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Citation: An, K.; Chen, L.; Liu, Y.; Wei,
H.; Chen, G. Seed Dormancy and
Germination Responses to Different
Temperatures of Leptochloa chinensis
(L.) Nees: A Case Study with 242
Populations Collected from Rice
Fields in East China. Agronomy 2024,
14, 2177. https://doi.org/10.3390/
agronomy14092177
Academic Editors: Shouhui Wei,
Judith Wirth and Agnieszka Lejman
Received: 2 August 2024
Revised: 14 September 2024
Accepted: 20 September 2024
Published: 23 September 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
agronomy
Article
Seed Dormancy and Germination Responses to Different
Temperatures of Leptochloa chinensis (L.) Nees: A Case Study
with 242 Populations Collected from Rice Fields in East China
Kai An 1,2, Ling Chen 1,2, Yiyang Liu 1,2, Haiyan Wei 1,2 and Guoqi Chen 1 ,2 ,*
1Jiangsu Key Laboratory of Crop Genetics and Physiology/Jiangsu Key Laboratory of Crop Cultivation and
Physiology, Agricultural College/Research Institute of Rice Industrial Engineering Technology of Yangzhou
University, Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou
University, Yangzhou 225009, China; 16638727457@163.com (K.A.); 15050787061@163.com (L.C.);
liuyiyang0636@163.com (Y.L.); wei_haiyan@163.com (H.W.)
2Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University,
Yangzhou 225009, China
*Correspondence: chenguoqi@yzu.edu.cn
Abstract: Leptochloa chinensis (L.) Nees is a troublesome rice weed. We collected 242 L. chinensis
populations from rice fields in eastern China and studied the duration of seed dormancy and the
seed germination ability at different temperatures. All L. chinensis populations studied exhibited
seed dormancy. The periods required to reach 50% germination under optimal conditions were
31–235 days,
with an average of 96 days. None of the populations germinated at 15
◦
C. Under
constant temperatures of 20, 25, 30, and 35
◦
C, the average germination percentages of 242 populations
were 0%, 71%, 79%, and 60% at 2 days after treatment (DAT), and were 56%, 84%, 88%, and 88% at
14 DAT,
respectively. The duration of seed dormancy, as well as the germination ability of seeds, were
significantly (p< 0.05) influenced by the agricultural region and the longitude and latitude of the
collection locations. Under constant temperatures of 20 to 35
◦
C, the average germination percentages
of seeds collected from transplanted rice fields were significantly higher than those collected from
direct-seeded fields. This is the first study on seed germination biology of L. chinensis with multiple
populations systematically collected from rice fields on a regional scale.
Keywords: agricultural region; Chinese sprangletop; intraspecific variation; rice planting method;
rice weed; sampling location; weed seedbank
1. Introduction
Chinese sprangletop [Leptochloa chinensis (L.) Nees] is a troublesome weed in paddy
fields worldwide that seriously affects rice quality and yield [
1
]. Leptochloa chinensis pro-
duces up to about 45,000 seeds per individual plant [
2
,
3
], and 25 plants m
−2
caused a
69% yield loss [
4
]. Chemical control is one of the main methods of L. chinensis manage-
ment in rice fields. Leptochloa chinensis populations in different rice-growing areas have
evolved resistance to the main post-emergence rice herbicides, such as cyhalofop-butyl and
metamifop [
5
,
6
]. It is insensitive to other herbicides used for grass control in rice, such as
florpyrauxifen-benzyl, bispyribac-sodium, pyribenzoxim, and penoxsulam [
5
]. Various
pre-emergence herbicides showed good efficacy against L. chinensis [
7
], which mainly target
the growing stage from germination to the two-leaf stage [
6
]. Knowledge of seed dormancy
and germination is the basis for predicting the occurrence of L. chinensis on-field.
To date, different conclusions on seed dormancy in similar studies with different
populations of L. chinensis have been reported. Benvenuti et al. reported that L. chinensis
seeds did not show seed dormancy [
8
]; however, Dong et al. reported that L. chinensis seeds
showed dormancy [
9
]. The heterogeneity of seed dormancy duration makes it difficult to
Agronomy 2024,14, 2177. https://doi.org/10.3390/agronomy14092177 https://www.mdpi.com/journal/agronomy
Agronomy 2024,14, 2177 2 of 13
determine the optimal application period of herbicides, which greatly decreases the efficacy
of applied herbicides. For example, pre-emergence herbicides are typically applied 3–5 days
after rice seeding, 3–5 days before rice transplanting, or 7–10 days after rice transplanting.
The seed dormancy of L. chinensis is reported to be physiological dormancy [
9
,
10
], which
might be overcome with different periods for different seeds. Thus, variations in seed
dormancy frequently result in the heterogeneity of seedling emergence. In eastern China,
L. chinensis seedlings frequently emerge continuously for one month in rice fields, which
causes great difficulties for pre-emergence chemical control practices [
11
]. Revealing the
variations in seed dormancy durations of L. chinensis with multiple populations could be
very important for its integrative management. Moreover, temperature is an important
climate factor that plays a fundamental role in the determination of the emergence of plants.
As a rice weed, seed germination of L. chinensis occurs in higher temperatures. Lower
temperatures may constrain or delay its seed germination, which should be managed with
different strategies accordingly. To date, germination patterns of L. chinensis responding to
different temperatures are still unclear.
In 2022, we collected 242 L. chinensis populations (Figure 1) from rice fields in eastern
China and compared their seed dormancy, germination percentage, and rate at differ-
ent temperatures. We aimed to (1) reveal the range and general characteristics of the
seed dormancy periods of L. chinensis, (2) reveal the range and general characteristics of
L. chinensis
seed germination at different temperatures, and (3) explore correlations among
seed dormancy, germination percentage at different temperature, and longitude, latitude,
agricultural region, and rice planting methods of the seed collecting rice fields.
