Comparison of rhizosphere microbial community of hybrid rice
cultivars based on phospholipids fatty acid analysis
Y. Zhu1#, G. Hu1#, B. Liu1*, H. Xie2, X. Zheng1 & J. Zhang
1 Agricultural Bio-resources Institute, Fujian Academy of Agricultural Sciences, Fujian, China
2 Fuzhou Sub-center of National Center of Rice Improvement and Rice Research Institute, Fujian
Academy of Agricultural Sciences, Fujian, China.
# These
authors contributed equally to this work.
* Correspondence
B. Liu, Agricultural Bio-resources Institute, Fujian Academy of Agricultural Sciences, Fuzhou 350003,
Fujian, China
Tel.: +86 591 87864601
Fax: +86 591 87864601
Email: liubofaas@163.com
Number of figures: 5
Number of tables: 3
1
Abstract
This paper deals with the variation in productive characteristics and rhizosphere microbial community structure
among Chinese hybrid rice cultivars. The intrinsic relationship between variety and the rhizosphere microbial
community structure of rice was also analyzed. Three series of new-breeding Chinese hybrid rice cultivars were
tested in the experiment, IIyouming86 (II-32A/Minghui86), IIyouhang1hao (II-32A/Hang1hao) and
IIyouhang2hao (II-32A/Hang2hao) with II-32A as female parent, XinyouHK02 (XinA/HK02) and YiyouHK02
(YXA/HK02)
with
HK02
as
male
parent,
Chuanyou167
(ChuanxiangA/MR167)
and
44you167
(Hunan44A/MR167) with MR167 as male parent. The microbial community structures in rhizosphere of
different hybrid rice cultivars were determined with phospholipid fatty acids (PLFA) analysis. The results
showed that 40 PLFA biomarks were detected in all, The PCA of the PLFA composition showed that the first
two principal components PC1 and PC2 account for 69.62% and 17.82% of the variation. Bacterial PLFAs were
more abundant than fungal and actinomycetic PLFAs in paddy soil of the hybrid rice tested. Both
sulfate-reduсing and methane-oxidizing bacteria PLFAs were found to exist in the hybrid rice rhizosphere. It was
observed that microbial biomass revealed as PLFAs amount had positive correlation with the rice grain number
per spike (R2=0.801**), seed setting rate (R2=0.845**) and yield (R2=0.909**), and had negative correlation
with the rice plant height (R2=-0.969**). Based on the characteristics of PLFAs and the biological traits of rice,
the results of cluster analysis suggested that microbial community structure and activity in rhizosphere were
associated with genetic background of the rice cultivar.
Key words Rice · Productive characteristics · Rhizosphere Microorganism · Phospholipid Fatty Acid
Running title
Introduction
Rice (Oryza sativa L.) is a staple food in China, contributing 40% to the total calorie intake of Chinese people.
However, rapid population growth and economic development had been posing a growing pressure for food
production (Zhang, 2007). In addition, the continuing reduction of cultivated land area and the serious lack of
water resource during the past three decades appealed to develop and extend super rice varieties or hybrids with
wide adaptation and super high yielding potential (Chen et al., 2007). To meet the need of the food consumption,
hybrid rice that has a yield advantage of 10-20% over conventional varieties was developed and commercially
grown, and has commanded about 50% of the total rice area in China. Now, the rice yield has risen to about 6.0 t
ha-2 that was 2.0 t ha-2 and 3.5 t ha-2 in the 1960s and 1970s (Cheng et al., 2007). Recently, super hybrid rice has
been generally exploited through systematic researches of growth and development, dry matter production,
tillering ability, root activity, optimum locations and seasons, high yielding path and the technique system for
super hybrid rice, the physiological basis of high yielding formation, cultivation environments and agronomy
techniques under the cultivation conditions of single seedling and sparse planting and increase in nitrogen
fertilizer (Wang et al., 2002).
It is well known that soil microorganisms that colonize the rhizosphere assist plants in the uptake of several
vital nutrients, such as phosphorous, potassium and nitrogen, from the soil (Cocking, 2003; Ikeda et al., 2006).
The rice field is a unique agro-ecosystem, where the field is maintained under flooded conditions during most of
the period of rice cultivation, and is left under drained conditions during the off-crop season (Kimura and
Asakawa, 2006). Numerous studies have been carried out on microbial community structure of irrigated rice
2
system, and most of them were focused on polluted soil, soil management practices, nutrient cycling and ecology,
and various habitats (Kong et al., 2008; Zhang et al., 2007). However, there were quite few studies on the soil
microbial community structure of different hybrid rice cultivars. The composition and amount of
microorganisms presented in the rhizospheres of rice cultivars might differ due to variations in the quantity and
quality of compounds exuded by the different plants.
Recent methodological advanced such as analysis of DNA and the phospholipid fatty acids (PLFAs) as well as
cultivation on Biolog Gram-negative (GN)-plates allowed us to obtain more detailed information on soil
microbial activities and community structures (Zhang, 2007). Polar lipids in soil microbes were primarily
phospholipids. Thus determination of PLFAs could provide a quantitative measurement of microbial biomass
and information on community structure composition of microorganism with specific PLFAs markers
(Mubyana-John et al., 2007). For example, (Zhang et al., 2007) investigated the microbial communities under
irrigated rice cropping with different fertilizer treatments by using PLFA profile method. Kimura and Asakawa
(2006) (Kimura and Asakawa, 2006) compared the community structures of microbiota at main habitats in rice
field ecosystems based on phospholipids fatty acid analysis. Lu et al. (2007) (Lu et al., 2007) revealed the spatial
variation of active microbiota in the rice rhizosphere by in situ stable isotope probing of phospholipid fatty acids.
