Yujing Zhu · Guiping Hu · Bo Liu · Xuefang Zheng · Jianfu Zhang · Huaan Xie
Variation in productive characteristics and rhizosphere microbial
community structure among new Chinese hybrid rice cultivars
Y. Zhu · G. Hu · B. Liu () · X. Zheng
Agricultural Bio-resources Institute, Fujian Academy of Agricultural Sciences, Fuzhou 350003, e-mail:
liubofaas@163.com
J. Zhang
Rice Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou, 350018
H. Xie
Subcenter of Fuzhou, National Center of Rice Improvement, Fujian Agricultural Academy of Sciences,
Fuzhou, 350018
Abstract To analyze the intrinsic relationship between rhizosphere microbial community structure and
variety of rice, the microbial community structures in rhizosphere of different hybrid rice cultivars
were determined with phospholipid fatty acids (PLFA) analysis. 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 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 · Rhizosphere Microorganism · Relationship · Phospholipid Fatty Acid · Diversity
Introduction
Rice (Oryza sativa L.) is a staple food in China, contributing 40% to the total calorie intake of
Chinese people (Cheng, Zhuang, Fan, Du, & Cao, 2007). 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(Ying-hui & Dong-yang, 2007). 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, Zhuang, Fan, Du, & Cao, 2007).
Super hybrid rice was 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 FY, Zhang HC, Dai QG,
Zhao XH, Duan XM, Xu J, Huo ZY and Xu K, 2002). The high yielding potential of super hybrid rice
was studied by using the methods of enzyme, hormone, and molecular level, as well as cultivation
techniques (Zhou Y b, Zhou S Y & Tang Q Y, 2003). Also, numerous researches had given an account
of the relationship between soil fertility and microbial biomass in rice field ecosystems(Brookes et al.,
2008; Q. ZHANG, 2007b). Most of the studies on microbial community structure were focused on
polluted soil, soil management practices, nutrient cycling and ecology, and various habitats of irrigated
rice system(Kong, Wang, Gu, Xu, & Wang, 2008) (P. Zhang et al., 2007). However, there were quite
few studies on the soil microbial biomass of different hybrid rice cultivars. The composition and
amount of microorganisms presented in the rhizospheres of different 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 structure(Q. ZHANG, 2007b). 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, Wutor, Yeboah, & Ringrose, 2007).
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 between the cultivar characteristics and
were the community structure 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.
Material 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 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 m2).
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 & 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 N 2. 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.
Statistical analysis
All the statistical analysis was performed using software SPSS version 17, Chicago, Ill. Analysis
of variance (ANOVA) was performed by using Fisher’s least significant difference comparison of
means (LSD).
The productive characteristics 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 or the detected fatty acids as indexes, and Lance-William distance as
similarity scale.
Principal component analysis (PCA) was completed using the content of individual fatty acid
methyl esters. 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.
The contents of PLFAs were subjected to the hierarchical cluster analysis to elucidate the
relationship between the PLFAs and the heredity. The process was carried out by using the seven rice
cultivars as samples, the contents of PLFAs as indexes, and Lance-William distance as similarity scale
with unweighted pair-group mean average method (UPGMA).
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 were introduced Spearman index as the correlation index.
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,
(Hill et al., 2000; Myers, Zak, White, & Peacock,
a17:0, 17:0, 18:1ω7t, 18:1ω5, i19:0, a19:0 18:0
2001;
WaldropBalser
&
Firestone,
2000)
i15:0, a15:0, 15:0, 16:0, 16:1ω5, 16:1ω9, i17:0,
(Frosteg Rd & B Th, 1996)
a17:0, 17:0, 18:1ω7t, 18:1ω5, i19:0, a19:0
Fungal
18:1ω9с,
18:3ω6с(6,9,12),
18:2ω6c,
18:3ω6c,
(Olsson, Thingstrup, Jakobsen, & B Th, 1999;
18:3ω3c, 16:1ω9с
KourtevEhrenfeld & H Ggblom, 2002)
Actinomycetic
10Me 17:0, 10Me18:0, 10Me16:0
(KourtevEhrenfeld & H Ggblom, 2002)
Gram-positive bacteria
i14:0, a14:0, i15:0, a15:0, i16:0, a16:0, i17:0, a17:0,
(WaldropBalser
10Me 17:0, i18:0
KourtevEhrenfeld & H Ggblom, 2002)
12:0, 14:0, , 15:0 3OH, i15:0 3OH, 15:0 2OH, 16:1
(WaldropBalser
2OH, i17:0 3OH, 17:0, 17:1ω8с, cy17:0, 18:1ω5с,
KourtevEhrenfeld & H Ggblom, 2002)
Gram-negative bacteria
&
&
Firestone,
Firestone,
2000;
2000;
18:1ω7с, i18:1 H, cy19:0ω8с
Mycorrhizal
16:1ω5, 16:1ω5c, 18:2ω6c, 18:2ω6c, 18:2ω9c
(Hinojosa, Carreira, Garcia-Ruiz, & Dick, 2005;
Sulfate-reducing bacteria
10Me16:0, i17:1ω7c, 17:1ω6c
(KourtevEhrenfeld & H Ggblom, 2002)
Methane-oxidizing bacteria
16:1ω8c, 16:1ω8t, 16:1ω5с, 18:1ω8c, 18:1ω8t,
(Hill et al., 2000)
Balser & Firestone, 2005)
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
(KourtevEhrenfeld & H Ggblom, 2002)
Desulfosporomusa
14:1ω5с
(Sass,
polytropa gen
Overmann,
Rütters,
Babenzien,
&
Cypionka, 2004)
20:1ω9
Arthropoda
(Haubert, H Ggblom, Scheu, & Ruess,
2008)
Results
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 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%, 596.56 kg/666m2 and
110 cm, while YiyouHk02 had those parameters as 123.50, 72.71%, 504.30 kg/666m2 and 130 cm.