Agronomy 2024, 14, x FOR PEER REVIEW 2 of 14
seeds showed dormancy [9]. The heterogeneity of seed dormancy duration makes it diffi-
cult to determine the optimal application period of herbicides, which greatly decreases
the efficacy of applied herbicides. For example, pre-emergence herbicides are typically
applied 3–5 days after rice seeding, 3–5 days before rice transplanting, or 7–10 days after
rice transplanting. The seed dormancy of L. chinensis is reported to be physiological dor-
mancy [9,10], which might be overcome with different periods for different seeds. Thus,
variations in seed dormancy frequently result in the heterogeneity of seedling emergence.
In eastern China, L. chinensis seedlings frequently emerge continuously for one month in
rice fields, which causes great difficulties for pre-emergence chemical control practices
[11]. Revealing the variations in seed dormancy durations of L. chinensis with multiple
populations could be very important for its integrative management. Moreover, temper-
ature is an important climate factor that plays a fundamental role in the determination of
the emergence of plants. As a rice weed, seed germination of L. chinensis occurs in higher
temperatures. Lower temperatures may constrain or delay its seed germination, which
should be managed with different strategies accordingly. To date, germination patterns
of L. chinensis responding to different temperatures are still unclear.
In 2022, we collected 242 L. chinensis populations (Figure 1) from rice fields in eastern
China and compared their seed dormancy, germination percentage, and rate at different
temperatures. We aimed to (1) reveal the range and general characteristics of the seed
dormancy periods of L. chinensis, (2) reveal the range and general characteristics of L.
chinensis seed germination at different temperatures, and (3) explore correlations among
seed dormancy, germination percentage at different temperature, and longitude, latitude,
agricultural region, and rice planting methods of the seed collecting rice fields.
Figure 1. Distribution of 242 L. chinensis populations (green dots) collected from rice fields in differ-
ent agricultural areas in eastern China. Note: JX: Jiaxing agricultural region, 20 populations; LXH:
Lixiahe agricultural region, 18 populations; NZY: Ningzhenyang agricultural region, 36 popula-
tions; TH: Taihu agricultural region, 54 populations; XH: Xuhuai agricultural region, 53 popula-
tions; YH: Yanhai agricultural region 14 populations; and YJ: Yanjiang agricultural region, 47 pop-
ulations.
2. Materials and Methods
2.1. Plant Material
In October 2022, we collected 242 L. chinensis populations from rice fields in eastern
China, including 222 populations collected from Jiangsu Province and 20 populations
Figure 1. Distribution of 242 L. chinensis populations (green dots) collected from rice fields in different
agricultural areas in eastern China. Note: JX: Jiaxing agricultural region, 20 populations; LXH: Lixiahe
agricultural region, 18 populations; NZY: Ningzhenyang agricultural region, 36 populations; TH: Taihu
agricultural region, 54 populations; XH: Xuhuai agricultural region, 53 populations; YH: Yanhai
agricultural region 14 populations; and YJ: Yanjiang agricultural region, 47 populations.
2. Materials and Methods
2.1. Plant Material
In October 2022, we collected 242 L. chinensis populations from rice fields in eastern
China, including 222 populations collected from Jiangsu Province and 20 populations
from Jiaxing City, Zhejiang Province. Panicles with mature seeds were randomly collected
from more than 100 individuals of each population with a pollen bag (100 mesh, 30 cm
Agronomy 2024,14, 2177 3 of 13
by 45 cm), and mature seeds were collected by hand. An interval of >5 km was set for
adjacent populations (Figure 1, Table S1). All counties cultivating rice in Jiangsu Province
were visited. Jiangsu Province was divided into six agricultural regions: Xuhuai (XH),
Yanhai (YH), Lixiahe (LXH), Ningzhenyang (NZY), Yanjiang (YJ), and Taihu (TH) [
12
]; and
Jiaxing City, which belongs to Zhejiang Province, was set as another agricultural region
(Figure 1; Table S1). The longitude, latitude, agricultural regions, and rice planting methods
of each L. chinensis population were recorded. The collected seeds were stored in kraft paper
envelopes at room temperature, fluctuating from 20 to 25
◦
C in a lab with air conditioners
to control the temperature. The lemmas of mature L. chinensis seeds were usually not closed
and were easily separated. Before each experiment, candidate seeds of each population
were put in a pollen bag and gently rubbed by hand to gain seeds without lemmas. Then,
plumb and intact seeds were used for the experiments.
The seed-collecting areas referred to 14 cities in eastern China. We checked the
temperatures during the ordinary rice growing season (1st June to 15th October) and
annual precipitation in 2022 of these 14 cities. Temperature data were cited from www.
tianqihoubao.com, and annual precipitation data were cited from http://tjj.zj.gov.cn/
(Jiaxing city, accessed on 10 September 2024) and http://www.jiangsu.gov.cn/col/col84736
(13 cities of Jiangsu Province, accessed on 10 September 2024). The average maximum
temperatures of the 14 cities ranged from 28.2 to 30.2
◦
C during the rice growing season in
2022, which were significantly and negatively correlated with latitude (Figure 2A) but not
significantly correlated with longitude (Figure 2B). Annual precipitations ranged from 633.5
to 1189.2 mm in 2022 (Figure 2), which were significantly and negatively correlated with
latitude (Figure 2C) and significantly and positively correlated with longitude (Figure 2D).
Agronomy 2024, 14, x FOR PEER REVIEW 3 of 14
from Jiaxing City, Zhejiang Province. Panicles with mature seeds were randomly collected
from more than 100 individuals of each population with a pollen bag (100 mesh, 30 cm by
45 cm), and mature seeds were collected by hand. An interval of >5 km was set for adjacent
populations (Figure 1, Table S1). All counties cultivating rice in Jiangsu Province were
visited. Jiangsu Province was divided into six agricultural regions: Xuhuai (XH), Yanhai
(YH), Lixiahe (LXH), Ningzhenyang (NZY), Yanjiang (YJ), and Taihu (TH) [12]; and Jiax-
ing City, which belongs to Zhejiang Province, was set as another agricultural region (Fig-
ure 1; Table S1). The longitude, latitude, agricultural regions, and rice planting methods
of each L. chinensis population were recorded. The collected seeds were stored in kraft
paper envelopes at room temperature, fluctuating from 20 to 25 °C in a lab with air con-
ditioners to control the temperature. The lemmas of mature L. chinensis seeds were usually
not closed and were easily separated. Before each experiment, candidate seeds of each
population were put in a pollen bag and gently rubbed by hand to gain seeds without
lemmas. Then, plumb and intact seeds were used for the experiments.
