Varied Susceptibility of Five Echinochloa Species to Herbicides and Molecular Identification of Species Using CDDP Markers

Abstract

Echinochloa spp. are among the most problematic malignant weeds in paddy fields. Under long-term herbicide selection pressure, they have developed resistances to multiple herbicides, leading to diminished control efficacy. Precision herbicide application, tailored to the susceptibility disparities among Echinochloa species, has emerged as a promising strategy to enhance weed control efficacy and decelerate herbicide resistance development. Nevertheless, the herbicide susceptibility variation across different Echinochloa taxa remain uncharted. Therefore, in this study, we determined the susceptibility of five Echinochloa species to 15 commonly used herbicides using the whole-plant bioassay method. Additionally, we explored the feasibility of employing the CDDP molecular marker technique for the rapid identification of distinct Echinochloa species. The results showing that five Echinochloa species exhibited differential susceptibility to 12 of the 15 herbicides tested underscore the necessity of personalized herbicide application strategies. Among the seven CDDP markers, KNOX-3 generated a specific band in the Echinochloa caudata population, which can be used to distinguish it from the other four Echinochloa species. The findings of this study will facilitate the precision application of herbicides for Echinochloa management in paddy fields.

1. Introduction

The genus Echinochloa (P.) Beauv. includes annual or perennial gramineous plants that are widely distributed between latitudes 50 N to 40 S [1]. There are approximately 50 species in the genus Echinochloa [2], and most of them act as field weeds, severely affecting crop growth, particularly in rice fields [3,4]. In paddy field weed communities, Echinochloa spp. (barnyard grass) always emerges as a dominant or sub-dominant weed. It commandeers substantial growth resources by virtue of its strong competitive ability, suppressing the growth and development of rice and causing yield reductions [5,6,7]. Research shows that at a density of 100 rice plants per m⁻², ten plants of Echinochloa spp. can reduce rice yields by up to 50% [5].

Using herbicides is a simple and effective method to control barnyard grass in paddy fields. However, the long-term use of herbicides has led barnyard grass to develop serious resistances to herbicides and the control effect has been greatly reduced [8,9,10]. Confronted with the escalating severity of herbicide resistance, researchers have initiated investigations into strategies for delaying resistance evolution. As early as 2004, the literature documented that Italian rice growers had found that the control efficacy of quinclorac against barnyard grass lacking red pigmentation was significantly higher than that against barnyard grass with reddish basal stem sections [11]. This indicates that there may be differences in sensitivity among different Echinochloa species. This observation was subsequently validated by additional studies [12,13,14]. Despite the fact that all tested species were controlled at the full field rate of cyhalofop-butyl (300 g a.i./ha⁻¹), E. colona was more sensitive (ED₅₀ = 34 g a.i./ha) than E. oryzicola (ED₅₀ = 170 g a.i./ha) [15]. At the recommended dose of 270 g a.i./ha tripyrizone, the fresh weight inhibition rate of two E. crus-galli var. crus-galli populations was over 90%, that of two E. glabrescens populations was 70–80%, and that of two E. crus-galli var. zelayensis populations was less than 40% [14]. Targeted application of herbicides based on sensitivity differences among Echinochloa species may be a method to delay the development of resistance. However, the current understanding of sensitivity differences among Echinochloa species to commonly used herbicides remains unclear.