Cluster analysis results of the seven hybrid rice cultivars based on productive characteristics
indicated that seven cultivars could be divided into two groups at 0.18 of Lance-William distance
(Fig1). The first group contained two subgroups. One subgroup was the IIyou series that included
IIyouming86, IIyouhang1hao and IIyouhang2hao; the other was HK02 series that obtained
XinyouHK02 and YiyouHK02. The second group was you167 series.
Table 2 Productive characteristics of seven new Chinese hybrid rice cultivars
Productive characteristics
Hybrid rice cultivars
IIyouming86
Grain number per spike
145.17± 8.38 ab
Seed setting rate (%)
79.96±4.62 ab
Plant height (cm)
123±7.10 a
Yield (kg/666 m2)
533.39±30.80 b
IIyouhang1hao
141.03± 8.14 ab
75.23±4.34 ab
125±7.22 a
526.72±30.41 b
IIyouhang2hao
143.14± 8.26 ab
74.72±4.31 ab
122±7.04 a
520.17±30.03 bc
XinyouHK02
128.22± 7.40 b
70.48±4.07 b
130±7.51 a
516.55±29.82 bc
YiyouHK02
125.50± 7.25 b
72.71±4.20 ab
125±7.22 a
504.30±29.11 c
Chuanyou167
172.78± 9.98 a
92.70±5.35 a
112±6.47 b
573.96±33.13 a
44you167
180.71±10.43 a
94.88±5.48 a
110±6.35 b
596.56±34.44 a
*The data in the table represented means, different letters in a column indicated significant difference among rice cultivars（LSD，
P<0.05）
Fig1. Cluster analysis of seven new Chinese hybrid rice cultivars based on productive characteristics by
using unweighted pair-group mean average (UPGMA) method
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
16:1ω9сindicative 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, cy19:0ω8сindicative of gram-negative
bacteria, 16:1ω5с indicative of methane-oxidizing bacteria, 10Me16:0 indicative of Sulfate-reducing
bacteria, 14:1ω5с indicative of Desulfosporomusa polytropa gen, 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 nonspecial microbial.
Twenty-six PLFAs existed in all the rice soil samples such as 14:0, i14:0 and 10Me17:0, whereas
the other 14 PLFAs presented only in part of the samples such as 14:1 ω5с, 15:0 2OH and i15:0 3OH.
Therefore, there are two types of distributing patterns were found for the PLFAs detected in the rice
rhizosphere, namely, complete distribution and incomplete distribution.
