwares to manage huge amounts of data and to validate them statistically, it is possible to get much more details about microbial communities characterized by high diversities.

In conclusion, in the case of the MESCOSAGR project here reported, it appears that only the amendments with a mature compost had a significant effect on the soil microbial communities, while the addition of a biomimetic catalyst, used to increase C sequestration, did not have an effect as well as traditional and minimum tillage. By investigating and comparing bacterial communities inhabiting bulk and rhizosphere soil, it was also clear that the role played by plant root deposition; very likely root exudates make the nutrients brought by the compost amendment more available for bacteria, enhancing their diversity.

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