Assessment of Differences in Ascomycete Communities in the Rhizosphere of Field-Grown Wheat and Potato

FEMS Microbiology Ecology 53 (2005) 245–253 www.fems-microbiology.org

Assessment of diﬀerences in ascomycete communities in the rhizosphere of ﬁeld-grown wheat and potato

Mareike Viebahn a, Christiaan Veenman a, Karel Wernars b, Leendert C. van Loon a, Eric Smit b, Peter A.H.M. Bakker a,*

a Section of Phytopathology, Faculty of Biology, Utrecht University, P.O. Box 80084, 3508 TB Utrecht, The Netherlands b National Institute of Public Health and the Environment, Bilthoven, The Netherlands

Received 1 June 2004; received in revised form 29 October 2004; accepted 22 December 2004

First published online 19 February 2005

Abstract

To assess effects of plant crop species on rhizosphere ascomycete communities in the field, we compared a wheat monoculture and an alternating crop rotation of wheat and potato. Rhizosphere soil samples were taken at different time points during the growing season in four consecutive years (1999–2002). An ascomycete-specific primer pair (ITS5–ITS4A) was used to amplify internal transcribed spacer (ITS) sequences from total DNA extracts from rhizosphere soil. Amplified DNA was analyzed by denaturing gradient gel electrophoresis (DGGE). Individual bands from DGGE gels were sequenced and compared with known sequences from public databases. DGGE gels representing the ascomycete communities of the continuous wheat and the rotation site were compared and related to ascomycetes identified from the field. The effect of crop rotation exceeded that of the spatial heterogeneity in the field, which was evident after the first year. Significant differences between the ascomycete communities from the rhizospheres of wheat in monoculture and one year after a potato crop were found, indicating a long-term effect of potato. Sequencing of bands excised from the DGGE gels revealed the presence of ascomycetes that are common in agricultural soils. 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Ascomycete communities; Crop rotation; DGGE; ITS; rDNA; Rhizosphere

1. Introduction On the other hand, they also comprise a large number of plant pathogens [3]. It is estimated that only 5% of all Fungi are a diverse group of microorganisms, playing fungal species are known [4] and that only 17% of the a fundamental role in terrestrial ecosystems. They are in- known species can be grown in culture [5]. This implies volved in nutrient cycling, transportation of nutrients to that the diversity of fungi is largely unrecognized. More- plants, plant growth stimulation and antagonism over, cultivating fungi from soil is not without bias, be- against plant pathogens [1,2]. The majority of plant- cause they can be dormant, occur as spores, as associated fungi show a beneﬁcial or neutral interaction mycelium, or both, and are often closely associated with with the host plant. Additionally, some fungi are used as other organisms [5]. Therefore, in recent studies cultiva- biological control agents against plant pathogenic fungi. tion-independent molecular approaches have been used to determine the diversity of fungal communities

* through analysis of ribosomal (r) RNA genes [6–8]. Corresponding author. Tel.: +31 30 2536861; fax: +31 30 2518366. E-mail address: [email protected] (P.A.H.M. Bakker). The rRNA genes are well suited for analyzing microbial URL: http://www.bio.uu.nl/~fytopath/ (P.A.H.M. Bakker). diversity, because they are present in all known