The present study compared productive characteristics of seven new Chinese hybrid rice cultivars, in which
three of them were from a same female parent and four from two male parents. The rhizosphere microbial
community structures of different rice cultivars were also studied by measuring the PLFAs compositions.
Furthermore, the inherent correlation among the cultivar characteristics, the community structure and cultivar
were evaluated, such as the rice growth ability and the genetic relationship. Our findings are helpful to our
further understanding of hybrid rice cultivation and varietal improvement in China.
Materials and Methods
Field experiment
The trials were conducted at No.3 field of Rice Experimental Station, Rice Research Institute, Fujian Academy
of Agricultural Sciences, Shaxian, Fujian, China. Shaxian (26°24’N, 117°48’E, 119 m above sea level) is in the
Subtropical Zone and continental monsoon area with the average annual temperature about 15.6°C-19.6°C and a
frost-free period of 270-300 days. The annual precipitation is about 1,661.9 mm with above 50% in May and
June. The field area was 2,001 m2 with 2 m protection rows around and the experiment was carried out from
May to September, 2008. Three series of hybrid rice cultivars were used in this experiments, IIyouming86
(IIyou/Minghui86), IIyouhang1hao (IIyou/Hang1hao) and IIyouhang2hao (IIyou/Hang2hao) with II-32A as
female parent, XinyouHK02 (XinAn/HK02) and YiyouHK02 (YiXiangA/HK02) with HK02 as male parent,
Chuanyou167 (ChuanxiangA/MR167) and 44you167 (Hunan44A/MR167) with MR167 as male parent.
The rice seeds were sown on sowing 16th May under dry condition in greenhouse, and the seedlings were
transplanted on 26th June. Plot size was 3.6 m2 with row length of 4 m, plant-to-plant of 13 cm and row-to-row
spacing of 30 cm in each plot. The plants were singly cultivated. The experiment was set up in a randomized
block design with three replications. Seven rice cultivars had total 21 plots. Standard cultivation practices as
commonly performed in the area were followed in all experimental plots. For yield estimate, early-maturing rice
cultivars were harvested on 25th September, while the middle-maturing cultivars on 22nd October and
3
late-maturing cultivars on 8th November. Ten plants at the center of each plot were selected and the agronomic
parameters were recorded, including grain number per spike, seed setting rate (%), plant height (cm) and paddy
yield (kg 666 m-2).
Soil sampling
For PLFAs analysis, sampling was conducted on 27th July at rice booting stage by root-shaking method. After
the plant with root was dug out, the soil combining loose on the root were removed by shaking. And then, the
soil tight attached on the root within 0-4 mm was brushed as rhizosphere soil sample. Five plants were sampled
by quincunx-sampling method in each plot. The soil samples of each plot were mixed and transported by plastic
bag to the laboratory at the same day. Each rice cultivar had three replications. The rhizosphere samples were
air-dried at room temperature till the soil moisture was in the range of 25-30%. Afterwards, the samples were
filtered with 2 mm sieve and then maintained at -80°C until phospholipid extraction.
PLFA analysis
The phospholipid fatty acids extraction procedure employs a mild alkaline methanolysis method developed by
Dr. Rhae Drijber, University of Nebraska, Lincoln, NE (Schutter and Dick, 2000). The particular steps were as
follow. In the first step, 15 ml of 0.2 M KOH in methanol were added to a 50-ml Teflon-lined, screw-cap glass
centrifuge tube containing 3 g of soil. The contents of the tubes were mixed and incubated at 37°C for 1 h,
during which ester-linked fatty acids were released and methylated. Samples were vortexed every 10 min during
the incubation period. In the second step, 3 ml of 1.0 M acetic acid were added to neutralize the pH of the tube
contents. PLFAs were partitioned into an organic phase by adding 10 ml of hexane followed by centrifugation at
2000 r min-1 for 15 min. and then the hexane was transferred to a clean glass test tube and evaporated under a
stream of N2. Finally, PLFAs were dissolved in 0.5 ml 1:1 hexane:methyl-tert butyl ether and transferred to a GC
via and kept at 4°C until analysis.
All samples were analyzed on automated Sherlock® Microbial Identification System (MIDI, Newark, DE,
USA). The system is based on GC-FID platform employing HP 6890 Series GC with equivalent column ULTRA
2 (25 m × 0.2 mm × 0.33 μm) operated under default conditions. The shorthand nomenclature common in
biochemistry and fully supported by the Sherlock® & MIDI was used for identification of the fatty acids in the
form as <number of carbon atoms>:<number of double bonds> ω <position of double bonds from methyl end of
molecule>. Prefixes i, a and cy are used for iso-, anteiso and cyclopropyl- fatty acids. Hydroxy groups are
indicated by ‘OH’. 10Me denotes a methyl group on the 10th carbon from carboxylic end of molecule. Methyl
nonadecanonate (C19:0) was used as the internal standard and the PLFAs were expressed as equivalent peak
responses to the internal standard. The total microbial biomass was expressed as µg PLFAs g-1 dry weight soil.
PLFAs that correspond to carbon chain lengths of 12-20 carbons are generally associated with microorganisms.
PLFAs used as markers for microorganism are list in Table 1.
