IJERD – International Journal of Environmental and Rural Development (2010) 1–1

Research article
erd

Diversity of the Actinomycetes Community Colonising Rice
Straw Residues in Cultured Soil Undergoing Various Crop
Rotation Systems in the Mekong Delta of Vietnam
TRAN VAN DUNG
Soil Science Department, College of Agriculture & Applied Biology,
Can Tho University, Vietnam
Division of Soil and Water Management, Faculty of Bioscience Engineering,
K.U. Leuven, Belgium
Email: tvandung@ctu.edu.vn

DUONG MINH VIEN, VO THI GUONG AND CAO NGOC DIEPCan Tho University, Vietnam

PATRICIA DOMINGUES, ROEL MERCKX AND DIRK SPRINGAELDivision of Soil and Water Management, Faculty of Bioscience Engineering,
K.U. Leuven, Belgium
Received 30 December 2009

Accepted 5 March 2010

Abstract Actinomycetes are involved in important environmental processes such as thedecomposition of organic matter. In this study, we examined the impact of crop rotation on
the actinomycetes community colonizing rice straw residues in soil over 3 field seasons bymeans of Denaturing Gradient Gel Electrophoresis (DGGE) fingerprinting analysis ofactinomycetes 16S rRNA gene fragments amplified from field-incubated rice straw
residues and analysis of actinomycetes 16S rRNA gene clone libraries from selected
samples. The studied yearly crop rotation systems were rice-rice-rice (CRS1), rice-ricebaby corn (CRS2), rice-rice-mungbean (CRS3) and baby corn-rice-mungbean (CRS4),
applied on different experimental plots of the same field location. Litter bags containing
rice stems were inserted into the soil and recollected at different time points forcomparison of the structure of the actinomycetes community colonizing the rice straw.
The actinomycetes community was significantly different in composition in the baby cornrice-mungbean rotation system (CRS4) compared to those in the 3 other systems during
the growth of the first crop and second crop. In contrast, during the cultivation of the third
crop, actinomycetes communities were significantly different in the rice-rice-rice (CRS1)
system compared to those in the 3 other systems. The analysis of the 16S rRNA genelibraries constructed from selected samples of rotation systems CRS1 and CRS4 during
growth of the first two crops confirmed the DGGE results. The diversity of actinomycetes
tended to be highest in the CRS4 system and lowest in the CRS1 system.
Keywords Actinomycetes community, diversity, crop rotation, Mekong delta

INTRODUCTION
Actinomycetes, phylogenetically defined as a number of taxa within the high-GC subdivision of thegram-positive phylum (Embley et al., 1994), represent a group of relatively abundant andmetabolically diverse bacteria in soils (Labeda and Shearer, 1990; McCarthy and Williams, 1992;
Holmalahti et al., 1994). They are involved in important processes in a wide range of habitats(Williams et al., 1984), such as the decomposition of organic materials in soil, including lignin andother recalcitrant polymers, and in the degradation of agricultural and urban wastes (Crawford,
1988; McCarthy, 1987).