0168-6496/$22.00 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsec.2004.12.014 246 M. Viebahn et al. / FEMS Microbiology Ecology 53 (2005) 245–253 organisms, contain conserved as well as variable regions Gaeumannomyces graminis var. tritici was kindly pro- and their sequences are collected in large public dat- vided by Dr. J.M. Raaijmakers (Phytopathology, abases [9]. While comparison of the small subunit Wageningen Agricultural University, The Netherlands). (SSU or 16S) rRNA is a well-accepted tool in bacterial Genomic DNA of Trichoderma harzianum B28 and Ver- community studies, the SSU or 18S rRNA genes of fun- ticillium longisporum was kindly provided by Dr. J. Post- gal communities are less informative and therefore less ma (Plant Research International, Wageningen, The suitable for fungal community studies [10]. Netherlands). All other isolates were derived from the Previous research has shown that the internal tran- experimental field and have been identified by Dr. W. scribed spacers (ITS) of the nuclear rRNA genes are bet- Gams (Centraal Bureau voor Schimmelcultures, Utr- ter targets for the molecular analysis of fungal echt, The Netherlands). communities than the 18S rRNA genes [11,12]. The ITS are variable, non-coding regions located between 2.2. Rhizosphere soil sampling the 18S and 5.8S subunit (ITS1) and between the 5.8S and 28S subunit (ITS2) of the rRNA [13]. Ribosomal Rhizosphere soil was sampled from an experimental RNA operons in fungi are often found as tandem re- field located at the Botanical Gardens of Utrecht Univer- peats of up to a hundred copies, which makes it possible sity, the Netherlands, as described earlier [18–20]. The to amplify specific fragments even from small environ- soil contains 12% clay, has an organic matter content mental samples [14]. Several primers have been designed of 4% and a pH (KCl) of 5, and was a grassland for more to target this region. Primers ITS1 to ITS5 cover the two than 20 years. From 1999 to 2002 wheat (Triticum aes- spacer regions and partial sequences of the 18S and 28S tivum cv. Baldus) was grown continuously in six 1 m2 rDNA [12]. Recently, Larena et al. [15] developed a pri- plots, while in another six plots wheat was grown in mer with enhanced specificity for ascomycetes. With this 1999, followed by potato (Solanum tuberosum L. cv. primer, in combination with ITS1 or ITS5, only the Modesta) in 2000, wheat in 2001, and potato in 2002. ascomycetes were successfully amplified from a mixture Wheat was sown and potatoes were planted in early of ascomycetes, basidiomycetes, oomycetes, zygomyce- spring every year, and samples were taken 11, 25, 39 tes and mitosporic fungi. and 54 days later [19,20]. The field was designed in such Ascomycetes are the largest group of the true fungi a way that three replicates of each treatment were located [15]. Most of them are saprophytic and live on dead or- on the left half of the field, the other three replicates on ganic material, which they help decompose. However, the right half. From each plot, three random samples ascomycetes also cause plant diseases, varying from pow- of plant roots with adhering soil were taken at each time dery mildews to rots, cancers and vascular wilts [3].In point, and excess soil was removed. The upper 10 cm of the present study, we compared the rhizosphere micro- the roots (3–5 g), with tightly adhering soil, were mixed flora in a long-term field trial between a crop rotation with 10 ml sodium phosphate buffer (120 mM, pH 8). of wheat and potato and continuous wheat cultivation. In our first attempt, we tried to analyze the total fungal 2.3. Total DNA extraction community. However, polymerase chain reaction (PCR) amplicons of 18S rDNA could not be adequately One gram of gravel was added and samples were vor- separated on a denaturing gel. Therefore, we decided to texed for 30 s. The supernatant was decanted into a new analyze a subgroup. The ascomycete communities were tube. One milliliter of the supernatant was used to ex- compared by performing an rDNA fingerprint analysis tract total DNA with the FastDNA SPIN Kit for Soil of the ITS region of the rRNA operon by denaturing gra- (Bio 101, Biogene, Vista, CA, USA) in combination dient gel electrophoresis (DGGE) [16,17]. Ascomycetes with a Ribolyser (Hybaid, Ashford, UK) [21]. The ex- were chosen because of their preponderance in the soil tracts were diluted 1:100 with Millipore-filtered distilled and because of the availability of specific primers. Finally, water before purification with the Wizzard DNA some of the predominant ascomycete bands revealed by Clean-Up System (Promega, Madison, WI, USA), DGGE were identified by cloning and sequencing. according to the manufacturerÕs protocol. DNA from the ascomycete isolates was extracted by using the Fast DNA Spin Kit (Bio 101) according to the supplierÕs 2. Materials and methods manual, and subsequently purified with the Wizzard DNA Clean-Up System. 