Table 3 PLFAs detected in rhizosphere of seven new Chinese hybrid rice cultivars
PLFAs*
12:0
14:0
14:1ω5с
i14:0
a14:0
15:0 2OH
15:0 3OH
15:1 ISO G
i15:0
i15:0 3OH
a15:0
16:0
16:0 N
ALCOHOL
i16:0
Contents of PLFAs in rhizosphere of hybrid rice (µg/g)
Ⅱyouhang1hao
Ⅱyouhang2hao
XinyouHK02
YiyouHK02
Ⅱyouming86
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
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
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
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
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
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
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
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
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
0.00±0.00 b
153.50±8.86 c 215.53±12.44 bc
212.20±12.25 bc 190.77±11.01 bc 248.69±14.36 b
830.64±47.96 b 967.73±55.87 ab 1088.41±62.84 a
815.26±47.07 b 336.72±19.44 c
42.52±2.45 e
61.10±3.53 de
142.42±8.22 b 186.12±10.75 b
129.53±7.48 b
180.65±10.43 b
90.70±5.24 c
81.51±4.71 cd
143.66±8.29 b 187.41±10.82 b
Chuanyou167
44you167
46.82±2.70 b
119.67±6.91 a
107.82±6.23 bc 412.03±23.79 a
0.00±0.00 c
90.39±5.22 a
46.03±2.66 bc
153.74±8.88 a
49.27±2.84 bc 274.40±15.84 a
0.00±0.00 b
0.00±0.00 b
48.48±2.80 b
355.54±20.53 a
0.00±0.00 d
110.44±6.38 b
282.18±16.29 ab
0.00±0.00 d
0.00±0.00 b
166.18±16.62 a
199.55±11.52 bc 1043.14±60.23 a
917.53±91.75 b
968.33±96.83 ab
90.10±5.20 c
258.34±14.92 a
164.40±9.49 b
782.76±45.19 a
PLFAs*
Ⅱyouming86
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
16:1 ISO G
0.00±0.00 c
17:0
43.26±2.50 d
i17:0
104.03±6.01 b
i17:0 3OH
0.00±0.00 b
a17:0
95.26±5.50 c
17:1ω8с
139.04±8.03 b
cy17:0
55.09±3.18 bc
10Me 17:0
42.75±2.47 c
18:0
179.34±10.35 c
18:1ω5с
0.00±0.00 c
18:1ω7с
559.92±32.33 a
18:1ω9с
482.63±48.26 c
18:3ω6с(6,9,12)
81.55±4.71 cd
i18:0
0.00±0.00 c
i18:1 H
0.00±0.00 b
10Me18:0
141.08±8.15 c
11Me 18:1ω7с 244.74±14.13 a
cy19:0ω8с
221.33±12.78 c
20:0
98.54±5.69 cd
20:1ω9с
0.00±0.00 b
20:4ω6,9,12,15с
85.39±4.93 bc
Total
5241.28
Contents of PLFAs in rhizosphere of hybrid rice (µg/g)
Ⅱyouhang1hao
104.83±6.05 cd
251.58±14.53 b
188.65±10.89 b
36.27±2.09 bcd
138.35±7.99 b
0.00±0.00 c
89.06±5.14 b
122.68±7.08 b
0.00±0.00 b
130.30±7.52 bc
36.71±2.12 d
60.93±3.52 bc
60.32±3.48 b
221.75±12.80 bc
0.00±0.00 c
271.94±15.70 cd
551.77±55.18 b
92.88±5.36 bcd
0.00±0.00 c
0.00±0.00 b
143.08±8.26 bc
40.82±2.36 cd
274.94±15.87 c
114.26±6.60 bc
34.12±3.41 a
93.13±5.38 b
5128.84
Ⅱyouhang2hao
95.03±5.49 cd
286.98±16.57 b
148.98±8.60 bc
43.19±2.49 bc
64.08±3.70 d
38.37±2.22 b
56.95±3.29 cd
118.47±6.84 b
0.00±0.00 b
126.98±7.33 bc
58.40±3.37 c
74.38±4.29 b
50.87±2.94 bc
262.93±15.18 b
0.00±0.00 c
352.76±20.37 b
673.05±67.31 a
118.57±6.84 b
0.00±0.00 c
0.00±0.00 b
187.88±10.85 b
48.40±2.79 c
248.08±14.32 c
130.58±7.54 b
0.00±0.00 b
77.28±4.46 bcd
5573.04
XinyouHK02
98.43±5.68 cd
182.18±10.52 b
158.71±9.16 bc
27.14±1.57 d
37.85±2.18 e
0.00±0.00 c
44.70±2.58 d
86.03±4.97 b
0.00±0.00 b
114.34±6.60 c
30.51±1.76 d
46.76±2.70 c
38.38±2.22 c
164.43±9.49 c
0.00±0.00 c
228.82±13.21 d
444.78±44.48 c
73.11±4.22 d
0.00±0.00 c
0.00±0.00 b
97.52±5.63 c
28.97±1.67 d
182.55±10.54 c
74.53±4.30 d
0.00±0.00 b
68.50±3.95 cd
4039.03
YiyouHK02
Chuanyou167
44you167
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
130.23±7.52 c
138.46±8.00 bc
660.11±38.11 a
44.32±2.56 b
41.02±2.37 bc 174.85±10.10 a
50.36±2.91 de
90.88±2.25 c
260.83±15.06 a
54.98±3.17 a
37.46±2.16 b
0.00±0.00 c
65.60±3.79 c
74.09±4.28 bc 243.56±14.06 a
122.39±7.07 b
103.69±5.99 b
584.31±33.74 a
0.00±0.00 b
0.00±0.00 b
161.92±16.19 a
182.30±10.53 b
115.08±6.64 c
857.78±49.52 a
46.07±2.66 cd
41.06±2.37 cd 214.20±12.37 a