4
Table 1 PLFAs used as markers for microorganism
microorganism
PLFAs maker
Reference
bacteria
i15:0, a15:0, 15:0, 16:0, 16:1ω5, 16:1ω9, i17:0, a17:0,
(Frostegfird and Baath, 1996; Hill et al., 2000; Myers
17:0, 18:1ω7t, 18:0, 18:1ω5, i19:0, a19:0
et al., 2001; Waldrop et al., 2000)
Fungal
18:1ω9с,
18:3ω6с(6,9,12),
18:2ω6c,
18:3ω6c,
(Kourtev et al., 2002; Olsson et al., 1999)
18:3ω3c, 16:1ω9с
Actinomycetic
10Me 17:0, 10Me18:0, 10Me16:0
(Kourtev et al., 2002)
Gram-positive bacteria
i14:0, a14:0, i15:0, a15:0, i16:0, a16:0, i17:0, a17:0,
(Kourtev et al., 2002; Waldrop et al., 2000)
10Me 17:0, i18:0
Gram-negative bacteria
12:0, 14:0, , 15:0 3OH, i15:0 3OH, 15:0 2OH, 16:1
(Kourtev et al., 2002; Waldrop et al., 2000)
2OH, i17:0 3OH, 17:0, 17:1ω8с, cy17:0, 18:1ω5с,
18:1ω7с, i18:1 H, cy19:0ω8с
Mycorrhizal
16:1ω5, 16:1ω5c, 18:2ω6c, 18:2ω6c, 18:2ω9c
(Balser and Firestone, 2005; Hinojosa et al., 2005)
Sulfate-reducing bacteria
10Me16:0, i17:1ω7c, 17:1ω6c
(Kourtev et al., 2002)
Methane-oxidizing bacteria
16:1ω8c,
16:1ω8t,
16:1ω5с,
18:1ω8c,
18:1ω8t,
(Hill et al., 2000)
18:1ω6c
Aerobes
16:1ω7t, 16:1ω7c
(Hill et al., 2000)
Anaerobes
cy19:0 cy17:0
(Hill et al., 2000)
Protozoa
20:2ω6,9,c, 20:3ω6,9,12c, 20:4ω6,9,12,15c
(Kourtev et al., 2002)
Desulfosporomusa polytropa gen
14:1ω5с
(Sass et al., 2004)
Arthropoda
20:1ω9
(Haubert et al., 2008)
Statistical analysis
Analysis of variance (ANOVA) was performed by using Fisher’s least significant difference comparison of
means (LSD). In order to find out the predominant PLFA in the rice rhizosphere, the content of individual fatty
acid methyl esters was analyzed with Principal component analysis (PCA). Mean coordinates of individuals were
calculated for the first two principal components (PC1 and PC2). PLFAs were assigned to the positive and
negative parts of the principal components according to the sign of their eigen values. To study the variation
among productive characteristics of different hybrid rice cultivars, the productive parameters of rice were
submitted to hierarchical cluster analysis with unweighted pair-group mean average method (UPGMA) by using
the seven rice cultivars as samples, the productive characteristics as indexes, and Lance-William distance as
similarity scale. To compare the rhizosphere microbial community structures of the tested rice, the same cluster
process was carried out by using the seven rice cultivars as samples and the contents of PLFAs as indexes. The
correlation analysis was conducted using the PLFAs’ content and the plant characteristics (grain number per
spike, seed setting rate, paddy yield and plant height) of rice as indexes. The procedure was introduced
Spearman index as the correlation index. All the statistical analysis was performed using software SPSS version
17, Chicago, Ill.
Results and Discussion
Productive characteristics
The productive characteristics of the seven new hybrid rice cultivars tested are listed in Table 2. Field
experiment showed that the rice cultivar Chuanyou167 and 44you167 of you167 series had significant higher
5
grain number per spike, seed setting rate and yield than three rice cultivars of IIyou series and two cultivars of
HK02 series. However, the plant heights of HK02 series were highest, following by IIyou series; the shortest
were those of you167 series. For example, 44you167 had grain number per spike, seed setting rate, yield and
plant height as 180.71, 94.88%, 8948.40 kg ha-1 and 110 cm, while YiyouHk02 had those parameters as 123.50,
72.71%, 7564.50 kg ha-1 and 130 cm.