2.1. Ascomycete isolates 2.4. PCR amplification of the ITS region Ascomycete isolates used to investigate the specificity of DGGE were grown on potato dextrose agar at 25 C Polymerase chain reaction on purified DNA extracts for 3–7 days. Alternaria brassicicola was kindly provided was performed with primers specific for ascomycetes, by Dr. J. Ton (Utrecht University, The Netherlands). amplifying the ITS1, 5.8S, and ITS2 region of the fungal M. Viebahn et al. / FEMS Microbiology Ecology 53 (2005) 245–253 247 ribosomal RNA operon. The forward primer ITS5 bined from samples taken at three to five time points (50GGAAGTAAAAGTCGTAACAAGG-30) [12] with (composite dendrograms). the reverse primer ITS4A (50-CGCCGTTACTGG- GGCAATCCCTG-30) [15] generates amplicons of about 2.6. Cloning and sequencing of DGGE bands 700 bp. ITS4A was extended with a 40-base-pair G+C rich sequence to stabilize the melting behavior of the Twelve dominant bands from gels loaded with sam- DNA fragments for DGGE analysis [22]. Primers were ples taken 11 days after sowing or planting were excised synthesized by Eurogentec, Maastricht, The Nether- from DGGE gels and eluted in 20 ll Millipore-filtered lands. The 50 ll reaction mixture contained 1 ll of appro- distilled water. One microliter was used for re-amplifica- priately diluted DNA extract, PCR buffer 2 (pH 9.2) tion with the primers mentioned above, using the follow- containing 2.2 mM MgCl2 (Roche Diagnostics, Mann- ing conditions: one cycle of 94 C for 5 min, followed by heim, Germany), 200 lM each of dATP, dTTP, dGTP 35 cycles of 94 C for 1 min, 63 C for 1 min and 72 C and dCTP, 200 nM primers ITS5 and ITS4A, and 1.5 U for 3 min. The reaction was terminated at 72 C for Expand Long Template enzyme (Roche Diagnostics, 10 min. After re-amplification the purity of the excised Mannheim, Germany). Amplification was carried out in DNA fragments was checked in a final DGGE under a Hybaid Thermocycler (Thermo Hybaid, Ashford, the conditions described above. Appropriate PCR prod- UK). The initial denaturing step was done at 94 C for ucts were ligated into the pGem-T Easy Vector (Pro- 5 min, followed by 35 cycles of 94 C for 1 min, 60 C mega, Madison, WI, USA) and transformed into for 1 min and 72 C for 3 min. The reaction was termi- Escherichia coli Ultra Competent Cells (Stratagene, nated at 72 C for 10 min. To check the size of the ampli- Cambridge, UK) as described by the manufacturers. cons, PCR products were separated on a 1% agarose gel Clones were directly resuspended in the PCR mixture in TAE buffer (0.04 M Tris–acetate, 0.001 M EDTA), and amplified with primer pair ITS5/ITS4A. The ampli- run for 40 min at 150 V, and stained with the nucleic acid fied DNA fragments were identified by DGGE as de- stain SYBR Green (Molecular Probes Inc., Leiden, The scribed. Only clones with a migration comparable to Netherlands) for 30 min. The gels were viewed under a the excised band were used for further sequence analysis. blue light transilluminator (Clare Chemical Research, Prior to sequencing, the PCR products were purified Dolores, CO, USA), and digitalized using a charged- with the QIAquick PCR Purification Kit (Qiagen, The coupled device (CCD) camera (Syngene, Cambridge, UK). Netherlands), according to the manufacturerÕs protocol. The sequencing reaction was carried out with the Big- 2.5. DGGE Dye Terminator v.3.1 Cycle Sequencing Kit in an AB3700 Sequencer (Applied Biosystems, Nieuwerkerk, PCR amplicons of the expected size from rhizosphere The Netherlands). Two clones per band were sequenced samples and ascomycete isolates were analyzed by in both directions with primers ITS5 and ITS4A, respec- DGGE. Denaturing gels were prepared with the gradi- tively. Sequences obtained were checked for chimeras ent former Bio-Rad Model 230 (Bio-Rad Laboratories, using the program CHIMERA_CHECK version 2.7 Veenendaal, The Netherlands) at a speed of 4.5 ml/min [24]. Sequences that most likely did not contain chimeric as described earlier [23]. The resulting gels consisted of regions were compared with known ascomycete se- 8% (wt/vol) polyacrylamide (0.5 · TAE buffer, 37.5:1 ra- quences of relevant databases with the Standard nucleo- tio of acrylamide/bisacrylamide, 20 · 20 cm) and had a tide Blast program [25], provided by the National gradient of 20–50% denaturant. A 100% denaturing Center for Biological Information (NCBI, USA). Se- acrylamide gel contained 7 M urea and 40% formamide. quences of the clones were submitted to GenBank with Up to 25 ll of PCR product per lane was loaded and Accession Nos. AY615877–AY615884. gels were run for 17 h at 80 V at a constant temperature of 60 C in a DCode Universal Mutation Detection 2.7. Comparing DGGE gels with a defined marker System (Bio-Rad Laboratories, Veenendaal, The Neth- erlands). For comparison of DNA patterns a reference Sequencing of the twelve excised DGGE bands re- marker was added in triplicate to each gel. After electro- vealed matches for ten bands with ascomycete sequences phoresis, gels were stained and digitalized as described from relevant databases, and these bands were used as a above for the agarose gels. Gels were analyzed with reference marker. DGGE was performed with rhizo- the BioNumerics program version 3.5 (Applied Maths, sphere samples taken 11, 25, 39 and 54 days after sowing Sint-Martens-Latem, Belgium). After normalizing the wheat in the experimental field in 2001. Prior to PCR, gel and background subtraction, PearsonÕs correlation samples from three replicate plots were pooled, yielding coefficient was calculated for each lane, followed by two replicates per treatment, and samples from six con- the UPGMA cluster analysis (unweighted pair group tinuous wheat plots were compared with samples from method using arithmetic averages). Dendrograms were six wheat–potato–wheat rotation plots. DNA was ex- generated from samples at a single time point, or com- tracted and DGGE was performed as described above. 248 M. Viebahn et al. / FEMS Microbiology Ecology 53 (2005) 245–253