To implement such targeted weed management, it is necessary not only to clarify the sensitivity variations in these species to herbicides but also to develop rapid and effective methods for identifying different Echinochloa species. At present, the main method to identify different barnyard grass species relies on the observation of morphological characteristics, with particular emphasis on the diagnostic features of spikelets and awns [15,16]. However, this method has significant limitations since some morphological features can only be observed during the reproductive stage. And apart from that, the morphology of spikelets and awns of barnyard grass is highly variable [17]. In addition to interspecific genetic exchange, the growing environment also significantly influences Echinochloa spp. morphology, leading to intraspecific phenotypic variation even within the same species [15,18,19]. Driven by advancements in molecular biology, molecular markers have become indispensable tools in taxonomic identification, genetic diversity, and other related research domains. Notably, some molecular marker techniques have been applied to address questions regarding genetic diversity and species delimitation in Echinochloa spp. For example, by using polymerase chain reaction–restriction fragment length polymorphism methods, E. oryzicola and E. crus-galli are distinguished [17]. Based on nucleotide sequence differences in the non-coding region trnT-L-F, Yamaguchi et al. reconstructed the molecular phylogeny of the nine Echinochloa species [17]. With the use of 21 random amplified polymorphic DNA (RAPD) markers, E. oryzicola and E. colona were clearly separated [15]. By applying amplified fragment length polymorphism (AFLP) analysis, 80 different Echinochloa spp. samples clustered into three main groups: barnyard grass, early watergrass, and late watergrass [20]. Simple sequence repeats (SSRs) have also been used [16,18,21,22,23]. Lee et al. distinguished the late watergrass from other Echinochloa species by the specific bands amplified by five pairs of SSR markers [16]. Chen et al. discovered 3081 new SSR markers of E. crus-galli by using Restriction Site-Associated DNA (RAD) Illumina sequencing technology [22]. Although these studies have provided some insights into the taxonomic identification of Echinochloa spp., they have not achieved the goal of enabling the effective identification of different barnyard grass species. CDDP (conserved DNA-derived polymorphism) is a target-gene-based molecular marker method. Primers are designed based on the conserved amino acid sequences of functional genes and gene families in plants, enabling the generation of markers with desirable stability, reproducibility, and linkages to target traits [24]. In view of these advantages, it has been widely applied in genetic diversity studies and species identification [24,25,26,27,28]. However, the effectiveness of CDDP markers in the identification of Echinochloa spp. has not yet been investigated.

As one of China’s most critical rice-producing regions, Jiangsu Province holds a pivotal role in China’s rice production. In Jiangsu Province’s rice fields, Echinochloa crus-galli var. crus-galli, E. crus-galli var. mitis, E. crus-galli var. zelayensis, E. glabrescens, and E. caudata are five of the most widespread and severely damaging species [29]. To explore the possibility of targeted herbicide application based on susceptibility differences among Echinochloa species, we first determined the susceptibility of these five Echinochloa species to commonly used herbicides in rice fields. However, in rice fields, different Echinochloa species often grow mixed together. If susceptibility differences exist among the co-occurring species, does there exist any correlation between the growth density of the mixed populations and the control efficacy of herbicides, so as to provide assistance for herbicide selection? Therefore, we further conducted differentiated control studies to explore the impact of the growth density of mixed Echinochloa on the control efficacy. Due to the lack of effective identification methods for the five Echinochloa species during their seedling stage—the critical control period—we employed the CDDP molecular marker method to investigate its potential for discriminating among the five species.

2. Materials and Methods

2.1. Plant Materials

A total of 22 Echinochloa spp. populations were collected from rice-growing areas in Jiangsu province. Among them, six populations were collected from fields that have never been treated with herbicides, including one population each of E. glabrescens, E. crus-galli var. crus-galli, E. caudata populations, E. crus-galli var. zelayensis, and two populations of E. crus-galli var. mitis. All seeds were collected when they naturally matured, then they were air-dried and stored for further testing.

2.2. Susceptibility Assays

A total of 15 herbicides were tested, including 8 postemergence herbicides and 7 pre-emergence herbicides. And details of the herbicide doses are shown in

Table 1. Herbicide doses applied.

Types	Herbicide	Recommended Application Dose (g a.i./ha)	Application Dose (g a.i./ha)
postemergence herbicides	Penoxsulam	15–30	0, 0.94, 1.88, 3.75, 7.50, 15.00
	bispyribac-sodium	30–45	0, 2.81, 5.63, 11.25, 22.50, 45.00
	propyrisulfuron	49.875–78.375	0, 3.44, 6.88, 13.75, 27.50, 55.00
	cyhalofop-butyl	112.5–157.5	0, 2.81, 5.63, 11.25, 22.50, 45.00
	metamifop	105–120	0, 1.88, 3.75, 7.50, 15.00, 30.00
	propanil	2025–2700	0, 56.25, 112.50, 225.00, 450.00, 900.00
	quinclorac	300–375	0, 5.86, 11.72, 23.44, 46.88, 93.75
	halauxifen-methyl	18–36	0, 1.13, 2.25, 4.50, 9.00, 18.00
pre-emergence herbicides	oxyfluorfen	36–72	0, 4.50, 9.00, 18.00, 36.00, 72.00
	pyraclonil	165–210	0, 7.50, 15.00, 30.00, 60.00, 120.00
	oxadiazon	256.5–342	0, 10.69, 21.38, 42.75, 85.50, 171.00
	oxadiargyl	75.6–113.4	0, 2.35, 4.69, 9.38, 18.75, 37.50
	pretilachlor	324.8–526.5	0, 5.06, 10.13, 20.25, 40.50, 81.00
	pendimethalin	866.25–990	0, 3.13, 6.25, 12.50, 25.00, 50.00
	mefenacet	375–450	0, 10.00, 20.00, 40.00, 80.00, 160.00