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The composition of a soil microbial community can be affected by various factors such as soil
characteristics, environmental conditions, plant growth and crop management strategies (Curl andTruelove, 1986). Numerous studies have compared microbial communities among different
ecosystems such as agricultural soils versus soils from forest or grassland systems (Ovreas andTorsvik, 1998; Waldrop et al., 2000; Yao et al., 2000), among soils undergoing different long-term
cropping and management regimes (Zelles et al., 1992, 1995; Bossio et al., 1998; Drijber et al.,2000), and among soils cultivated by different plant species (Grayston et al., 1998; Siciliano et al.,
1998; Ibekwe and Kennedy, 1999; Miethling et al., 2000; Marschner et al., 2001). However, there
are few studies documenting the effects of crop rotation systems on specific soil microbialcommunities (Lupwayi et al., 1998). Especially, the effect of crop rotation on soil actinomycetes
has been poorly studied despite the important role of this group of micro-organisms in degradationof organic matter and nutrient cycling.
Rice is the most important crop in the Mekong Delta of Vietnam. The total area cultivatedwith rice in that region occupies nearly 4 million hectares per year. The introduction of highyielding rice varieties and intensive rice cultivation has resulted into numerous constraints, such as
high plant disease pressure, the extensive use of fertilizers and pesticides and soil degradation. In
order to reduce chemical inputs, to improve profitability, and to increase sustainability, newcropping systems and management practices for rice production in the Mekong Delta are being
assessed. In this context, we have initiated different field experiments in which we assessed theeffect of crop rotation on rice productivity and soil health. As a part of this study, we examined
whether the type of crop rotation affected microbial communities playing a role in soil functioning.
In this paper, we assessed the effect of crop rotation system on the actinomycetes community
colonizing and degrading rice straw residues in soil which had been historically cultivated forcontinuous rice production for more than 10 years.
METHODOLOGY
Set-up of field experiment and sampling approach
The experimental field used in this study is located in Cay Lay district, Tien Giang province,
Vietnam. The field was designed as a complete randomized block of experimental plots undergoing
4 different crop rotation systems (CRS) with 3 replicate plots per system since the year 2001. Eachplot covered an experimental area of 90 m2 (6 by 15m). The 4 applied rotation systems were (1)CRS1: rice (Crop I) - rice (Crop II) - rice (Crop III), (2) CRS2: rice (Crop I) - rice (Crop II) - baby
corn (Crop III), (3) CRS3: rice (Crop I) - rice (Crop II) - mungbean (Crop III) and (4) CRS4: baby
corn (Crop I) - rice (Crop II) - mungbean (Crop III).

Fig. 1 Time schedule showing the recovery of litter bags from the Cay Lay field experiment
Arrow indicated with 1 refers to litter bags inserted before the start of the experiment. Arrows indicated with 2,
5 & 8 refer to sampling of litter bags after 14 days of cultivation of crops I, II and III, respectively. Arrows
indicated with 3 & 6 refer to sampling of litter bags after 50 days of cultivation of crops I and II, respectively.
Arrows indicated with 4, 7 & 9 refer to sampling of litter bags at harvest of crops I, II and III, respectively. The
cropping calendars of three cropping patterns were as follow: crop I (between 20/6/2006 and 1/10/2006), crop
II (between 1/11/2006 and 10/2/2007) and crop III (between 30/2/2007 and 10/6/2007).

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Litter bags (nylon material with a pore size of 200 ηm) were filled with 5 g of dried rice straw
residues and buried, prior to seeding of the first crop, into the soil at a depth of around 10 cm on 20
June 2006.Before inserting, the bags with rice straw were sterilized at 121oC for 20 minutes. The
litter bags were periodically recovered from the soil for 16S rRNA gene based DGGE analysis of
the actinomycetes community colonizing rice straw according to the time schedule shown in Fig 1.
At each time point, 3 litter bags were recovered from each plot.
DNA extraction and DGGE analysis
DNA was extracted from the rice straw as described by Boon et al. (2000). 30µl of the extract wascleaned from humic acids by adding 2mg acid-washed PVPP (Sigma-Aldrich Chemie GmbH,
Steinheim, Germany) and 30µl TE-buffer. The mix was vortexed and centrifuged at 11,000 rpm
during 5 min. The supernatant was recovered and subjected to PCR amplification of the target 16SrRNA gene. To amplify actinomycetes specific 16S rRNA gene fragments, a nested PCR approach
was used in which in the first PCR the actinomycete specific forward primer F243 (Heuer et al.,1997) was used together with the bacterial reverse primer R1378 (Heuer et al., 1997). The productof this PCR was used as template in a second PCR with primers F984GC (Nubel et al., 1996) andR1378. PCR reactions were performed in a Mastercycler apparatus (Eppendorf; Hamburg,
Germany), according to van Dillewijn et al. (2002). DGGE of amplified actinomycetes 16S rRNA
gene fragments was performed on an Ingeny phor U-2 system (Leiden, The Netherlands). 30 µl
portions of the PCR product were loaded onto an 8% (w/v) polyacrylamide gel with a denaturing
gradient ranging from 35% denaturant to 65% denaturant in Tris–acetate–EDTA (TAE) buffer.
Electrophoresis was performed for 15 h at 60°C and 120 V. The gels were stained for 30 min with
1xSYBR Gold (Molecular Probes, Leiden, and The Netherlands) and photographed on a UVtransilluminator with a GeneLink camera system (SYNGENE, Cambridge, UK). Gelcompar II
version 3.5 (Applied Math’s, Sint-Martens-Latern, Belgium) was used for UPGMA cluster analysis
of DGGE fingerprints. Dendrograms were constructed by using the Pearson correlation index foreach pair of lanes within a gel and cluster analysis by the unweighted pair group method using
arithmetic averages.
Cloning and sequencing of 16S rRNA gene fragments
PCR products were cloned into pCR®2.1-TOPO®, using the TOPO TA Cloning kit (Invitrogen,Merelbeke, Belgium) as described by the manufacturer. To check transformants for theincorporation of the actinomycetes 16S rRNA gene, a nested PCR was performed consisting of afirst PCR with M13f and M13r primers as described by Invitrogen followed by a second PCR withbacterial primers GC-984F and 1378R. DGGE fingerprints from fragments amplified from theclones were compared with the actinomycetes 16S rRNA gene DGGE fingerprints obtained fromenvironmental DNA and appropriate clones were chosen for sequence analysis. The PCR products
obtained from the clones were purified with the PCR purification kit (Promega) as described by
Promega and subjected to DNA sequencing reactions performed with the QuickStart DNA
sequencing kit (Beckman) and analysed on an automatic sequencer (CEQTM8000, Beckman
Coulter, Fullerton, CA, USA). Resulting partial 16S rRNA gene sequences (about 400 bp) wereanalyzed by BLASTN search (Altschul et al., 1997).
RESULTS AND DISCUSSION
DGGE analysis of actinomycetes community colonizing rice straw incubated under various
crop rotation systems
Fig. 2 shows the actinomycetes 16S rRNA gene DGGE profiles obtained from rice straw residues
incubated under the different crop rotation systems at 14 days of cultivation, at 50 days ofcultivation and at harvest of crop I, crop II and crop III while Fig. 3 shows the corresponding© ISERD