Table 2 Productive characteristics of seven new Chinese hybrid rice cultivars
Hybrid rice cultivars
Productive characteristics
Yield (kg ha-1)
Grain number per spike
Seed setting rate (%)
Plant height (cm)
IIyouming86
145.17± 8.38 ab
79.96±4.62 ab
123±7.10 a
8000.85±462.00 b
IIyouhang1hao
141.03± 8.14 ab
75.23±4.34 ab
125±7.22 a
7900.80±456.15 b
IIyouhang2hao
143.14± 8.26 ab
74.72±4.31 ab
122±7.04 a
7802.55±450.45 bc
XinyouHK02
128.22± 7.40 b
70.48±4.07 b
130±7.51 a
7748.25±447.30 bc
YiyouHK02
125.50± 7.25 b
72.71±4.20 ab
125±7.22 a
7564.50±436.65 c
Chuanyou167
172.78± 9.98 a
92.70±5.35 a
112±6.47 b
8609.40±496.95 a
44you167
180.71±10.43 a
94.88±5.48 a
110±6.35 b
8948.40±516.60 a
*The data in the table represented means, different letters in a column indicated significant difference among rice cultivars（LSD，P<0.05）
PLFAs detected in rhizosphere
The rhizosphere of each hybrid rice cultivar contained various PLFAs composed of saturated, unsaturated,
methyl-branched and cyclopropane fatty acids (Table 3). Forty PLFAs with chain lengths ranging from C12 to
C20 were identified, including the PLFA biomarks of 16:0 and 18:0 indicative of bacteria, 10Me18:0, 10Me16:0
indicative of actinomycetes, 18:1ω9с, 18:3ω6с(6,9,12) and 16:1ω9с indicative of fungi, i14:0, a14:0, i15:0,
a15:0, i16:0, a16:0, i17:0, a17:0, 10Me 17:0, i18:0 indicative of gram-positive bacteria, 12:0, 14:0, 15:0 3OH,
i15:0 3OH, 15:0 2OH, 16:1 2OH, i17:0 3OH, 17:0, 17:1ω8с, cy17:0, 18:1ω5с, 18:1ω7с, i18:1 H, cy19:0ω8с
indicative of gram-negative bacteria, 16:1ω5с indicative of methane-oxidizing bacteria, 10Me16:0 indicative of
sulfate-reducing bacteria, 14:1ω5с indicative of Desulfosporomusa polytropagen, 20:1ω9 indicative of
Arthropoda, 20:4ω6,9,12,15с indicative of protozoa, and 15:1 ISO G, 16:0 N ALCOHOL, 16:1 ISO G, 11Me
18:1ω7с, 20:0 indicative of all non-special microbial.
Table 3 PLFAs detected in rhizosphere of seven new Chinese hybrid rice cultivars
PLFAs*
Contents of PLFAs in rhizosphere of hybrid rice (µg/g)
Ⅱyouming86
Ⅱyouhang1hao
Ⅱyouhang2hao
XinyouHK02
YiyouHK02
Chuanyou167
44you167
12:0
34.10±1.97 c
46.83±2.70 b
54.02±3.11 b
26.71±1.54 c
35.39±2.04 c
46.82±2.70 b
119.67±6.91 a
14:0
82.80±4.78 c
100.18±5.78 bc
121.68±7.03 b
96.37±5.56 bc
116.04±6.70 b
107.82±6.23 bc
412.03±23.79 a
0.00±0.00 c
0.00±0.00 c
0.00±0.00 c
26.77±1.55 b
0.00±0.00 c
0.00±0.00 c
90.39±5.22 a
i14:0
31.63±1.83 c
42.36±2.45 bc
54.15±3.13 b
40.33±2.33 bc
46.40±2.68 bc
46.03±2.66 bc
153.74±8.88 a
a14:0
30.25±1.75 c
60.77±3.51 b
59.99±3.46 b
59.60±3.44 b
67.85±3.92 b
49.27±2.84 bc
274.40±15.84 a
15:0 2OH
25.65±2.57 a
0.00±0.00 b
0.00±0.00 b
0.00±0.00 b
0.00±0.00 b
0.00±0.00 b
0.00±0.00 b
14:1ω5с
15:0 3OH
57.46±3.32 b
61.91±3.57 b
71.35±4.12 b
51.70±2.98 b
0.00±0.00 c
48.48±2.80 b
355.54±20.53 a
15:1 ISO G
211.03±12.18 a
29.80±1.72 c
32.05±1.85 c
32.45±1.87 c
38.95±2.25 c
0.00±0.00 d
110.44±6.38 b
i15:0
241.94±13.97 ba
298.14±17.21 a
306.80±17.71 a
236.47±13.65 c
302.35±17.46 a
0.00±0.00 b
0.00±0.00 b
0.00±0.00 b
0.00±0.00 b
i15:0 3OH
0.00±0.00 b
a15:0
153.50±8.86 c
215.53±12.44 bc
16:0
830.64±47.96 b
967.73±55.87 ab
212.20±12.25 bc
1088.41±62.84 a
6
282.18±16.29 ab
0.00±0.00 b
190.77±11.01 bc
248.69±14.36 b
199.55±11.52 bc
815.26±47.07 b
336.72±19.44 c
917.53±91.75 b
0.00±0.00 d
166.18±16.62 a
1043.14±60.23 a
968.33±96.83 ab
PLFAs*
16:0 N
ALCOHOL
Contents of PLFAs in rhizosphere of hybrid rice (µg/g)
Ⅱyouming86
42.52±2.45 e
i16:0
142.42±8.22 b
a16:0
309.14±17.85 b
10Me16:0
232.16±13.40 b
16:1ω5с
149.62±8.64 bc
16:1ω9с
30.71±1.77 cd
16:1 2OH
61.76±3.57 d
0.00±0.00 c
17:0
i17:0
16:1 ISO G
i17:0 3OH
a17:0
17:1ω8с
cy17:0
18:0
61.10±3.53 de
186.12±10.75 b
104.83±6.05 cd
251.58±14.53 b
188.65±10.89 b
Ⅱyouhang2hao
XinyouHK02
129.53±7.48 b
90.70±5.24 c
180.65±10.43 b
143.66±8.29 b
95.03±5.49 cd
98.43±5.68 cd
286.98±16.57 b
182.18±10.52 b