. Six populations that have never been treated with herbicides were used for assays. Twenty seeds were sown in each plastic pot mixed with sand and organic matter (1:2), and then cultured in greenhouses (day/night temperature regime of 30 °C/25 °C and 12 h photoperiod) and herbicide spraying was carried out at an appropriate time by a laboratory sprayer (machine model: 3WP-2000, Nanjing Research Institute for Agricultural Mechanization, Nanjing, National Ministry of Agriculture of China). For postemergence herbicides, herbicide application was conducted when plants reached the 3–4 leaf stage. For pre-emergence herbicides, application was performed within 24–48 h post-sowing of barnyard grass seeds. After herbicide spraying, all the plants were moved back to the greenhouse for continuous cultivation. After 21 days of spraying, the aboveground parts of the plants were collected and the fresh weights were weighed.

According to the measured fresh weight data of the aboveground parts, the inhibition rate of fresh weight was calculated using the following equation.

$$\mathrm{P}\mathrm{e}\mathrm{r}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{g}\mathrm{e} \mathrm{i}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}(\%)={\displaystyle \frac{\mathrm{control\; treatment}}{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}}\times 100$$

(1)

Then the data were subjected to Linear regression equation analysis to determine the effective herbicide dose that causes the 50% inhibition of fresh weight (ED₅₀) through DPS software v20.05 using Equation (2).

$$\mathrm{Y}=\mathrm{A}+\mathrm{B}\mathrm{X}$$

(2)

where Y represents the probit value; A is the intercept; B is the regression coefficient; and X is the base 10 logarithm of herbicide doses.

To compare the differences in ED₅₀ among different species, Duncan’s multiple range test (p < 0.05) was used. This experiment was conducted twice in a completely randomized design with three replicates.

2.3. Differentiated Management Strategy Based on the Varied Susceptibility

According to the results of the susceptibility assays, the susceptibility of different Echinochloa species varied under the treatments of penoxsulam, propyrisulfuron, cyhalofop-butyl, metamifop, propanil, and quinclorac. Therefore, Echinochloa species showing sensitivity differences to these postemergence herbicides were paired up for differential control assays. We set seven density ratios (relatively insusceptible: susceptible) of 0:1, 1:4, 1:2, 1:1, 2:1, 4:1, and 1:0 to explore the relationship between the density ratio of two Echinochloa species and the susceptibility of mixed populations. Seeds of two Echinochloa species were separated by small plastic cards and sown in the same plastic pot at density ratios of 1:0, 4:1, 2:1, 1:1, 1:2, 1:4, and 0:1. Herbicides were applied when the seedlings of the two Echinochloa species reached the 3–4 leaf stag. Plant cultivation and herbicide spraying followed the same methods as those described in Section 2.2. After 21 days of herbicide treatment, the fresh weight of the aboveground part was weighed. The ED₅₀ values of different mixed Echinochloa populations were calculated using DPS software v20.05, and Duncan’s multiple range test (p < 0.05) was performed to analyze the significant differences. This experiment was conducted twice in a completely randomized design with three replicates.

2.4. CDDP Analysis

2.4.1. DNA Extraction

The plants were raised up to the 3-to-4-leaf stage for the extraction of DNA. Total genomic DNA was extracted using the CTAB method, according to Lee et al. (2016) [16]. Quality of isolated DNA samples was checked on a 1% agarose gel electrophoresis. The concentrations and relative purities were checked using an ultraviolet spectrophotometer. All the DNA samples were diluted with sterile distilled water and adjusted to 100 ng/µL and stored at −20 °C for further use.