The seed-collecting areas referred to 14 cities in eastern China. We checked the tem-
peratures during the ordinary rice growing season (June 1st to 15th October) and annual
precipitation in 2022 of these 14 cities. Temperature data were cited from www.tianqihou-
bao.com, and annual precipitation data were cited from http://tjj.zj.gov.cn/ (Jiaxing city,
accessed on 10 September 2024.) and http://www.jiangsu.gov.cn/col/col84736 (13 cities of
Jiangsu Province, accessed on 10 September 2024.). The average maximum temperatures
of the 14 cities ranged from 28.2 to 30.2 °C during the rice growing season in 2022, which
were significantly and negatively correlated with latitude (Figure 2A) but not significantly
correlated with longitude (Figure 2B). Annual precipitations ranged from 633.5 to 1189.2
mm in 2022 (Figure 2), which were significantly and negatively correlated with latitude
(Figure 2C) and significantly and positively correlated with longitude (Figure 2D).
Figure 2. Regressions among latitude, longitude, and average maximum temperatures (A,B) during
rice growing season and annual precipitation (C,D) in 2022 of the 14 cities where L. chinensis seeds
were collected.
2.2. Seed Dormancy
The experiments were conducted in the laboratory of Yangzhou University
(E119.423; N32.388) from October 2022 to August 2023. Six treatments were set up in the
Figure 2. Regressions among latitude, longitude, and average maximum temperatures (A,B) during
rice growing season and annual precipitation (C,D) in 2022 of the 14 cities where L. chinensis seeds
were collected.
2.2. Seed Dormancy
The experiments were conducted in the laboratory of Yangzhou University (E119.423;
N32.388) from October 2022 to August 2023. Six treatments were set up in the experiment,
which was tested at 3, 15, 30, 120, 210, and 300 days of seed storage. Storage conditions were
the same as mentioned above, and storage duration (seed age) was calculated according
to the period from seed collecting to the day of incubation in Petri dishes. Seeds of
Agronomy 2024,14, 2177 4 of 13
43 populations were not transported to the lab within 3 days and were not used in this
experiment. Thus, 199 populations were used in the dormancy experiments. For each
treatment, thirty-five intact and undamaged seeds were selected and placed in a 9 cm
diameter Petri dish with two layers of filter paper moistened with 5 mL distilled water.
Each treatment was replicated with three Petri dishes. Each Petri dish was sealed with
cling film, and water was added as needed. The seed germination percentage for each
treatment was then determined in illumination boxes (CFG-400CH, Changzhou Haibo
Instrument Equipment Co., Ltd., Changzhou, China) with a constant temperature of 30
◦
C
(light 12 h/dark 12 h, 12,000 lx). The number of seeds that germinated in each Petri dish
was recorded every 2 days until 14 days after treatment.
2.3. Seed Germination under Different Temperatures
The experiments were conducted in the laboratory of Yangzhou University (E119.423359;
N32.388486) in August 2023. Seeds (300 days of seed aging) of 242 L. chinensis populations
were used for testing seed germination at different temperatures, using the same method as
mentioned above. Dishes were placed in illumination boxes (two of CFG-400CH, Changzhou
Haibo Instrument Equipment Co., Ltd.; three of HP1000GS-B, Wuhan Ruihua Instruments
& Equipment Co., Ltd., Wuhan, China) with temperatures of 15, 20, 25, 30, and 35
◦
C (light
12 h/dark 12 h, 12,000 lx). Each treatment was replicated with three Petri dishes.
2.4. Statistical Analysis
Data are presented as mean
±
standard error. To analyze variations among popula-
tions in different indices, the coefficient of variation (CV) was calculated [
13
]. To analyze
the influences of the seed storage period, treated temperature, agricultural region, and rice
planting method of seed collecting sites on germination percentage, a generalized linear
model (GLM) univariate analysis in SPSS 16.0 was applied. The relationship between longi-
tude, latitude, and germination percentage was analyzed by linear regression of SPSS 16.0.
To analyze the significant influences of seed storage, temperatures, and agricultural regions
on average germination percentages, data were subjected to a one-way analysis of variance
using SPSS 16.0, which was checked for normality and constant variance before analysis.
The germination percentage data were log-transformed before analysis. Non-transformed
means for germination percentages are reported, with statistical interpretation based on
the transformed data. Treatment means were separated using Fisher’s protected LSD
test at
p= 0.05.
An independent sample t-test was used to compare the seed germination
percentages of L. chinensis populations collected from direct-seeded rice fields and those
collected from transplanted rice fields.
A three-parameter logistic function was fitted to test the response of germination
percentage to the seed storage period. A four-parameter logistic function was fitted to test
the response of germination percentage to temperature treatment, which both using the
‘drc’ add-on package in R 3.1.3 [14]:
logistic, 3 parameter: Y 1 = (a −d)/[1 + (x/e1)b] (1)
logistic, 4 parameter: Y 2 = d + (a −d)/[1 + (x/e2)b] (2)
Y 1 and Y 2 represent seed germination percentage; x is the seed storage period or
treatment temperature for germination tests; a is the upper limit; d is the lower limit; e
1
is
the number of days of seed storage when the germination percentage reaches 50% (DR
50
);
e
2
is the germination temperature at which the germination percentage reaches 50% (TR
50
);
and b is the slope.