YiyouHK02
81.51±4.71 cd
Chuanyou167
90.10±5.20 c
258.34±14.92 a
187.41±10.82 b
164.40±9.49 b
782.76±45.19 a
143.21±8.27 c
86.74±5.01 d
634.49±36.63 a
243.29±14.05 b
214.24±12.37 b
1108.86±64.02 a
158.71±9.16 bc
130.23±7.52 c
138.46±8.00 bc
660.11±38.11 a
43.19±2.49 bc
27.14±1.57 d
44.32±2.56 b
41.02±2.37 bc
174.85±10.10 a
138.35±7.99 b
64.08±3.70 d
37.85±2.18 e
50.36±2.91 de
90.88±2.25 c
260.83±15.06 a
0.00±0.00 c
38.37±2.22 b
0.00±0.00 c
54.98±3.17 a
37.46±2.16 b
43.26±2.50 d
89.06±5.14 b
56.95±3.29 cd
44.70±2.58 d
65.60±3.79 c
74.09±4.28 bc
104.03±6.01 b
122.68±7.08 b
86.03±4.97 b
122.39±7.07 b
36.27±2.09 bcd
118.47±6.84 b
0.00±0.00 c
243.56±14.06 a
103.69±5.99 b
584.31±33.74 a
0.00±0.00 b
0.00±0.00 b
0.00±0.00 b
0.00±0.00 b
0.00±0.00 b
0.00±0.00 b
161.92±16.19 a
95.26±5.50 c
130.30±7.52 bc
126.98±7.33 bc
114.34±6.60 c
182.30±10.53 b
115.08±6.64 c
857.78±49.52 a
139.04±8.03 b
36.71±2.12 d
58.40±3.37 c
30.51±1.76 d
46.07±2.66 cd
41.06±2.37 cd
214.20±12.37 a
60.93±3.52 bc
74.38±4.29 b
46.76±2.70 c
69.00±3.98 bc
60.79±3.51 bc
324.02±18.71 a
236.09±13.63 a
42.75±2.47 c
179.34±10.35 c
60.32±3.48 b
221.75±12.80 bc
38.38±2.22 c
53.85±3.11 bc
46.08±2.66 bc
262.93±15.18 b
50.87±2.94 bc
164.43±9.49 c
220.81±12.75 bc
201.51±11.63 bc
0.00±0.00 c
0.00±0.00 c
18:1ω5с
0.00±0.00 c
18:1ω7с
559.92±32.33 a
271.94±15.70 cd
352.76±20.37 b
228.82±13.21 d
296.56±17.12 bc
297.92±17.20 bc
18:1ω9с
482.63±48.26 c
551.77±55.18 b
673.05±67.31 a
444.78±44.48 c
457.36±45.74 c
540.40±54.04 b
118.57±6.84 b
73.11±4.22 d
112.69±6.51 bc
109.04±6.30 bc
18:3ω6с(6,9,12)
44you167
148.98±8.60 bc
55.09±3.18 bc
10Me 17:0
Ⅱyouhang1hao
0.00±0.00 c
0.00±0.00 c
2465.77±246.58 a
875.16±50.53 a
1149.82±114.98 b
0.00±0.00 e
0.00±0.00 d
81.55±4.71 cd
92.88±5.36 bcd
i18:0
0.00±0.00 c
0.00±0.00 c
0.00±0.00 c
0.00±0.00 c
36.69±3.67 b
29.44±2.94 b
111.90±11.19 a
i18:1 H
0.00±0.00 b
0.00±0.00 b
0.00±0.00 b
0.00±0.00 b
0.00±0.00 b
0.00±0.00 b
106.65±10..67 a
187.88±10.85 b
97.52±5.63 c
141.94±8.19 bc
112.08±6.47 c
604.80±34.92 a
10Me18:0
141.08±8.15 c
11Me 18:1ω7с
244.74±14.13 a
cy19:0ω8с
221.33±12.78 c
20:0
20:1ω9с
20:4ω6,9,12,15с
Total
143.08±8.26 bc
40.82±2.36 cd
274.94±15.87 c
48.40±2.79 c
28.97±1.67 d
51.77±2.99 c
248.08±14.32 c
182.55±10.54 c
238.23±13.75 c
40.99±2.37 cd
825.61±47.67 b
420.61±24.28 a
116.44±6.72 b
1062.93±61.37 a
98.54±5.69 cd
114.26±6.60 bc
130.58±7.54 b
74.53±4.30 d
75.54±4.36 d
90.70±5.24 cd
0.00±0.00 b
34.12±3.41 a
0.00±0.00 b
0.00±0.00 b
0.00±0.00 b
0.00±0.00 b
304.19±17.56 a
0.00±0.00 b
85.39±4.93 bc
93.13±5.38 b
77.28±4.46 bcd
68.50±3.95 cd
64.69±3.73 d
60.99±3.52 d
186.26±10.75 a
5241.28
5128.84
5573.04
4039.03
4363.19
7776.22
15134.74
*The data in the table represented means, different letters in a row indicated significant difference among soils of rice cultivars（LSD，P<0.05）
Rhizosphere Soil microbial community structure
An amount of bacteria that obtained gram-positive bacteria, gram-negative bacteria, sulfate-reducing bacteria
and methane-oxidizing bacteria, also a number of fungi, actinomycetes, arthropoda and protozoa were living in
the rhizosphere of rice based on the outcome of PLFAs above. PLFAs data were processed using the principal
component analysis (PCA). Results presented in Fig. 1. PCA produced two principal components (PC) which
accounted for 87.44% of the total variability. PC1 accounted for 69.62% of the variance and PC2 was
responsible for explaining 17.82% of the variation, respectively. PLFAs 16:0, 18:1ω7с, 18:1ω9с and 10Me16:0
were positively correlated with PC1. PLFAs a15:0, i16:0, a16:0, i17:0 3OH, i18:0 and i18:1 H were highly
negatively correlated with PC1. PLFAs 15:0 2OH, i15:0 3OH, i17:0 3OH, i18:0 and i18:1 H were highly
positively correlated with PC2. PLFAs 12:0, 14:1ω5с, 15:0 2OH, i15:0, 16:1 ISO G and 20:1ω9с were highly
negatively correlated with PC2.