2.4.2. PCR Amplification

Seven primers with clear amplified bands and high polymorphism were selected from 21 CDDP primers for the identification of five barnyard grass species [24]. PCR amplification was performed in a 20 µL reaction, containing 10 µL of 2xTaq Master Mix (with dye), 1 µL of 100 ng/µL DNA template, 0.8 µL of 10 µmol/mL primer, and 8.2 µL of ddH₂O. Then the reaction was carried out as follows: an initial denaturation step at 94 °C for 3 min, followed by 35 cycles of 94 °C for 1 min, the corresponding annealing temperature for 1 min, and 72 °C for 2 min. A final extension cycle at 72 °C for 10 min followed. PCR products were separated by 1.5% agarose gel electrophoresis at 100 Volt for 1 h. The fragment patterns were photographed under UV light for further analysis.

2.4.3. Data Analyses

To perform statistical analysis on the amplification results, 0 represents band absence, while 1 represents the presence of a band, and the data were transformed into a 1/0 binary character matrix. The genetic similarity coefficients between the materials were calculated using NTSYS 2.10 software, and cluster analysis was conducted using Unweighted Pair Group Mean Arithmetic (UPGMA) and followed by bootstrap analysis with 1000 permutations.

3. Results

3.1. Susceptibility Assays

3.1.1. Susceptibility to Postemergence Herbicides

We evaluated the sensitivity of five Echinochloa species to eight commonly used postemergence herbicides, including pentaflusulam, bispyribac-sodium, propyrisulfuron, cyhalofop-butyl, metamifop, propanil, quinclorac, and halauxifen-methyl. The results are shown in

Table 2. Sensitivity to postemergence herbicides. Different lowercase letters indicate significant differences (p < 0.05) among Echinochloa populations under herbicide treatment.