3. Results
3.1. Seed Dormancy Duration
All 199 L. chinensis populations collected from paddy fields in eastern China showed
seed dormancy 30 days after collection. At seed storage for 3–30 days, the mean germination
Agronomy 2024,14, 2177 5 of 13
percentages of 199 populations were 0.3–1.3% (Figure 3A–C). Among the 199 populations,
38.7–66.3% of the populations showed germinated seeds, with germination percentages all
<25%. One population showed 24.6% seed germination at 30 days after collection.
Agronomy 2024, 14, x FOR PEER REVIEW 5 of 14
3. Results
3.1. Seed Dormancy Duration
All 199 L. chinensis populations collected from paddy fields in eastern China showed
seed dormancy 30 days after collection. At seed storage for 3–30 days, the mean germina-
tion percentages of 199 populations were 0.3–1.3% (Figure 3A–C). Among the 199 popu-
lations, 38.7–66.3% of the populations showed germinated seeds, with germination per-
centages all <25%. One population showed 24.6% seed germination at 30 days after col-
lection.
Figure 3. Seed germination at 14 days (d) after treatment with constant 30 °C (12/12 h light/dark) for
L. chinensis populations stored for 3 (A), 15 (B), 30 (C), 120 (D), 210 (E), and 300 (F) days after col-
lecting and DR50 of different populations (G). Note: DR50 = days of storage after collection, at which
the germination percentage reaches 50%.
The mean germination percentage of 199 L. chinensis populations increased signifi-
cantly (p < 0.05) with extending seed storage (Figure 3), and meanwhile, the CVs among
populations decreased. After storing for 120 days, seed germination percentages of the
199 populations ranged from 6.7 to 100%, averaging 64.6% (Figure 3D), and 74% of popu-
lations showed seed germination percentages >50%. After storing for 210 d, 95% of the
populations showed seed germination percentages >50% (Figure 3E). After storing for 300
days, the mean germination percentage of 199 populations was 88.0%, and 98% of
0
1
2
3
4
5
6
7
030 60 90 120 150 180
% of germination
Number of populations
3 d
0.3±0.1
A
0
4
8
12
16
20
030 60 90 120 150 180
% of germination
Number of populations
15 d
1.3±0.2
B
0
5
10
15
20
25
30
030 60 90 120 150 180
% of germination
Number of populations
30 d
0.9±0.2
C
0
20
40
60
80
100
030 60 90 120 150 180
% of germination
Number of populations
120 d
64.6±1.6
D
0
20
40
60
80
100
030 60 90 120 150 180
% of germination
Number of populations
210 d
75.8±1.0
E
0
20
40
60
80
100
030 60 90 120 150 180
% of germination
Number of populations
300 d
88.0±0.7
F
0
50
100
150
200
250
030 60 90 120 150 180
Days
Number of populations
DR50
95.7±2.9
G
A B C
DEF
G
Figure 3. Seed germination at 14 days (d) after treatment with constant 30
◦
C (12/12 h light/dark)
for L. chinensis populations stored for 3 (A), 15 (B), 30 (C), 120 (D), 210 (E), and 300 (F) days after
collecting and DR
50
of different populations (G). Note: DR
50
= days of storage after collection, at
which the germination percentage reaches 50%.
The mean germination percentage of 199 L. chinensis populations increased significantly
(p< 0.05) with extending seed storage (Figure 3), and meanwhile, the CVs among populations
decreased. After storing for 120 days, seed germination percentages of the 199 populations
ranged from 6.7 to 100%, averaging 64.6% (Figure 3D), and 74% of populations showed seed
germination percentages >50%. After storing for 210 d, 95% of the populations showed seed
germination percentages >50% (Figure 3E). After storing for 300 days, the mean germination
percentage of 199 populations was 88.0%, and 98% of populations showed seed germination
percentages >50% (Figure 3F). The DR
50
of the
191 L. chinensis populations
was 31–235 days,
with an average of 96 days (Figure 3G), and the germination percentages of the other
eight populations did not conform to the logistic model used. DR
50
periods for 16.8%
of populations showed DR
50
periods <50 days, 62.8% of L. chinensis populations ranged from
70 to 120 days, and 8.9% showed DR50 periods >150 days.
Agronomy 2024,14, 2177 6 of 13
In May 2024, another experiment suggested that the average germination percentage
of 89 populations (randomly selected from the seven agricultural regions, stored for 570 d,
and treated with a constant temperature of 30
◦
C, light 12 h/dark 12 h, and 12,000 lx for
14 days) ranged from 56.4 to 98.8%, averaging 88.0% (Figure S3A; unpublished data).
3.2. Influences of Location and Planting Methods on Seed Dormancy Durations
The seed storage, agricultural region, and seed storage
×
agricultural region showed
significant (p< 0.05) influences on germination percentages of L. chinensis seeds (Table 1).
The average germination percentage of populations collected from the YH agricultural
region was the highest among all seed dormancy experimental treatments
(Tables 2and S2).
At seed storage for 120 days, the average germination percentage of populations collected
from the YH agricultural region was significantly the highest among the seven regions,
followed by the XH agricultural region, and the average germination percentage of popula-
tions collected from the JX agricultural region was significantly the lowest, 38.6% lower
than that of YH. At seed storage for 210 days, the NZY and JX agricultural regions were
significantly the lowest, and the TH and JX agricultural regions were significantly the
lowest at 300 days of seed storage. The variation in germination percentages among the
different agricultural regions decreased significantly. Thus, the variations in germina-
tion percentages among different agricultural regions were mainly due to the duration of
seed dormancy.
Table 1. Results (F-values) of GLMs among germination percentages of Leptochloa chinensis (L.) Nees
seeds at different seed storage, agricultural regions, and rice planting methods of seed collecting fields.
Influencing Factors F
Seed storage 128.5 *
Agricultural region 21.1 *
Rice planting method 0.1
Seed storage ×agricultural region 7.4 *
Seed storage ×rice planting method 2.1
Agricultural region ×rice planting method 0.8
Seed storage ×agricultural region ×rice planting method 1.6
Note: The incubating temperature was constant at 30
◦
C (12/12 h light/dark). Seed storage treatments included
120, 210, and 300 days after collecting; agricultural regions were the same as in Figure 1; rice planting methods
include direct-seeded and transplanting. *: p< 0.05. The same as below.