69.00±3.98 bc
60.79±3.51 bc 324.02±18.71 a
53.85±3.11 bc
46.08±2.66 bc 236.09±13.63 a
220.81±12.75 bc
201.51±11.63 bc 875.16±50.53 a
0.00±0.00 c 2465.77±246.58 a 1149.82±114.98 b
296.56±17.12 bc
297.92±17.20 bc
0.00±0.00 e
457.36±45.74 c
540.40±54.04 b
0.00±0.00 d
112.69±6.51 bc
109.04±6.30 bc 420.61±24.28 a
36.69±3.67 b
29.44±2.94 b
111.90±11.19 a
0.00±0.00 b
0.00±0.00 b
106.65±10..67 a
141.94±8.19 bc
112.08±6.47 c
604.80±34.92 a
51.77±2.99 c
40.99±2.37 cd
116.44±6.72 b
238.23±13.75 c
825.61±47.67 b 1062.93±61.37 a
75.54±4.36 d
90.70±5.24 cd 304.19±17.56 a
0.00±0.00 b
0.00±0.00 b
0.00±0.00 b
64.69±3.73 d
60.99±3.52 d
186.26±10.75 a
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
A 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 aboved.
PLFAs data were processed using the principal component analysis (PCA). Results presented in Fig. 2
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 (Fig. 2). 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. In general, 16:0, 18:1ω9с and 10Me16:0 which represented
bacteria, fungi and actinomycetes respectively were chiefly responsible for the variances. it showed
that 16:0, 18:1ω9с and 10Me16:0 were the primary compositions on the microbial community.
Fig.2 Principal component analysis of individual PLFA
In order to analyse the structure of the microbial community in the rhizosphere of rice. We
compared with the contents of bacteria(16:0), fungi(18:1ω9с) and actinomycetes(10Me16:0) ,the
results was presented in Fig. 3. The results showed that PLFAs of the three main kinds of microbe
varied among different hybrid rice cultivars plots. In general, bacteria were most predominant followed
by fungi and actinomycetes as the second and third abundant microorganism in the rice rhizosphere,
compared with 10Me16:0 and 16:1ω5с as indicators respectively. Moreover, the sulfate-reduсing
bacteria and methane-oxidizing bacteria in rhizosphere of different rice cultivars were compared with
10Me16:0 and 16:1ω5с as indicators respectively (Fig. 4). 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 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.
Fig.4 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 between soil microbial biomass and productive characteristics of rice cultivar
Cluster analysis of seven hybrid rice cultivars was conducted based on PLFAs by using unweighted
pair-group mean average (UPGMA) method. The statistic analysis showed that the seven tested hybrid
rice cultivars could be clustered into two groups at 3.68 of Lance-William distance (Fig. 5). The first
group consisted of two subgroups: one subgroup included the two rice cultivars of HK02 series with
characteristics of lowest PLFAs biomasses (>5000 µg g-1), grain number per spike (<130), seed setting
rate (<72.72%) and yields (<520.00 kg) as well as highest plant height (>125 cm), and the other
subgroups comprised the three rice cultivars of IIyou series with characteristics of middle values of the
PLFAs biomasses and the biological characteristics mentioned above. The hybrid rice Chuanyou167
and 44you167 were involved in the second group with characteristics of highest PLFAs biomasses
(>7000 µg g-1), grain umber per spike (>172), seed setting rate (>92%) and yields (>573 kg/666 m2) as
well as lowest plant height (<112 cm).
On the other hand, the amount of PLFAs biomasses in the rhizosphere of hybrid rice 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**). For example,
XinyouHK02 had grain number per spike, seed setting rate, yield and plant height as 128.22, 70.48%,
516.55 kg and 130 cm, while 44you167 had 180.71, 94.70%, 573.96 kg and 112 cm for the same
biological characteristics, respectively (Table 2).