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UPGMA cluster analysis. The DGGE profiles revealed that the composition of the actinomycetes
community in colonizing rice straw residues was strongly affected by crop rotation. The clearest
difference was observed between plots undergoing system CRS4 on the one hand and the 3 othersystems on the other hand during growth of crop I and II, and between CRS1 plots on the one hand
and the other 3 systems during growth of crop III.
During growth of crop I, at 14 days of cultivation, at 50 days of cultivation and at harvest, theprofiles of the replicates of systems CRS1, CRS2 and CRS3 clustered as a separate group from theprofiles of replicates of system CRS4 in the UPGMA clustering analysis (Fig. 3A). Profiles from
replicate plots of systems CRS1, CRS2 and CRS3 were highly similar with a similar level amongthe 3 systems of 48.2, 42 and 76% for samples taken at 14 days of cultivation, at 50 days ofcultivation and at harvest, respectively. In contrast, profiles from system CRS4 were 36.5%, 31 and72.8% similar to profiles originating from the other 3 rotation systems at 14 days, at 50 days ofcultivation and at harvest, respectively.
During growth of crop II, which was rice in all systems, at 14 days of cultivation and at 50
days of cultivation, profiles for systems CRS1, CRS2 and CRS3 were still more similar to eachother than to profiles obtained for system CRS4, i.e., the profiles of CRS1, CRS2 and CRS3 wereabout 70.4 % and 71.5% similar to the profiles of CRS4 at 14 days and 50 days of cultivation,
respectively, while the values of similarity were 87 and 87.5% among the profiles of CRS1, CRS2
and CRS3 at day 14 and day 50, respectively. However, at harvest of crop II, the profiles clusteredinto two main groups (Fig. 3B). One group consisted of the profiles of CRS1 and CRS2 and theother group of profiles of CRS3 and CRS4. The similarity between the group containing CRS1 and
CRS2 and the group containing CRS3 and CRS4 was 59.5% while the similarity of profiles within
the two groups was 69% and 66.9%, respectively.
During cultivation of crop III, at day 14 of crop cultivation and at harvest, the actinomycetes
community profiles clustered into two main groups, i.e., a first group consisting of profiles fromsystems CRS2, CRS3 and CRS4 and a second group consisting of profiles derived from system
CRS1 (Fig. 3C). Profiles from CRS1 were 42.1% and 34.5% similar to profiles originating fromthe other rotation systems at day 14 and at harvest, respectively. The similarity among the CRS2,
CRS3 and CRS4 profiles was 50.9% at day 14 day and 45.0% at harvest.
Analysis of 16S rRNA gene clone libraries
16S rRNA gene clone libraries were constructed from two representative samples of the CRS1 and
CRS4 system, i.e., a sample taken at 50 days of cultivation of crop I and a sample taken at 50 daysof cultivation of crop II. For each library 15 to 20 clones were analyzed by DNA sequence analysis.
Figure 4 shows the phylogenetic distribution of the 16S rRNA gene sequences within each library.
In the sample taken at 50 days of cultivation of crop I in system CRS1, most of the 16S rRNA
sequences matched with 16S rRNA gene sequences of actinomycetes especially with the family
Nocardioidaceae (22.3%) and uncultured actinomycetes (33.3%).Unexpectedly, other clones
carried sequences which were not related to 16S rRNA gene sequences of actinomycetes but rather
to sequences associated with the phyla verrucomicrobia and fimircutes (44.4%). In contrast, in thecorresponding samples taken from the CRS4 rotation system, almost all sequences were associatedwith the actinomycetes (84.75%) while only a minority was non-actinomycetes sequences such as
sequences associated with the phylum verrucomicrobia (15.3%). Interestingly, the actinomycetes
present in the CRS4 system were apparently more diverse than those in the CRS1 system, showing
the presence of four different families of actinomycetes, i.e., Microbacteriaceae,
Promicromonosporaceae, Nocardioidaceae, and Mycobacteriaceaee. The family Microbacteriaceae
constituted the most abundant family (53.8%).
Diversity in the actinomycetes community was also found at day 50 of cultivation of crop II.
Moreover, although in both systems, rice was the cultivated crop, the samples from the two systems
apparently contained two different actinomycetes communities. The community in the CRS4
system was more diverse and included members of five actinomycetes different families, i.e.,
Streptomycetaceae,
Catenulisporaceae,
Nocardioidaceae,
Mycobacteriaceae
andPromicromonosporaceae while the CRS1 sample contained members of two families, i.e.,© ISERD