3. Results lyzed. Fig. 1 shows the separation of the ITS amplicons of eight different ascomycetes. Most isolates 3.1. DGGE analysis of ascomycete isolates and showed a single discrete band. However, the bands in predominant ascomycete communities in the wheat and lanes 2, 8 and 9 are fuzzy. potato rhizospheres Total DNA from rhizosphere samples of six wheat plots and six rotation plots was extracted and analyzed To evaluate the specificity of DGGE, DNA ex- by DGGE. Samples of the three plots from each half tracted from different ascomycete isolates were ana- were pooled for analysis, resulting in two replicates per treatment. Fig. 2 shows the results from the cluster analysis of the four consecutive years (1999–2002). In 1999, wheat was sown in both the control and the rotation plots. Samples from the left half of the field (marked a) clustered apart from samples of the right half (b) at a similarity index of only 30% (Fig. 2A). The similarity of both samples within each cluster was 40% and 52%, respectively. In 2000, with potato planted in the rotation plots, there is a clear differentiation of the ascomycete communities in the rhizospheres of wheat and potato (Fig. 2B). All samples from the potato rhizosphere clustered separate from those of wheat. The 2001 samples clustered in the same way as in 2000, even though now wheat was sown again in the rotation plots (Fig. 2C). The similarity between the two clusters was only 2%. In 2002, the dendrogram from the wheat and potato rhizosphere samples was again comparable, with Fig. 1. DGGE profile of ITS rDNA fragments of ascomycete species only about 2% similarity between the potato and the generated by PCR with ascomycete specific primers. Lane 1: Alternaria wheat clusters (Fig. 2D). brassicicola, lane 2: Acremonium murorum, lane 3: Gaeumannomyces The pronounced differences in the ascomycete com- graminis var. tritici (G.g.t.), lane 4: G.g.t. (PCR product 1:10 diluted), lane 5: Chaetomium bostrychodes, lane 6: Trichoderma harzianum B28, munities of the rhizospheres of wheat and potato led us lane 7: Verticillium longisporum, lane 8: Muratella elegans, lane 9: to a more thorough investigation of samples taken at Monocillium mucidum. M, marker. 11 and 25 days after sowing in 2002. Fig. 3A and B shows