Herbicide	Population	Virulence Regression Equation	Correlation Coefficient	ED50 (g a.i./ha)	95% Confidence Interval
Penoxsulam	E. crus-galli var. mitis 1	Y = 5.19 + 1.47X	0.99	0.75 d	0.56~1.00
	E. crus-galli var. mitis 2	Y = 5.21 + 1.27X	0.99	0.68 d	0.51~0.90
	E. glabrescens	Y = 5.02 + 1.30X	0.99	0.97 cd	0.75~1.25
	E.caudata	Y = 4.71 + 2.37X	0.98	1.32 b	1.00~1.75
	E. crus-galli var. crus-galli	Y = 4.84 + 2.81X	0.99	1.14 bc	0.92~1.43
	E. crus-galli var. zelayensis	Y = 4.51 + 1.85X	0.96	1.83 a	1.30~2.59
bispyribac-sodium	E. crus-galli var. mitis 1	Y = 4.28 + 1.71X	0.98	2.65 b	1.85~3.78
	E. crus-galli var. mitis 2	Y = 4.39 + 1.34X	0.99	2.87 b	2.26~3.63
	E. glabrescens	Y = 4.9 + 1.04X	0.98	1.24 d	0.62~2.46
	E.caudata	Y = 3.22 + 3.11X	1.00	3.73 a	3.73~3.74
	E. crus-galli var. crus-galli	Y = 2.83 + 3.53X	0.99	4.13 a	3.44~4.96
	E. crus-galli var. zelayensis	Y = 4.17 + 2.53X	1.00	2.12 c	1.85~2.42
propyrisulfuron	E. crus-galli var. mitis 1	Y = 4.22 + 1.14X	0.98	4.84 b	3.62~6.47
	E. crus-galli var. mitis 2	Y = 4.27 + 0.97X	1.00	5.74 b	4.96~6.65
	E. glabrescens	Y = 4.23 + 1.02X	0.98	5.66 b	4.16~7.71
	E.caudata	Y = 3.35 + 1.58X	0.98	11.12 a	9.17~13.49
	E. crus-galli var. crus-galli	Y = 3.14 + 1.68X	0.96	12.70 a	9.32~17.30
	E. crus-galli var. zelayensis	Y = 3.93 + 0.99X	0.99	12.01 a	10.30~14.00
cyhalofop-butyl	E. crus-galli var. mitis 1	Y = 1.23 + 2.77X	0.97	23.03 a	17.22~30.81
	E. crus-galli var. mitis 2	Y = 0.38 + 3.28X	0.99	25.45 a	20.96~30.89
	E. glabrescens	Y = 1.25 + 2.76X	0.98	22.92 a	18.82~27.90
	E.caudata	Y = 1.96 + 2.77X	0.98	12.49 b	9.84~15.85
	E. crus-galli var. crus-galli	Y = 3.49 + 1.60X	0.99	8.70 c	7.59~9.97
	E. crus-galli var. zelayensis	Y = 2.12 + 2.38X	0.98	16.10 b	12.62~20.55
metamifop	E. crus-galli var. mitis 1	Y = −2.93 + 6.00X	0.98	21.01 a	16.05~27.49
	E. crus-galli var. mitis 2	Y = −0.06 + 3.98X	1.00	18.65 a	17.84~19.50
	E. glabrescens	Y = 3.36 + 1.53X	0.97	11.72 b	8.89~15.46
	E.caudata	Y = 0.75 + 5.04X	0.99	6.97 c	5.55~8.75
	E. crus-galli var. crus-galli	Y = 2.92 + 2.67X	0.93	6.03 c	2.66~13.67
	E. crus-galli var. zelayensis	Y = 0.82 + 4.85X	0.99	7.28 c	5.42~9.76
propanil	E. crus-galli var. mitis 1	Y = −2.86 + 3.44X	0.96	192.65 b	136.23~272.43
	E. crus-galli var. mitis 2	Y = −1.52 + 2.87X	0.95	185.71 b	125.83~274.09
	E. glabrescens	Y = −2.85 + 3.21X	0.96	280.21 a	197.73~397.07
	E.caudata	Y = −0.84 + 2.64X	0.97	162.78 bc	117.48~225.54
	E. crus-galli var. crus-galli	Y = 1.62 + 1.55X	0.98	149.65 c	117.81~190.09
	E. crus-galli var. zelayensis	Y = 0.33 + 2.15X	0.98	150.08 c	117.89~191.05
quinclorac	E. crus-galli var. mitis 1	Y = 1.73 + 3.10X	0.98	11.34 c	9.07~14.18
	E. crus-galli var. mitis 2	Y = 2.62 + 2.15X	1.00	12.79 c	11.89~13.75
	E. glabrescens	Y = 2.37 + 2.11X	0.99	17.63 b	14.64~21.23
	E.caudata	Y = 0.70 + 2.94X	0.96	28.96 a	21.18~39.6
	E. crus-galli var. crus-galli	Y = 1.32 + 2.57X	0.99	26.99 a	24.07~30.25
	E. crus-galli var. zelayensis	Y = 2.46 + 1.98X	0.99	19.20 b	17.03~21.65
halauxifen-methyl	E. crus-galli var. mitis 1	Y = 4.53 + 1.40X	0.99	2.17 a	1.80~2.60
	E. crus-galli var. mitis 2	Y = 4.47 + 1.55X	0.99	2.20 a	1.74~2.79
	E. glabrescens	Y = 4.45 + 1.58X	0.99	2.24 a	1.76~2.85
	E.caudata	Y = 3.62 + 3.88X	0.99	2.27 a	2.01~2.56
	E. crus-galli var. crus-galli	Y = 4.56 + 1.97X	0.99	1.68 a	1.34~2.11
	E. crus-galli var. zelayensis	Y = 4.53 + 2.09X	0.96	1.68 a	1.20~2.37