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Microbacteriaceae and Mycobacteriaceae. The family Streptomycetaceae was dominant (43.2%) in
the CRS4 system while in the CRS1 system, the Microbacteriaceae family was dominant (46.7%).
In both systems, the actinomycetes were still the most highly represented sequences (53.3% in
CRS1 and 88.3% in CRS4) but also non- actinomycetes 16S rRNA gene sequences were found(46.7% in CRS1 and 14.2% in CRS4), which were associated with the groups of proteobacteria,
verricomicrobia and firmicutes. Xuan et al (2007) found that the family Microbacteriaceae waspresent in rice straw decomposing in soil of two experimental sites in the Mekong Delta while
Hesham et al (2006) showed Streptomyces as the dominant actinomycetes genus in rice straw
decomposition although also members of the genera Nocardiopsis, Micromonospora andNocardioides were present.
Three main conclusions can be taken from our study. First, the actinomycetes community
seems to be dynamic in function of time, both during the growth of a particular crop and ofdifferent crops. This can be explained by changes in substrate (i.e., the rice straw) composition
during decomposition (Lynch and Harper, 1985; Aulakh et al., 1991).

A

B

C

Fig. 2 16S rRNA gene DGGE fingerprints of the actinomycetes community colonizing ricestraw residues in 3 replicate plots undergoing the studied crop rotation systems at 14days (left), 50 days (middle) and harvest (right) of crops I (A), II (B) and III (C)Lanes 1-3: replicate plots undergoing system CRS1; lanes 4-6: replicate plots undergoing system CRS2; lanes
7-9: replicate plots undergoing system CRS3; lanes 10-12: replicate plots undergoing system CRS4. Lane L:
reference bacterial 16S rRNA gene ladder.

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Alternatively, the different environmental conditions implemented when different crops arecultivated can be a reason. Different crop species might produce different types of root exudates
which supply nutrients to the microbial community (Miethling et al., 2000; Smalla et al., 2001;
Marschner et al., 2001).

A

B

C

Fig. 3 UPGMA clustering of actinomycetes 16S rRNA gene DGGE profiles recovered fromrice straw residues in replicate plots undergoing the different crop rotation systems
during cultivation of crops I (A), II (B) and III (C) at 14 days of incubation (left), 50
days of incubation (middle) and harvest (right), respectively
Indicated numbers correspond with lane numbers used in Fig. 3.