7
Fig.1 Principal component analysis of individual PLFA
In general, 16:0, 18:1ω9с and 10Me16:0 which represented bacteria, fungi and actinomycetes were chiefly
responsible for the variances, which indicated they were the primary compositions on the microbial community.
Therefore, the contents of PLFAs 16:0, 18:1ω9с and 10Me16:0 could be used as the abundant indexes of
bacteria, fungi and actinomycetes to analyze the structure of the microbial community in rice rhizosphere. The
rhizosphere microbial community structures of different hybrid rice cultivars were showed in Fig. 2. On the
whole, bacteria were most predominant followed by fungi and actinomycetes as the second and third abundant
microorganism in the rice rhizosphere.
Fig.2 The microbial community structure in rhizosphere of seven hybrid rice cultivars by using 16:0,
18:1ω9с and 10Me l7:0 as measures of biomasses for bacteria, fungi and actinomycetes,
respectively.
8
Rice field soils represent anaerobic freshwater habitats. Shortly after flooding of the fields the oxygen in the
bulk soil is consumed by respiration. Subsequently anaerobic processes such as denitrification, ferric iron
reduction, sulfate reduction and methanogenesis are the terminal steps in the degradation of organic matter
(Scheid and Stubner, 2001). Therefore, the sulfate-reducing bacteria and methane-oxidizing bacteria in
rhizosphere of different rice cultivars were compared with 10Me16:0 and 16:1ω5с as indicators (Fig. 3). The
sulfate reducers were more plentiful than methabitriphs in all the treatments. The rhizosphere of MR167 series
rice contained higher amounts of both of sulfate-reducing and methane-oxidizing bacteria than HK02 and
MR167 series.
Fig.3 The specific microbe in rhizosphere of seven hybrid rice cultivars by using 10Me 16:0 and
16:1ω5с as measures of biomasses for sulfate-reduсing bacteria and methane-oxidizing bacteria,
respectively.
Relationship among cultivar, productive characteristics and rhizosphere microbial community structure
of rice
By using unweighted pair-group mean average (UPGMA) method, cluster analysis results of the seven hybrid
rice cultivars based on productive characteristics (Table 2) and on soil PLFAs analysis (Table 3) were performed
in Figure 4. The seven rice cultivars could be divided into two groups at 0.18 of according to their biological
features (Fig. 4a). The first group contained the two subgroups. One subgroup was the IIyou series that included
IIyouming86, IIyouhang1hao and IIyouhang2hao with middle grain number per spike (143-146), seed setting
rate (74-80%) and yields (7800-8000 kg ha-1) as well as middle plant height (122-125 cm); the other subgroup
was HK02 series that obtained XinyouHK02 and YiyouHK02 with grain number per spike (<130), seed setting
rate (<72.72%) and yields (<7800 kg ha-1) as well as highest plant height (>125 cm). The second group was
you167 series with highest grain umber per spike (>172), seed setting rate (>92%) and yields (>8600 kg ha-1) as
well as lowest plant height (<112 cm). The results indicated that the productive characteristics of rice in relation
to plant variety and their genetic background.
Similarly, the statistic results based on PLFAs analysis showed that the tested rice cultivars could be also
9
clustered into two groups at 3.68 of Lance-William distance (Fig. 4b). The first group consisted of two
subgroups, included rice cultivars of HK02 series and IIyou series, respectively. The hybrid rice Chuanyou167
and 44you167 of you167 series were involved in the second group. Because the PLFAs composition reflected
the microbial status in soil, it could be concluded that the microbial biomass and community structure in
rhizosphere of rice cultivars in relate to hybrid genetic background. For example, HK02 series rice had of lowest
PLFAs biomasses (<5000 µg g-1), while those of IIyou series had middle values of PLFAs biomasses
(5100-5600 µg g-1) and you167 series and highest PLFAs biomasses (>7000 µg g-1).
On the other hand, based on the data showed in Table 2 and 3, the amount of PLFAs biomasses in the
rhizosphere of hybrid rice was found to had positive correlation with the rice grain number per spike
(R2=0.801**), seed setting rate (R2=0.845**) and yield (R2=0.909**), and had negative correlation with the rice
plant height (R2=-0.969**).
a
b
Lance-William distance
Lance-William distance
Fig4. Cluster analysis of seven new Chinese hybrid rice cultivars based on productive characteristics (a) and
PLFAs (b) by using unweighted pair-group mean average (UPGMA) method
Discussion
Heterosis has been successfully exploited on a large scale in rice, which is a self-pollinated crop. The selection
of parental lines plays a vital role in developing ideal combinations (Wang et al., 2006). In this study, the seven
hybrid rice cultivars that bred by different parents performed high varied in their productive traits, which
indicated the genetic diversity in hybrid breeding. However, the cluster analysis mentioned that genetic
background had remarkable effect on the rice agronomic characteristics. For example, rice cultivars of
Chuanyou167 and 44you167 derived from male parent MR167 had significant higher yield and lower plant
height than the other three rice cultivars bred from female parent IIyou and two rice cultivars from male parent
HK02.