. Except for halauxifen-methyl, the five Echinochloa species exhibited differences in susceptibility to the remaining seven herbicides. For example, under the treatment with bispyribac-sodium, the ED₅₀ values of the five Echinochloa species from highest to lowest are as follows: E. crus-galli var. zelayensis (1.83 g a.i./ha), E.caudata(1.32 g a.i./ha), E. crus-galli var. crus-galli (1.14 g a.i./ha), E. glabrescens (0.97 g a.i./ha), and E. crus-galli var. mitis(0.68 and 0.75 g a.i./ha). The five Echinochloa species were classified into four groups based on susceptibility differences: E. crus-galli var. zelayensis was the most susceptible, followed by E. crus-galli var. mitis, then E. glabrescens, with E. caudata being the least susceptible. However, under the treatment with bispyribac-sodium, the ED₅₀ values of the five Echinochloa species from highest to lowest are as follows: E. crus-galli var. crus-galli (4.13 g a.i./ha), E.caudata (3.73 g a.i./ha), E. crus-galli var. mitis (2.87 and 2.65 g a.i./ha), E. crus-galli var. zelayensis (2.12 g a.i./ha), and E. glabrescens (1.24 g a.i./ha). The five Echinochloa species were classified into four groups based on susceptibility differences: E. glabrescens was the most susceptible, followed by E. crus-galli var. zelayensis, then E. crus-galli var. mitis, E. caudata, and E. crus-galli var. crus-galli are the least susceptible.

3.1.2. Susceptibility to Pre-Emergence Herbicides

We evaluated the sensitivity of five Echinochloa species to seven commonly used pre-emergence herbicides, including oxyfluorfen, pyraclonil, oxadiazon, oxadiargyl, pretilachlor, pendimethalin, and mefenacet. The results are shown in

Table 3. Susceptibility to preemergence herbicides. Different lowercase letters indicate significant differences (p < 0.05) among Echinochloa populations under herbicide treatment.

Herbicide	Population	Virulence Regression Equation	Correlation Coefficient	ED50 (g a.i./ha)	95% Confidence Interval
oxyfluorfen	E. crus-galli var. mitis 1	Y = 1.70 + 3.47X	0.98	8.95 b	7.20~11.11
	E. crus-galli var. mitis 2	Y = 0.84 + 4.58X	0.97	8.10 b	6.03~10.89
	E. glabrescens	Y = 1.90 + 2.72X	0.97	13.79 a	9.99~19.04
	E. caudata	Y = 2.32 + 3.27X	1.00	6.58 c	5.82~7.45
	E. crus-galli var. crus-galli	Y = −1.46 + 7.47X	1.00	7.33 bc	6.55~8.19
	E. crus-galli var. zelayensis	Y = −1.12 + 7.27X	1.00	6.95 c	6.80~7.11
pyraclonil	E. crus-galli var. mitis 1	Y = 1.47 + 3.01X	0.95	14.87 a	10.27~21.54
	E. crus-galli var. mitis 2	Y = 1.60 + 3.05X	0.99	13.02 a	11.43~14.84
	E. glabrescens	Y = 0.51 + 3.96X	0.93	13.62 a	8.40~22.11
	E. caudata	Y = 1.23 + 3.50X	0.93	11.92 a	7.52~18.88
	E. crus-galli var. crus-galli	Y = 0.50 + 3.86X	0.98	14.63 a	11.50~18.62
	E. crus-galli var. zelayensis	Y = 0.96 + 3.80X	0.99	11.61 a	9.45~14.26
oxadiazon	E. crus-galli var. mitis 1	Y = 1.55 + 2.52X	0.99	23.35 a	19.95~27.32
	E. crus-galli var. mitis 2	Y = 0.45 + 3.05X	0.97	30.93 a	23.35~40.96
	E. glabrescens	Y = 2.63 + 1.94X	0.96	16.70 b	10.80~25.83
	E. caudata	Y = 1.14 + 3.04X	1.00	18.66 b	16.71~20.84
	E. crus-galli var. crus-galli	Y = 1.53 + 2.90X	1.00	15.68 b	15.63~15.73
	E. crus-galli var. zelayensis	Y = −0.27 + 4.31X	0.97	16.69 b	12.26~22.74
oxadiargyl	E. crus-galli var. mitis 1	Y = 3.26 + 2.12X	0.95	6.63 a	4.57~9.63
	E. crus-galli var. mitis 2	Y = 3.08 + 2.34X	0.97	6.63 a	4.93~8.93
	E. glabrescens	Y = 2.21 + 3.03X	0.97	8.32 a	6.22~11.13
	E. caudata	Y = 3.25 + 2.03X	0.97	7.29 a	5.69~9.35
	E. crus-galli var. crus-galli	Y = 2.18 + 3.79X	0.98	5.55 b	4.53~6.78
	E. crus-galli var. zelayensis	Y = 3.45 + 2.25X	0.97	4.87 b	3.65~6.52
pretilachlor	E. crus-galli var. mitis 1	Y = 2.59 + 1.87X	0.99	19.51 cd	17.20~22.14
	E. crus-galli var. mitis 2	Y = 2.31 + 2.12X	0.98	18.58 cd	14.65~23.57
	E. glabrescens	Y = 2.09 + 2.19X	0.98	21.22 bc	17.12~26.31
	E. caudata	Y = −0.24 + 3.58X	1.00	28.97 a	27.25~30.79
	E. crus-galli var. crus-galli	Y = 0.14 + 3.56X	0.96	23.08 b	17.01~31.31
	E. crus-galli var. zelayensis	Y = −0.28 + 4.3X	1.00	16.93 d	16.07~17.83
pendimethalin	E. crus-galli var. mitis 1	Y = 2.20 + 2.84X	0.93	9.70 b	6.11~15.42
	E. crus-galli var. mitis 2	Y = 0.74 + 4.01X	0.98	11.52 b	8.84~15.02
	E. glabrescens	Y = 2.28 + 2.79X	0.93	9.45 b	5.94~15.04
	E. caudata	Y = −0.58 + 4.51X	0.97	17.27 a	13.39~22.27
	E. crus-galli var. crus-galli	Y = 3.37 + 2.59X	1.00	4.25 c	4.11~4.39
	E. crus-galli var. zelayensis	Y = 1.52 + 4.54X	0.99	5.86 c	4.92~6.98
mefenacet	E. crus-galli var. mitis 1	Y = −4.24 + 5.33X	0.92	54.25 a	30.24~97.32
	E. crus-galli var. mitis 2	Y = −2.3 + 4.28X	0.98	50.93 a	37.06~70.00
	E. glabrescens	Y = −3.42 + 4.83X	1.00	55.16 a	53.36~57.03
	E. caudata	Y = 2.04 + 1.73X	1.00	51.83 a	45.51~59.03
	E. crus-galli var. crus-galli	Y = −0.51 + 3.19X	0.96	53.36 a	36.37~78.28
	E. crus-galli var. zelayensis	Y = −5.03 + 5.66X	0.94	59.07 a	37.29~93.56