Second, the main factor which drives actinomycetes community composition in the different
crop rotation systems seems to be whether the soils were saturated and unsaturated with water, i.e.,
whether aerobic and anaerobic conditions were implemented. Indeed, during growth of crop I,
profiles from systems CRS1, CRS2 and CRS3 clustered in one group different from profiles ofsystems CRS4. Crop I was rice for systems CRS1, CRS2 and CRS3 cultivated under saturated soil
conditions while it was an upland crop for system CRS4 cultivated under unsaturated soil
conditions.
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100%
90%
80%
Percentage of clones

Nonactinomycetes
70%

Catenulisporaceae
Streptomycetaceae

60%

Mycobacteriaceae

50%

Promicromonosporaceae

40%

Microbacteriaceae
Uncultured Actinomycetes

30%

Nocardioidaceae
20%
10%
0%
A

B

C

D

Crops and CRSs typeFig. 4 Fractions of recovered 16S rRNA gene sequences divided among different relevantbacterial families
The codes A and B indicate 16S rRNA gene libraries recovered from samples taken at 50 days of cultivation ofcrop I in rotation system CRS1 and CRS4, respectively. Codes C and D indicate 16S rRNA gene libraries
recovered from samples taken at 50 days of cultivation of crop II in rotation systems CRS1 and CRS4,
respectively.

Previously, Reichardt et al. (2001) showed a negative correlation between the biomass of fungi,
as primary microbial decomposers of crop residues, in soil and the soil-water content. Moreover,micro-site differences of aerobic or anaerobic conditions such as those influenced by therhizosphere and residue decomposition were previously shown to affect the makeup of a soil
microbial community (Lynch and Harper, 1985; Aulakh et al., 1991). However, no data exist on theeffect of soil moisture content on Actinomycetes community structure.
As a third conclusion, differently structured actinomycetes communities were also observed
between systems CRS4 on the one hand and systems CRS1, CRS2 and CRS3 on the other hand,during growth of crop II which was rice cultivated in water saturated soil in all systems. This was
especially the case at day 14 of sampling while at day 50 and especially at harvest, majordifferences in community profiles disappeared. The differences in actinomycetes community
profiles between systems CR4 and CRS1 during growth of crop II was also shown by the 16S
rRNA gene library analysis. This might indicate that the type of rotation system affects theactinomycetes community profile despite similar cultivation conditions. It is well-accepted thatcrop rotations have an important effect on soil microbiology. Several studies indicated that crop
rotation tend to result in a higher microbial diversity and/or biomass (Zelles et al., 1992, 1995;
Drijber et al., 2000; Larkin , 2003; Collins et al., 1992; Kirchner et al., 1993; Olsson andGerhardson, 1992). However, studies on the effect of crop rotation directed to the actinomycetes
are rare. Martyniuk and Wagner (1978) found a higher number of actinomycetes in a rotation
system including maize, oats, wheat and red clover than in continuous systems of maize or wheat.
Similarly, Kirchner et al. (1993) found that the number of actinomycetes is significantly higher insoil undergoing maize-crimson clover rotation than in soil undergoing continuous maize cultivation.
On the other hand, Collins et al. (1992) reported that actinomycetes biomass was significantly
higher under monoculture wheat than under wheat-fallow rotation. On the other hand, it is possiblethat DNA of actinomycetes communities colonizing the rice straw during cultivation of crop I andwhich died off when anaerobic conditions where implemented, did remain under those conditions
and resulted into amplification.
CONCLUSIONS
Overall, our results show that the actinomycetes communities were affected by the crop rotation
systems, but that the implemented environmental conditions rather than the rotation system as such
affected the community composition. Further studies will identify more specific changes associated© ISERD

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with particular rotations and relate these changes to potential effects on disease management, crop
health, and crop productivity.
ACKNOWLEDGEMENTS
We thank all the staff of the Soil Science and Land Management Department of Cantho University forhelp in experiment design and execution and the staff of the Division of Soil and Water Management of
K.U. Leuven for advice in molecular techniques. We thank to the farmer’s extension agency for support
at the field locations. This work was supported by a grant from the VLIR-IUC CTU program (ProjectR3) and a grant from the Vietnam Overseas Scholarship Program (Project 322).

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