Owing to leakage of O2 and organic substances from roots, the rice roots and the rhizosphere provide niches for
diverse organisms performing various biogeochemical processes (Kimura and Asakawa, 2006). The productivity
of soil system is known to depend greatly upon the structure and functions of soil microbial communities, which
regulate and influence many ecosystem processes such as nutrient transformation, litter decomposition, soil
structure and plant health (Garbeva et al., 2004). By using PLFAs as specific markers, gram-positive bacteria,
gram-negative bacteria, methane-oxidizing bacteria, sulfate-reducing bacteria, fungi, actinomycetes, Arthropoda
（节肢动物也算微生物？）and protozoa were detected at paddy soil in this experiment. In generally, bacteria
10
were most abundant in rice rhizosphere than fungi and actinomycetes. Both fungi and actinomycetes play a
major role in decomposition of organic residues high in cellulose and lignin (Mubyana-John et al., 2007).
Therefore, it could be concluded that bacteria was crucial in nutrient holding capacity for hybrid rice.
Bacterial communities in root-associated habitats respond with respect to density, composition and activity to
the abundance and great diversity of organic root exudates, eventually yielding plant species-specific microfloras
(Griffiths et al., 2007). (Arab et al., 2001) used PLFA method to determine the rhizosphere specific microbial
communities of two wheat cultivars, and the results notified the rhizosphere wheat cultivar Bohouth-6 involved
larger amount of Pseudomons spp. than cultivar Salamouni. (Siciliano et al., 1998) reported that the differences
in the microbial communities were associated with the roots of different cultivars of canola (rape, Brassica spp.)
and wheat (Triticum spp.). (Briones et al., 2002) compared the activities and diversities of ammonia-oxidizing
bacteria (AOB) in the root environment of different cultivars of rice and the results indicated marked difference
despite identical environment conditions during growth (Briones et al., 2002). The composition and amount of
microorganisms present in the rhizospheres of the hybrid rice cultivars tested also differed significantly, which
may due to variations in the quantity and quality of compounds exuded by the different plants (Söderberg et al.,
2002). Furthermore, because the selection of parental lines played a vital role in developing ideal hybrid rice
combinations, the identification of heterotic groups and patterns among breeding populations and lines provides
fundamental information in order to help the plant breeders to gain more information on heterosis (Chen et al.,
2007). The difference of rhizosphere microbial communities indicated by PLFAs contents was found to be in
relation to the rice hybrid genetic background, which might be used as a new approach to identify their parental
lines.
Soil microbial biomass is considered to act both as the agent of biochemical changes in soil and as a
repository of plant nutrients in agricultural ecosystems (Zhang and Wang, 2005). For instance, (Lu et al., 2006)
studied the structure and activity of bacterial community inhabiting rice roots and the rhizosphere and suggested
that cycling of iron and sulfur is active in the rhizosphere. The rice cultivar 44you167 that had highest yield was
revealed to possess most amount of microbial biomass in rhizosphere. Beside, the sulfate-reduсing bacteria and
methane-oxidizing bacteria existed more plentifully in rhizosphere of than other cultivars. Therefore, it could be
presumed that a great variety of abiotic and biotic factors shape soil- and plant-associated habitats and modify
the compositions and activities of their microbial communities, which in turn bear upon the quality of their
environment, the growth of plants, and the production of root exudates (Wieland et al., 2001). Consequently,
different rhizosphere microbial communities are associated with different plants (Kremer et al., 1990).
Acknowledgements The present investigation was supported by the National High Technology Research and
Development Program ("863" Program) of China (2006AA100101 and 2006AA10A211).
References
Arab H.G.D.E., Vlich V., Sikora R.A. (2001) The use of phospholipids fatty acids (PLFA) in the determination of
rhizosphere specific microbial communities of two wheat cultivars. Plant and Soil 228:291-297.
Balser T.C., Firestone M.K. (2005) Linking microbial community composition and soil processes in a California
annual grassland and mixed-conifer forest. Biogeochemistry 73:395-415.
11
Briones A.M., Okabe S., Umemiya Y., Ramsing N.B., Reichardt W., Okuyama H. (2002) Influence of different
cultivars on populations of ammonia-oxidizing bacteria in the root environment of rice. Appl Environ
Microbiol 68:3067-75.
Chen L.Y., Xiao Y.H., Tang W.B., Lei D.Y. (2007) Practices and Prospects of Super Hybrid Rice Breeding. Rice
Science 14:71-77.
Cheng S.H., Zhuang J.Y., Fan Y.Y., Du J.H., Cao L.Y. (2007) Progress in research and development on hybrid
rice: a super-domesticate in China. Ann Bot 100:959-66.
Cocking E.C. (2003) Endophytic colonisation of plant roots by nitrogen-fixing bacteria Plant Soil Environment
252:169-175.
Frostegfird A., Baath E. (1996) The use of phospholipid fatty acid analysis to estimate bacterial and fungal
biomass in soil. Biology and Fertility of Soils 22:59-65.
Garbeva P., van Veen J.A., van Elsas J.D. (2004) Microbial diversity in soil: selection microbial populations by
plant and soil type and implications for disease suppressiveness. Annu Rev Phytopathol 42:243-70.
Griffiths B.S., Heckmann L.H., Caul S., Thompson J., Scrimgeour C., Krogh P.H. (2007) Varietal effects of eight
paired lines of transgenic Bt maize and near-isogenic non-Bt maize on soil microbial and nematode
community structure. Plant Biotechnol J 5:60-8.