. Except for oxadiargyl and mefenacet, the five Echinochloa species exhibited susceptibility differences to the remaining five pre-emergence herbicides.

To more intuitively illustrate the susceptibility differences in five Echinochloa species to 15 herbicides, we summarized the ED₅₀ value results (

Table 4. Susceptibility of five Echinochloa species to 15 commonly used herbicides. Green indicates the most susceptible status of Echinochloa species to herbicides, orange represents the least susceptible, light green denotes sub-susceptible, and light orange signifies the sub-least susceptible. Different lowercase letters indicate significant differences (p < 0.05) among Echinochloa populations under herbicide treatment.

Herbicides	ED50 (g a.i./ha)
	E. crus-galli var. mitis	E. glabrescens	E. caudata	E. crus-galli var. crus-galli	E. crus-galli var. zelayensis
Penoxsulam	0.715 d	0.97 cd	1.32 b	1.14 bc	1.83 a
bispyribac-sodium	2.76 b	1.24 d	3.73 a	4.13 a	2.12 c
propyrisulfuron	5.29 b	5.66 b	11.12 a	12.70 a	12.01 a
cyhalofop-butyl	24.24 a	22.92 a	12.49 b	8.70 c	16.10 b
metamifop	19.83 a	11.72 b	6.97 c	6.03 c	7.28 c
propanil	189.18 b	280.21 a	162.78 bc	149.65 c	150.08 c
quinclorac	12.065 c	17.63 b	28.96 a	26.99 a	19.20 b
halauxifen-methyl	2.185 a	2.24 a	2.27 a	1.68 a	1.68 a
oxyfluorfen	8.525 b	13.79 a	6.58 c	7.33 bc	6.95 c
pyraclonil	13.945 a	13.62 a	11.92 a	14.63 a	11.61 a
oxadiazon	27.14 a	16.70 b	18.66 b	15.68 b	16.69 b
oxadiargyl	6.63 a	8.32 a	7.29 a	5.55 b	4.87 b
pretilachlor	19.045 cd	21.22 bc	28.97 a	23.08 b	16.93 d
pendimethalin	10.61 b	9.45 b	17.27 a	4.25 c	5.86 c
mefenacet	52.59 a	55.16 a	51.83 a	53.36 a	59.07a