Fig. 2. Dendrograms representing the percentage genetic similarity of rhizosphere ascomycete communities in continuous wheat (d) and in a crop rotation of wheat and potato (%). In the rotation plots, wheat was grown in 1999 (A) and 2001 (C), and potatoes were planted in 2000 (B) and 2002 (D). Each dendrogram is a combination of multiple dendrograms from samples taken at the earliest three to four time points per year. Similarities were based on DGGE patterns generated from ITS1/ITS2 rDNA fragments using PearsonÕs correlation coeﬃcient. Cluster analysis was done with UPGMA. Samples from three of six plots of continuous wheat culture and a wheat–potato rotation, respectively, were pooled, resulting in two replicates per treatment, from the left (a) and from the right half of the ﬁeld (b): cw, continuous wheat plots; rp, rotation plots. M. Viebahn et al. / FEMS Microbiology Ecology 53 (2005) 245–253 249

Fig. 3. Dendrograms representing the percentage genetic similarity of the ascomycete communities from the rhizosphere of wheat () and potato ( ), 11 (A) and 25 (B) days after sowing in 2002. In (A), the corresponding gel is shown. Samples originated from plots of continuous wheat culture and a wheat–potato rotation. Cluster analysis was based on the UPGMA linkage of PearsonÕs correlation coefficients of DGGE profiles obtained from six replicate samples of wheat and potato. The arrows indicate the similarity level at which clusters were defined. the dendrograms based on the DGGE patterns of all six migration compared to that of the excised band. Two replicate samples of the rhizosphere of wheat and potato. clones per band with exactly the same position in the After 11 days, three main clusters were defined at a cut- DGGE gel were used for further sequence analysis. off value of 50% similarity. One cluster contained only the rhizosphere samples from wheat (Fig. 3A). Five of the six replicates from the wheat rhizosphere were at least 65% similar. The remaining two clusters contained only the samples from the potato rhizosphere. The three replicates from the left half of the field (potato 1, 2, 3) formed one cluster and were at least 63% similar. The three replicates from the right half (potato 4, 5, 6) formed the other cluster and differed by no more than 15%. Analysis of samples taken at a later time point revealed a largely comparable clustering (Fig. 3B). Again, overall similarity was less than 10%. At a cut-off value of 50%, five clusters were distinguished. One cluster contained only one replicate from wheat, with almost no similarity to any of the other samples. The other clusters each contained only rhizosphere samples from either wheat or potato. Furthermore, DGGE gels profiling the ascomycete communities revealed only a small number of bands per treatment, varying between four and twelve.

3.2. Sequence analysis of ITS fragments recovered from DGGE gels

To further characterize the differences in the ascomycete communities of the wheat and potato rhizospheres, Fig. 4. DGGE profile of DNA fragments from rhizosphere samples twelve differentially migrating DGGE bands were ex- using ascomycete specific primers. DNA was directly extracted from the rhizosphere of wheat and potato 11 days after sowing in 2002. cised (Fig. 4), re-amplified and cloned into a vector. Excised and sequenced bands are labeled (*): W1–W6 refer to DNA At least four clones per band were subjected to DGGE isolated from wheat after 4-year monoculture, bands P7–P12 refer to under the same standardized conditions, and their DNA isolated from potato after alternating wheat–potato culture. 250 M. Viebahn et al. / FEMS Microbiology Ecology 53 (2005) 245–253

Table 1 Characterization of partial ITS sequences recovered from bands of DGGE profiles with DNA extracted from wheat and potato rhizosphere 11 days after sowing in 2002 Band No.a Most similar sequence Genbank Accession No. Similarity (%) 1 n.d.b 2 Verticillium nigrescens Seq.c 1 AJ292440 100 3 Podospora sp. AF126819 97 4 Verticillium nigrescens Seq. 2 AJ108473 97 5 Plectosphaerella cucumerinad Seq. 1 AJ492873 99 6 Issatchenkia orientalise AF455401 97 7 Plectosphaerella cucumerinad Seq. 2 AJ246154 99 8 n.d.c 9 Unidentified 10 Chaetomium sp. AJ279468 95 11 Unidentified 12 Ericoid mycorrhizal sp. AY046400 93 a Band No. refers to the excised DGGE bands. Band No. 1–6 originated from the wheat rhizosphere, No. 7–12 from the potato rhizosphere. b n.d., Not determined. c Seq., Sequence. d The current valid name is Monographella cucumerina (Lindf.) Arx. e The current valid name is Candida krusei (Castell.) Berkhout.