Haubert D., Ggblom M.M.H., Scheu S., Ruess L. (2008) Effects of temperature and life stage on the fatty acid
composition of Collembola. Eur J Soil Biol 44:213-219.
Hill G.T., Mitkowski N.A., Aldrich-Wolfe L., Emele L.R., Jurkonie D.D., Ficke A., Maldonado-Ramirez S.,
Lynch S.T., Nelson E.B. (2000) Methods for assessing the composition and diversity of soil microbial
communities. Appl Soil Ecol 15:25-36.
Hinojosa M.B., Carreira J.A., R. G.-R., Dick R.P. (2005) icrobial response to heavy metal-polluted soils:
community analysis from phospholipid-linked fatty acids and ester-linked fatty acids extracts. J Environ
Qual 34:1789-1800.
Ikeda S., Ytow N., Ezura H., Minamisawa K., Fujimura T. (2006) Soil microbial community analysis in the
environmental risk assessment of transgenic plants. Plant Biotechnology 23:131-151.
Kimura M., Asakawa S. (2006) Comparison of community structures of microbiota at main habitats in rice field
ecosystems based on phospholipid fatty acid analysis. Biology and Fertility of Soils 1:20-29.
Kong C.H., Wang P., Gu Y., Xu X.H., Wang M.L. (2008) Fate and impact on microorganisms of rice
allelochemicals in paddy soil. J Agric Food Chem 56:5043-9. DOI: 10.1021/jf8004096.
Kourtev P.S., Ehrenfeld J.G., Ggblom M.H. (2002) Exotic plant species alter the microbial community structure
and function in the soil. Ecology 83:3152-3166.
Kremer R.J., Begonia M.F., Stanley L., Lanham E.T. (1990) Characterization of Rhizobacteria Associated with
Weed Seedlings. Appl Environ Microbiol 56:1649-1655.
Lu Y., Abraham W.R., Conrad R. (2007) Spatial variation of active microbiota in the rice rhizosphere revealed by
in situ stable isotope probing of phospholipid fatty acids. Environ Microbiol 9:474-81.
Lu Y., Rosencrantz D., Liesack W., Conrad R. (2006) Structure and activity of bacterial community inhabiting
rice roots and the rhizosphere. Environ Microbiol 8:1351-60.
Mubyana-John T., Wutor V.C., Yeboah S.O., Ringrose S. (2007) Fire and its influence on microbial community
structure and soil biochemical properties in the Okavango Delta, Botswana. Scientific Research and
12
Essay 2:47-54.
Myers R.T., Zak D.R., White D.C., Peacock A. (2001) Landscape-level patterns of microbial community
composition and substrate use in upland forest ecosystems. Soil Sci Soc Am J 65:359-367.
Olsson P.A., Thingstrup I., Jakobsen I., Baath E. (1999) Estimation of the biomass of arbuscular mycorrhizal
fungi in a linseed field. Soil Biology and Biochemistry 31:1879-1887.
Söderberg K.K., Olsson P.A., Bååth E. (2002) Structure and activity of the bacterial community in the
rhizosphere of different plant species and the effect of arbuscular mycorrhizal colonisation. FEMS
Microbiol Ecol 40:223-231.
Sass H., Overmann J., Rütters H., D. B.H., Cypionka H. (2004) Desulfosporomusa polytropa gen. nov., sp. nov.,
a novel sulfate-reducing bacterium from sediments of an oligotrophic lake. Arch Microbiol
182:204-211.
Scheid D., Stubner S. (2001) Structure and diversity of Gram-negative sulfate-reducing bacteria on rice roots.
FEMS Microbiol Ecol 36:175-183.
Schutter M.E., Dick R.P. (2000) Comparison of fatty acid methyl ester (FAME) methods for characterizing
microbial communities. Soil Science Society of America Journal 2000. 64:5, 1659-1668.
Siciliano S.D., Theoret C.M., Freitas J.R.d., Hucl P.J., Germida J.J. (1998) Differences in the microbial
communities associated with the roots of different cultivars of canola and wheat. Canadian Journal of
Microbiology 1998. 44:9, 844-851.
Waldrop M.P., Balser T.C., Firestone M.K. (2000) Linking microbial community composition to function in a
tropical soil. Soil Biology and Biochemistry 32:1837-1846.
Wang F., Zhang H., Dai Q., Zhao X., Duan X., Xu J., Huo Z., Xu K. (2002) Study on environmental sensitivity
of variety in rice. Agricultural Sciences in China 2002. 1:6, 612-617.
Wang S., Lu Z., Wan J. (2006) Genetic diversity among parents of hybrid rice based on cluster analysis of
morphological traits and simple sequence repeat markers. Rice Science 2006. 13:3, 155-160.
Wieland G., Neumann R., Backhaus H. (2001) Variation of microbial communities in soil, rhizosphere, and
rhizoplane in response to crop species, soil type, and crop development. Appl Environ Microbiol
67:5849-54.
Zhang P., Zheng J., Pan G., Zhang X., Li L., Tippkotter R. (2007) Changes in microbial community structure and
function within particle size fractions of a paddy soil under different long-term fertilization treatments
from the Tai Lake region, China. Colloids Surf B Biointerfaces 58:264-70.
Zhang Q. (2007) Strategies for developing Green Super Rice. Proc Natl Acad Sci U S A 104:16402-9.
Zhang Q.C., Wang G.H. (2005) Studies on nutrient uptake of rice and characteristics of soil microorganisms in a
long-term fertilization experiments for irrigated rice. J Zhejiang Univ Sci B 6:147-54.
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