Table 2. Germination percentages of L. chinensis seeds stored for different periods and collected from
different agricultural regions.
Agricultural Region Days after Storage
120 210 300
XH 71.0 ±2.9 ab 80.3 ±1.3 b 92.0 ±0.6 a
YH 80.5 ±5.7 a 89.2 ±1.5 a 92.2 ±0.8 a
LXH 59.7 ±3.9 b 78.9 ±2.0 b 90.2 ±1.0 ab
NZY 66.5 ±3.1 b 68.5 ±2.4 d 87.6 ±1.1 bc
YJ 65.4 ±3.6 b 72.6 ±1.3 cd 87.4 ±1.0 bc
TH 62.8 ±3.1 b 77.6 ±1.4 bc 83.8 ±1.1 c
JX 41.9 ±6.4 c 67.2 ±2.5 d 85.7 ±1.4 c
Note: Different letters within the same column indicate significant differences at p< 0.05. The incubating
temperature was constant at 30 ◦C (12/12 h light/dark).
The collection sites of the 199 L. chinensis populations ranged from 4.1
◦
longitude
to 4.1
◦
latitude. The latitude of the collection site significantly (p< 0.05) influenced seed
germination percentages after being stored for different periods (Table 3; Figure S1). The
germination percentages at 120, 210, and 300 days of seed storage were positively correlated
with the latitude. Seed dormancy of the L. chinensis population in the low longitudinal
Agronomy 2024,14, 2177 7 of 13
(western) and high latitudinal (northern) areas was released faster, and the germination
percentage was higher after overcoming dormancy.
Table 3. Regressions between longitude, latitude, and germination percentages of L. chinensis seeds
stored for different periods.
Seed Storage Period
(Day)
Longitude Latitude
Formula R2Formula R2
120 y = −2.92x + 414.59 0.014 y = 5.35x −109.21 0.077 *
210 y = −0.54x + 140.22 0.001 y = 3.30x −31.37 0.069 *
300 y = −0.94x + 200.55 0.008 y = 2.39x + 10.33 0.081 *
Note: The longitude and latitude ranges of the 199 L. chinensis populations studied were 117.5–121.6
◦
and
30.4–34.6◦, respectively. *: p< 0.05. The incubating temperature was constant at 30 ◦C (12/12 h light/dark).
Among seed storage with 120, 210, and 300 days, the average germination percentage
of L. chinensis seeds collected from transplanted rice fields (76.1%) was slightly and signifi-
cantly higher than that of L. chinensis seeds collected from direct-seeded rice fields (74.8%).
3.3. Seed Germination Percentage under Different Temperatures
The optimal germination temperature was 30
◦
C, and seeds germinated well under
25
◦
C and 35
◦
C, according to germination percentages on 2, 4, and 14 days after treat-
ment. All 242 populations did not germinate at 15
◦
C (Figure 4A). The seed germination
percentage of L. chinensis increased significantly (p< 0.05) with the temperature increasing
from 20 to 30
◦
C, and the CV of germination percentages of 242 populations decreased
significantly with the increasing temperature (Figure 4). At 20
◦
C, germination percent-
ages of 242 populations ranged from 3.4 to 96.9%, averaging 56.1% (Figure 4B), and 63%
of populations showed seed germination percentages >50%. No seed from any of the
populations germinated two days after treatment, and four days after treatment, the av-
erage germination percentage of 242 populations was 22.4%. At 25
◦
C, the germination
percentage of 96% of populations was >50%, and the average germination percentage
of all populations was 84.3% (Figure 4C). Under 30
◦
C treatment, the mean germination
percentage of all populations was 88%, ranging from 41.5% to 100% (Figure 4D). At 25 and
30
◦
C, the average germination percentage of all populations at two days was >70%. At
30–35
◦
C, the germination percentage did not increase significantly (Figure 4D,E). Seed
germination of L. chinensis was inhibited in the first two days at 35
◦
C, compared with
those at 30
◦
C. The TR
50
of 240 L. chinensis populations ranged from 15.1 to 33.8
◦
C, with an
average of 19.7
◦
C (Figure 4F). The germination data for the two populations did not fit
the logistics model. Among the 240 L. chinensis populations, 82% showed a TR
50
between
18 and 21 ◦C.
3.4. Influences Factors on Seed Germination under Different Temperatures
The temperature, agricultural region, rice planting method, and temperature
×
agricul-
tural regions of the L. chinensis seed collection sites showed significant influences
(p< 0.05)
on the germination percentage 14 days after treatments (Table 4). At 20
◦
C, the germination
percentages of seeds collected from the YH agricultural region were significantly the highest
among the seven regions, and the TH agricultural region was the lowest
(Tables 5and S3).
The differences in seed germination of L. chinensis populations among different agricultural
regions were substantially lower at 25–35 ◦C.
Agronomy 2024,14, 2177 8 of 13
Agronomy 2024, 14, x FOR PEER REVIEW 8 of 14
Figure 4. Seed germination percentage at 2, 4, and 14 days (d) under 15 (A), 20 (B), 25 (C), 30 (D)
and 35 °C (E) and TR50 (F) of L. chinensis populations. Note: TR50 = The temperature at which the
germination percentage reaches 50%.
3.4. Influences Factors on Seed Germination under Different Temperatures
The temperature, agricultural region, rice planting method, and temperature × agri-
cultural regions of the L. chinensis seed collection sites showed significant influences (p <
0.05) on the germination percentage 14 days after treatments (Table 4). At 20 °C , the ger-
mination percentages of seeds collected from the YH agricultural region were significantly
the highest among the seven regions, and the TH agricultural region was the lowest (Ta-
bles 5 and S3). The differences in seed germination of L. chinensis populations among dif-
ferent agricultural regions were substantially lower at 25–35 °C.