Lance-William distance
Fig.5
Cluster analysis of seven hybrid rice cultivars based on PLFAs by using unweighted pair-group mean
average (UPGMA) method
Discussion
The microbial communities in the rhizpshere is affected by biotic and abiotic factors ,especially
plant characteristics. Siciliano et al (1998) found that the differences in the microbial communities
were associated with the roots of different cultivars of canola and wheat (Siciliano, Theoret, De Freitas,
Hucl, & Germida, 1998). Di Cello et al(1997) also discovered that biodiversity of a Burkholderia
cepacia population isolated from the maize rhizosphere were relation with different plant growth stages
(Di Cello et al., 1997). Briones et al(2002b) compared the activities and diversities of
ammonia-oxidizing bacteria(AOB) in the root environment of different cultivars of rice (Oryza sativa
L.) and the results indicated marked difference despite indetical environment conditions during
growth(Briones et al., 2002b).
But The reported showed that the growth of above- and under-ground organs in rice were
regulated by specific gene (Suzaki et al., 2004; Itoh, Kitano, Matsuoka, & Nagato, 2000)
Morphological and RFLP markers are associated in rice(Yu, Kinoshita, Sato, & Tanksley, 1995).
Chromosome number and plant characters in aneuploids of Oryza glaberrima presenced the
relationship (ISHIKI). Koga et al. (1967) and Watanabe et al. (1969) reported a close negative relation
between chromosome number and seed fertility(Watanabe, Ono, Mukai, & Koga, 1969;
KogaNagamatsu & Inoue, 1967). The more the extra chromosomes, the lower the seed fertility,
although there was a wide variation in plants with 2n=25 and 26. Most aneuploids with more than 27
chromosomes had nearly 0% seed fertility. Ramanujam (1937) and Watanabe et al. (1969) reported a
close negative relation between chromosome number and plant height or culm length. The more the
extra chromosomes, the shorter the plants(Watanabe, Ono, Mukai, & Koga, 1969) Two sequence
alterations, a 136 bp InDel and an A/C polymorphic site, in the S5 locus are associated with spikelet
fertility of indica-japonica hybrid in rice (Ji et al., 2010)
rhizosphere microbial communities can vary in structure and species composition in different root
locations or in relation to soil type, plant species, nutritional status, age, stress, disease, and other
environmental factors(Griffiths, Ritz, Ebblewhite, & Dobson, 1998; Yang & Crowley, 2000) Variation
of microbial rhizosphere communities in response to crop species, soil origin, and inoculation with
Sinorhizobium meliloti L33(Miethling, Wieland, Backhaus, & Tebbe, 2000) Taxonomic diversity of
bacteria associated with the roots of modern, recent and ancient wheat cultivars (Germida & Siciliano,
2001) Plant-dependent genotypic and phenotypic diversity of antagonistic rhizobacteria isolated from
different Verticillium host plants (Berg et al., 2002) Effect of host genotype on indigenous bacterial
endophytes of cotton (Gossypium hirsutum L.) (Adams & Kloepper, 2002)
Arab et al. (2001) used
PLFAs 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.
We found the bacteria(16:0), fungi(18:1ω9с) and
actinomycetes(10Me16:0) were the main microbial organisms in the rhizosphere of rice by PCA
analysis using PLFAs method and the the structure of rhizosphere microbial were relation with the rice
cutivars traits. The content of three microorganism (bacteria, fungi, actinomycete ) of MR167 series
with higher than the II-32 and HK02 series and the production of MR167 series were higher than the
II-32 and HK02 series.
Plant-growth-promoting rhizobacteria and kinetin as ways to promote corn growth and yield in a
short-growing-season area(Pan, Bai, Leibovitch, & Smith, 1999). It is also reported that
sulfate-reduсing bacteria could simulate the growth of plant(蒋先军 & 黄昭贤, 2000). Maria et al
(2005) discovered the population dynamics, genotypic diversity and activity of naturally-occurring
2,4-diacetylphloroglucinol (DAPG)-producing Pseudomonas spp. of four plant species (wheat, sugar
beet, potato, lily) were different(Bergsma VlamiPrins & Raaijmakers, 2005).We found the contents of
sulfate-reducing bacteria of rice cultivars with higher productiongs were higher. It could be tried If we
can develop beneficial microorganism communities for creating yield enhancing associations with
crops from the rhizosphere microbes.
In my experiment, based on the characteristics of PLFAs biomarks and the productive traits, the
rice cultivars could be divided into there groups by cluster analysis, in accordance with their parent
origins. This results indicted than the rhizosphere microbial communities were identical to the plant
evolutionary relationgships. It might be presumed that the microbial community also reflected the
genetic background of hybrid rice, which provided useful information in the cultivation of the super
rice.
Acknowledgements The present investigation was supported by the National High Technology
Research and Development Program ("863" Program) of China (2006AA100101 and 2006AA10A211).
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