Two of the twelve bands were not successfully recov- 3.3. Ascomycete communities of the wheat rhizosphere ered and excluded from further analysis. Table 1 shows after monoculture and rotation the best match of the ten sequences of the recovered bands with known ascomycete sequences. Two se- Ascomycete communities from the rhizospheres of quences (bands 9 and 11) did not match any of the wheat from the preceding three-year monoculture and known sequences and, therefore, could not be identified. from the wheat–potato–wheat rotation were compared The other sequences all represent ascomycetes that are to the ten ascomycetes identified from the 2002 samples common in agricultural soils [26]. Six bands showed (Table 1). Given the fact that the samples were derived high similarities (at least 97%) with the genera Issatchen- from the same field, there is a high probability that cor- kia, Plectosphaerella, Podospora and Verticillium. Two responding bands represent the same species. Based on bands showed a modest similarity of 95% and 93% to this assumption, Table 2 shows the different ascomycetes Chaetomium sp. and Ericoid mycorrhiza, respectively. from the continuous wheat and the wheat–potato–wheat In two cases, two bands with different melting behavior rotation plots in the season of 2001 that were identified and thus different sequences resembled the same species. according to their position in the DGGE gel in compar- Verticillium nigrescens sequences 1 and 2 differed by 1% ison with the characterized bands. Two species, Chaeto- of their sequence, Plectosphaerella cucumerina sequences mium sp. and Issatchenkia orientalis, were found only in 1 and 2 by 3%. the rotation plots. V. nigrescens sequence 2 and one