At 20–25 °C, the germination percentage at 4 and 14 days after treatment were signif-
icantly and negatively correlated with the longitude of the seed collection site and signif-
icantly positively correlated with the latitude (Table 6; Figure S2). At 30 °C , the germina-
tion percentage determined at 2 and 14 days after treatment was significantly and posi-
tively correlated with the latitude of the seed-collection sites. There was a significant and
negative correlation between germination percentage and longitude at 35 °C for 2 days.
Moreover, the germination percentages of seeds collected from transplanted rice fields
(81.5%) were significantly higher than those collected from direct-seeded fields (78.7%).
Table 4. Results (F-values) of GLMs among germination percentages of L. chinensis seeds at different
temperatures, agricultural regions, and rice planting methods of seed collecting fields.
Influencing Factors
F
Temperature
205.6 *
Agricultural region
21.0 *
Rice planting method
4.3 *
Temperature × agricultural region
10.3 *
Temperature × rice planting method
0.8
Agricultural region × rice planting method
1.6
Temperature × agricultural region × rice planting method
0.8
0
20
40
60
80
100
040 80 120 160 200 240
% of germination
Number of populations
15 ℃2d 4d 14d
0.0±0.0
A
0
20
40
60
80
100
040 80 120 160 200 240
% of germination
Number of populations
20 ℃ 2d 4d 14d
56.2±1.6
0.0±0.0
22.4±1.4
B
0
20
40
60
80
100
040 80 120 160 200 240
% of germination
Number of populations
25 ℃ 2d 4d 14d
84.3±0.8
71.3±1.2
81.2±0.9
C
0
20
40
60
80
100
040 80 120 160 200 240
% of germination
Number of populations
30 ℃ 2d 4d 14d
87.8±0.6
79.1±1.0
86.1±0.7
D
0
20
40
60
80
100
040 80 120 160 200 240
% of germination
Number of populations
35 ℃ 2d 4d 14d
88.2±0.6
60.0±1.3
85.0±0.7
E
0
5
10
15
20
25
30
35
040 80 120 160 200 240
Temperature (C )
Number of populations
TR50
19.7±0.1
F
AB C
DEF
Figure 4. Seed germination percentage at 2, 4, and 14 days (d) under 15 (A), 20 (B), 25 (C), 30 (D)
and 35
◦
C (E) and TR
50
(F) of L. chinensis populations. Note: TR
50
= The temperature at which the
germination percentage reaches 50%.
Table 4. Results (F-values) of GLMs among germination percentages of L. chinensis seeds at different
temperatures, agricultural regions, and rice planting methods of seed collecting fields.
Influencing Factors F
Temperature 205.6 *
Agricultural region 21.0 *
Rice planting method 4.3 *
Temperature ×agricultural region 10.3 *
Temperature ×rice planting method 0.8
Agricultural region ×rice planting method 1.6
Temperature ×agricultural region ×rice planting method 0.8
Note: Seeds were stored for 300 days before the experiment. The temperature treatments include 20, 25, 30, and
35
◦
C. Seeds of L. chinensis did not germinate at 15
◦
C. The agricultural regions are the same as in Figure 1. The
planting methods include direct-seeded and transplanting. *: p< 0.05.
Table 5. Germination percentages of L. chinensis seeds under different temperatures and collected
from different agricultural regions.
Agricultural
Region
Treated Temperature (◦C)
20 25 30 35
XH 55.5 ±3.2 b 87.7 ±1.9 ab 90.1 ±1.0 ab 91.8 ±0.8 a
0.8YH 84.6 ±2.2 a 90.8 ±1.5 a 92.8 ±0.8 a 91.9 ±1.0 a
LXH 65.7 ±6.6 b 89.6 ±1.4 a 89.3 ±1.4 ab 90.6 ±1.3 a
NZY 63.0 ±3.0 b 85.7 ±2.3 ab 86.5 ±1.4 b 86.9 ±1.4 ab
YJ 57.9 ±3.6 b 81.4 ±1.8 bc 85.5 ±1.5 b 88.4 ±1.5 a
TH 37.8 ±2.7 c 79.8 ±2.3 bc 88.7 ±1.1 ab 82.4 ±1.8 b
JX 63.4 ±3.2 b 76.2 ±3.7 c 87.2 ±2.6 b 86.3 ±2.1 ab
Note: Different letters within the same column indicate significant differences at p< 0.05. Note: Seeds were stored
for 300 days before the experiment.
Agronomy 2024,14, 2177 9 of 13
At 20–25
◦
C, the germination percentage at 4 and 14 days after treatment were sig-
nificantly and negatively correlated with the longitude of the seed collection site and
significantly positively correlated with the latitude (Table 6; Figure S2). At 30
◦
C, the
germination percentage determined at 2 and 14 days after treatment was significantly and
positively correlated with the latitude of the seed-collection sites. There was a significant
and negative correlation between germination percentage and longitude at 35
◦
C for 2 days.
Moreover, the germination percentages of seeds collected from transplanted rice fields
(81.5%) were significantly higher than those collected from direct-seeded fields (78.7%).
Table 6. Regressions between latitude or longitude and germination percentages of L. chinensis seeds
at 2, 4, and 14 days after treatment with different temperatures.
Temperature ◦CDays Longitude Latitude
Formula R2Formula R2
20 ◦C4 y = −3.06x + 388.89 0.016 * y = 2.78x −67.99 0.020 *
14 y = −4.96x + 649.83 0.035 * y = 2.87x −36.96 0.018 *
25 ◦C
2 y = −6.18x + 811.15 0.093 * y = 6.57x −142.16 0.160 *
4 y = −3.11x + 453.99 0.039 * y = 3.90x −45.51 0.092 *
14 y = −3.04x + 448.15 0.044 * y = 3.78x −38.50 0.104 *
30 ◦C
2 y = −0.12x + 92.98 0.000 y = 3.52x −35.19 0.058 *
4 y = −0.10x + 97.49 0.000 y = 2.57x + 2.60 0.055 *
14 y = −0.48x + 145.46 0.002 y = 2.64x + 1.84 0.089 *
35 ◦C
2 y = −2.85x + 401.40 0.017 * y = 4.47x −85.23 0.064 *
4 y = −0.40x + 132.80 0.001 y = 0.86x + 57.13 0.009
14 y = −0.24x + 116.90 0.001 y = 0.98x + 56.51 0.020
Note: The longitude and latitude ranges of the 242 populations studied were 117.5–121.6
◦
and 30.4–34.6
◦
,
respectively; seeds were stored for 300 days before the experiment. Seeds did not germinate after 2 days under
20 ◦C. *: p< 0.05.