Table 2 Ascomycetes of the rhizosphere of wheat as identified by comparison of DGGE bands Band No. Ascomycetes Days after sowing cw wpw 11 25 39 54 11 25 39 54 6p I. Orientalis xxxx 10 p Chaetomium sp. xxxx 5p P. cucumerina seq. 1 x xxxx 11 p unidentified x x xxxx 7p P. cucumerina seq. 2 x xxxx xx 2wV. nigrescens seq. 1 x xxxx xx 12 w Ericoid mycorrhiza x x x x x 3 w Podospora sp. x xxxx 4wV. nigrescens seq. 2 9 w unidentified Samples were taken at 11, 25, 39 and 54 days after sowing during the season of 2001 from continuous wheat plots (cw) and from rotation plots of wheat–potato–wheat (wpw) cultivation. w and p indicate that samples, from which the excised DGGE bands were recovered, originated from the rhizosphere of wheat (w) or potato (p) in 2002. M. Viebahn et al. / FEMS Microbiology Ecology 53 (2005) 245–253 251 unidentified species did not occur at all prior to 2002. of the same plant species, positioned in the left or the All other species were detected in both treatments. With- right half of the field. Crop rotation from wheat to po- in the season some changes became apparent. In the tato in the second year led to a marked difference be- continuous wheat plots, P. cucumerina sequence 1 was tween the ascomycete communities of the two plant detected only at 11 days, one of the unidentified species species. This effect was still evident after cultivating (band 9) at the first two sampling dates, and the Ericoid wheat again in the rotation plots, suggesting that a sin- mycorrhiza at 11, 39 and 54 days after sowing. In the gle change of wheat to potato has a long-term impact rotation plots, Podospora occurred at the first sampling on the microflora. Thus, the effect of crop rotation date only, the Ericoid mycorrhiza at the last two sam- clearly exceeded that of spatial heterogeneity in the pling dates and P. cucumerina sequence 2 and V. nigres- field. cens sequence 1 intermittently at 11, 39 and 54 days after In addition, DGGE was performed on six non- sowing. pooled replicate rhizosphere samples from wheat and potato taken 11 and 25 days after sowing in 2002. Re- sults from both DGGE analyses were consistent and 4. Discussion in agreement with the earlier results. Although DGGE can be used successfully to assess the diversity of fungal Currently, various genetic fingerprint techniques are communities [7,33–35], the method provides no infor- used in microbial ecology. They have provided patterns mation about the species composition. This information of the microbial community diversity in different habi- can be obtained by sequencing individual bands of the tats, but it is not clear which technique is yielding better DGGE gels and comparison with known sequences resolution or sensitivity. Torsvik et al. [27] analyzed from the public databases. Sequencing of DGGE bands microbial diversity by comparing different fingerprinting revealed only members of the phylum Ascomycota, con- methods with DNA melting profiles and reassociation firming the specificity of the primers. Ten of the twelve kinetic studies. They found that ARDRA (amplified excised bands showed unambiguous results and were in- rDNA restriction analysis) must be considered too cluded in the analysis. Two of the ten bands could not be sensitive for a reliable characterization of complex identified. One likely explanation is that the number of microbial communities, which contain too much infor- sequences of the ITS region in the public databases is mation to be reliably assessed, and that fingerprinting limited. Two clearly distinct bands in the DGGE gel methods such as DGGE are better suited for that resembled V. nigrescens, whereas two other bands purpose. Liesack and Dunfield [28], on the other hand, resembled P. cucumerina. The sequences differed by 1% argued that DGGE is often too sensitive for the determi- and 3%, respectively. This indicates that the described nation of total community fingerprints. species can be heterogeneous or that the names depos- Previously, it has been demonstrated that crop spe- ited in the databases are inconsistent. cies have a pronounced effect on the composition of All identified ascomycetes are common in agricultural the microbial communities [29–32]. In a recent study, soils. Three of these ascomycetes are reported to be using ARDRA, we could not detect that a crop rotation potentially pathogenic to plants. Chaetomium species from wheat to potato influenced the predominant bacte- are often found as decomposers of herbaceous and ligni- rial and fungal communities [20]. However, by re-exam- fied plant material and have been recognized as the cau- ining the samples using DGGE for comparing genetic sal agent of soft rot in wood [26]. P. cucumerina has been fingerprints of PCR-amplified ITS regions of ascomy- almost exclusively associated with arable soil and has cete communities, distinct clusters were evident: one been isolated from the rhizosphere of flax, grass, potato, comprising all samples from the potato rhizosphere sugar beet and wheat [26]. It can produce necrotic spots and one containing all wheat rhizosphere samples. Asco- on potato stalks and cause wilting in beets [26]. V. mycete communities were analyzed since the separation nigrescens is a common soil fungus in arable and non- of fungal 18S rDNA amplicons on a denaturing gradient cultivable soil. It has been found to attack spearmint did not lead to proper gels. It demonstrates that the two and peppermint [26]. plant species did have an effect on the composition of Using DGGE, we compared patterns of the ascomy- the rhizosphere communities. It also illustrates that the cete communities of the continuous wheat and the rota- use of different molecular techniques can lead to differ- tion sampling sites with ascomycete reference markers ent conclusions. resembling species identified from the same sites, but In the present study, using DGGE, we have shown in different years. Bands that correspond to bands of that in the first year of wheat cultivation the ascomy- the markers were assumed to be the ascomycete repre- cete communities differed according to their location sented by the marker band. We are aware that this is a in the field. Apparently, the soil and its environment very simplified method to assess differences in the micro- are not homogeneous, which caused a differentiation bial communities. However, it seems to be justified, since between ascomycete communities from the rhizosphere all material was sampled from the same site, and only 252 M. Viebahn et al. / FEMS Microbiology Ecology 53 (2005) 245–253 clones with unambiguous positions in the gel before and [2] Thorn, G. (1997) The fungi in soil In: Modern Soil Microbiology after cloning and with high sequence identity were used (van Elsas, J.D., Treves, D.S. and Wellington, E.M.H., Eds.), pp. as marker bands. 63–127. Marcel Dekker, Inc., New York. [3] Agrios, G.N. (1997) Plant Pathology. Academic Press, San Diego, Two species, I. orientalis and Chaetomium sp., were CA, USA. detected only in the rhizosphere of wheat after crop [4] Hawksworth, D.L. and Rossman, A.Y. 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