4. Discussion
4.1. Different Periods Were Required to Release Seed Dormancy in Different Populations
Together our study suggested that all L. chinensis populations collected from eastern
China showed physiological dormancy, which required different after-ripening periods.
Moreover, L. chinensis populations continuously and gradually released seed dormancy.
Thus, L. chinensis generally continuously emerges seedlings in rice fields, which effectively
facilitates the escape of this weed species from chemical control strategies. It is impor-
tant for rice growers to know the seed dormancy duration of L. chinensis in their own
fields. For example, in fields with a great part of L. chinensis seeds holding dormancy
durations >210 days, the seeds may possibly germinate continuously for more than two
months; and pre-emergence herbicides targeting this weed species should be applied
several times, such as before rice planting, 3–5 days after planting, and 15–20 days after
planting. Whereas, in fields with L. chinensis holding shorter durations of seed dormancy,
most seeds may germinate in 2 days after irrigation or rainy weather; thus, one-time
pre-emergence chemical control targeting this weed species may be enough. Leptochloa
chinensis biotypes show more significant variation in germination timing, which could
be an adaptation to variable climates [
15
]. Many weed species, such as Halenia elliptica
D. Don [
16
],
Convolvulus arvensis L. [17]
, and Capsella bursa-pastoris (L.) Medik. [
18
], can
potentially germinate under a wide range of dormancy durations.
The lemmas of mature L. chinensis seeds were usually not closed. Germination percent-
ages increased with the release of dormancy. Thus, we supposed that ungerminated seeds
were mostly under dormancy. Regarding seed germination experiments with temperature
treatments, the germination of seeds was constrained by unsuitable conditions. In another
experiment, the average germination percentage of L. chinensis seeds (seed storage for
570 days) of 31 populations treated with a water potential of
−
0.3 MPa for 14 days was
31.3%, and the average germination percentage of ungerminated seeds of these populations
Agronomy 2024,14, 2177 10 of 13
increased to 80.1% (Figure S3B) after incubation under optimal conditions for another
14 days (unpublished data).
4.2. Different Populations Showed Different Seed Germination Patterns under
Different Temperatures
Under optimal conditions, a majority of L. chinensis populations tested showed ger-
mination percentages >50% in 2 days, which could be sufficient for infesting rice fields.
Specifically, farmers frequently finish soil preparation of all paddy fields and then finish
transplanting or direct-seeding of rice in several days, which is frequently named “field
waiting for planting period”. Many L. chinensis seedlings may emerge and grow quickly
during the “field waiting for planting period”, which might cause failures of pre-emergence
chemical control. For example, pretilachlor, pendimethalin, clomazone, pyraclonil, and
mefenacet exhibit good control effects on various L. chinensis populations [
7
]. However,
these five pre-emergence herbicides are ineffective in managing L. chinensis seedlings af-
ter the two-leaf stage [
5
]. Moreover, applying post-emergence herbicides at early stages
after rice seeding or transplanting frequently causes serious rice injury. Therefore, in
rice-planting areas infested by L. chinensis, pre-emergence herbicides should be applied
in periods between soil preparation and rice planting. For example, in transplanted rice
fields, applying pre-emergence herbicides during the “field waiting for planting period”
and transplanting rice seedlings 3–5 days after pre-emergence chemical control frequently
results in good control efficacy against L. chinensis. In direct-seeded rice fields, modern
seeding methods with high efficiency to shorten the “field waiting for planting period”
could be important for L. chinensis management, such as mechanized seeding with drills or
unmanned aerial vehicles [19].
Benvenuti et al. found that the optimum temperature for L. chinensis seed germination
was 25–35
◦
C [
8
]. Our study suggested that the optimal temperature was 30
◦
C [
20
], while
different populations showed different seed germination patterns responding to tempera-
tures. Intraspecific differences in seed germination in response to different temperatures
have also been observed in Arabidopsis thaliana [
21
]. Liu et al. [
22
] found that four Dal-
bergia odorifera populations exhibited different seed germination percentages and speeds
at different temperatures. Zhang et al. [
23
] found that seed germination of temperature
requirements of Bidens pilosa differed significantly among populations.
4.3. Intraspecific Variations in Seed Dormancy and Germination
Overall, the experiments found intraspecific variations in seed dormancy and germina-
tion biology under different temperatures, among which the agricultural region, longitude,
latitude, and rice planting methods of seed-collecting fields all showed significant (p< 0.05)
influences. Seed germination can be affected by environmental conditions experienced
by mother plants, and differences in germination of seeds from different maternal envi-
ronments might be due to the epigenetic memory inherited from mother plants [
24
,
25
].
Brainard et al. found that in the same area, Amaranthus powellii seeds from vegetable fields
typically have more extended dormancy than those from dairy farms [
26
]. Gafni found that
the germination percentages of nine Amaranthus albus populations from different regions of
Israel were significantly different at different temperatures [
27
]. There are differences in
rice planting habits, terrain distribution, soil type, cultivated land quality, and irrigation
conditions between different agricultural regions [
28
,
29
], which might show different influ-
ences on L. chinensis seed dormancy and germination under different temperatures. The
YH and XH agricultural regions are located in the northern part of the seed-collection area,
and the TH and JX agricultural regions are located in the southern part. Thus, it appears
that L. chinensis populations collected from southern locations had a longer duration of
seed dormancy. Intraspecific variations in the dormancy characteristics of weed species at
different latitudes and longitudes have been reported [
15
]. Similar to the findings in this
study, Cheng et al. found that Spartina alterniflora populations collected from high-latitude
locations needed cold stratification to release dormancy, while low-latitude populations did
Agronomy 2024,14, 2177 11 of 13
not [
30
]. Shihan et al. found that the duration of summer dormancy in Dactylis glomerata
tended to decrease from South to North [
31
]. Variations in the duration of L. chinensis seed
dormancy might be due to the adaptive phenotypic plasticity of maternal plants or the
gene dominance and recessiveness of seeds [
32
,
33
]. In addition, geographically related
variations in seed germination percentage and speed have been observed in different
plant species [
34
]. Dwiyanti et al. found that seeds of Miscanthus sinensis, a grassy weed
species, collected from locations at higher latitudes germinated faster under 30
◦
C/20
◦
C
(day/night) and 15
◦
C/10
◦
C (day/night) [
35
]. In our study, L. chinensis seeds collected
from locations at higher latitudes and lower longitudes germinated rapidly, with higher
germination percentages at a lower temperature (20
◦
C). Interestingly, in seed collecting
areas of this study, areas with higher latitudes and lower longitudes tended to hold lower
precipitations with lower temperatures. Weng and Hsu found that the seed germination
percentage of Lilium formosanum collected at lower latitudes was higher than those collected
at higher latitudes [
36
]. Zhou et al. found that the seed germination periods of Ambrosia
artemisiifolia were significantly delayed with increasing latitude and longitude of collection
sites, whereas the germination percentages increased with increasing latitude and longitude
of collection sites [15].
Our results suggested that germination percentages of L. chinensis seeds collected
from transplanted rice fields were significantly and slightly higher than those collected
from direct-seeded fields, which may be related to the maternal effect [
27
]. There are
many differences in applying periods and dosages of herbicides between the two kinds
of rice fields [5]. In transplanted rice fields, rice seedlings at the four- to five-leaf stage are
transplanted, which have a higher tolerance to many herbicides; thus, chemical herbicides
with higher toxicity and efficacy against weeds could be used with higher doses and
acceptable weed control efficacies could be frequently achieved with an application of pre-
emergence herbicides and an application of post-emergence herbicides. Whereas, in direct-
seeded rice fields, rice seedlings emerge from seeds together with weed seedlings. Seedlings
of direct-seeded rice suffer longer periods and higher pressures of weed competition at early
growing stages, which are frequently injured by various herbicides. Farmers usually apply
chemical herbicides more times and with higher total dosages to effectively control weeds
on the basis of avoiding rice seedling injury, frequently applying various herbicides three
to five times. Hence, we suppose that differences in germination percentages among
L. chinensis
populations collected from the two different kinds of rice fields might be
related to the differences in chemical control strategies. The mechanisms under the above
differences between L. chinensis seeds collected from direct-seeded and transplanting rice
fields need more studies.
5. Conclusions
Together, our results suggested that L. chinensis seeds of all populations studied
exhibited physiological dormancy, while the dormancy duration of different populations
showed high intraspecific variation. The periods required to reach 50% germination under
optimal conditions were 31–235 days, with an average of 96 days. The temperature required
for 50% seed germination ranged from 15.1 to 33.8
◦
C among different populations, with an
average of 19.8
◦
C. None of the populations germinated at 15
◦
C. The optimal temperatures
for L. chinensis seed germination were 25 to 30
◦
C. Under optimal conditions, a majority of
L. chinensis populations tested showed germination percentages >50% in 2 days and >80%
in 4 days, which could be sufficient for infesting rice fields. Moreover, the agricultural
region, longitude, latitude, and rice planting methods (direct-seeded or transplanting) of
seed-collecting fields all showed significant (p< 0.05) influences on seed dormancy and
germination percentages under different temperatures. The commonness and uniqueness
of L. chinensis populations are valuable for improving its integrative management strategies
and are worth further investigation.
Agronomy 2024,14, 2177 12 of 13
Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/agronomy14092177/s1, Figure S1: Correlations between longitude
and latitude, and germination percentages of Leptochloa chinensis seeds stored for different periods;
Figure S2: Correlations between latitude, longitude, and germination percentages at 2, 4, and
14 days (d) of Leptochloa chinensis seeds treated with different temperatures; Figure S3: Leptochloa
chinensis seed germination of populations stored for 570 days after treated with constant temperature
(12/12 h light/dark) at 30
◦
C for 14 days (A), and germination percentages of Leptochloa chinensis (L.)
Nees seeds treated with
−
0.3 MPa water potential for 14 days and then continuously treated with
optimal conditions (0 MPa) for 14 days (B). Table S1: Collection information of 242 Leptochloa
chinensis populations; Table S2: The ANOVA table of the influence of different agricultural regions
on germination percentages of Leptochloa chinensis seed at different storing periods; Table S3: The
ANOVA table of the influence of different agricultural regions on the germination percentage of
Leptochloa chinensis under different temperature treatments.
Author Contributions: K.A. and G.C. designed the study, K.A., G.C. and L.C. performed material
seed collection, K.A., L.C. and Y.L. performed the germination treatment work, K.A. and L.C. per-
formed data processing work. K.A. wrote the first draft of the manuscript, G.C., K.A. and H.W.
contributed substantially to revisions. All authors have read and agreed to the published version of
the manuscript.
Funding: This study was funded by the Key R&D Plan of Shandong Province (2021LZGC020), the
Postgraduate Research & Practice Innovation Program of Jiangsu Province (SJCX23_1969), Jiangsu
Key R&D Plan (BE2022338), and a project funded by the Priority Academic Program Development of
Jiangsu Higher Education Institutions (PAPD).
Data Availability Statement: The original contributions presented in the study are included in the
article and Supplementary Materials.
Acknowledgments: We thank Hongcheng Zhang for his guidance on this study.
Conflicts of Interest: The authors declare that they have no competing financial interests or personal
relationships that could have influenced the work reported in this study.
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