). Based on the results of significance difference analysis, different susceptibility groups were color-coded to facilitate herbicide selection for field control. For example, to control E. crus-galli var. mitis, pentaflusulam, propyrisulfuron, and quinclorac are recommended, while cyhalofop-butyl, metamifop, oxadiazon, and oxadiargyl are not advised.

3.2. Differentiated Management

Based on the susceptibility of five Echinochloa species to postemergence herbicides, we conducted differentiated management, and the results are shown in

Table 5. Susceptibility of mixed populations under different density ratios of two Echinochloa species. Different lowercase letters indicate significant differences (p < 0.05) among Echinochloa populations under herbicide treatment.

Herbicide-Mixed Population (Insusceptible: Susceptible)	Density Ratio	Virulence Regression Equation	Correlation Coefficient	ED50 (g a.i./ha)	95% Confidence Interval
Penoxsulam E. crus-galli var. zelayensis: E. crus-galli var. mitis	1:0	Y = 4.2533 + 2.2983X	0.9964	2.1130 a	1.8327~2.4362
	4:1	Y = 4.8008 + 1.5794X	0.9984	1.3369 c	1.2582~1.4205
	2:1	Y = 4.5455 + 2.5502X	0.9862	1.5074 b	1.2055~1.8849
	1:1	Y = 5.1554 + 2.4560X	0.9817	0.8644 d	0.6955~1.0743
	1:2	Y = 4.9708 + 2.4405X	0.9903	1.0279 cd	0.8774~1.2042
	1:4	Y = 4.8717 + 3.2007X	0.9729	1.0967 cd	0.7628~1.5767
	0:1	Y = 5.2297 + 3.1821X	0.9467	0.8468 d	0.5771~1.2426
propyrisulfuron E. crus-galli var. crus-galli: E. glabrescens	1:0	Y = 3.3650 + 1.2136X	0.9892	22.2466 a	18.8446~26.2628
	4:1	Y = 2.4674 + 1.9346X	0.9954	20.3754 a	17.2928~24.0075
	2:1	Y = 1.9718 + 2.1943X	0.9662	23.9900 a	17.7325~32.4556
	1:1	Y = 3.0537 + 1.4776X	0.9946	20.7589 a	18.4775~23.3220
	1:2	Y = 3.2557 + 1.4138X	0.9954	17.1313 b	15.3807~19.0811
	1:4	Y = 4.3006 + 0.6796X	0.9919	10.6952 c	8.6040~13.2947
	0:1	Y = 4.2305 + 0.7403X	0.9991	10.9501 c	10.2080~11.7461
cyhalofop-butyl E. crus-galli var. mitis: E. crus-galli var. crus-galli	1:0	Y = 2.0743 + 2.4074X	0.9990	16.4170 a	15.3109~17.6029
	4:1	Y = 2.7572 + 2.1053X	0.9511	11.6226 b	7.9473~16.9976
	2:1	Y = 2.2521 + 2.4991X	0.9857	12.5760 b	10.3683~15.2538
	1:1	Y = 1.8835 + 3.0530X	0.9943	10.4903 c	9.173~11.9968
	1:2	Y = 2.3532 + 2.7892X	0.9886	8.8907 d	7.1504~11.0545
	1:4	Y = 2.7045 + 2.7606X	0.9992	6.7845 e	6.3025~7.3034
	0:1	Y = 3.0005 + 2.8553X	0.9861	5.0151 e	4.0355~6.2326
cyhalofop-buty E. crus-galli var. mitis: E.caudata	1:0	Y = 3.1231 + 1.3517X	0.9594	24.4655 a	17.6461~33.9203
	4:1	Y = 2.9259 + 1.7391X	0.9910	15.5830 b	13.4034~18.117
	2:1	Y = 3.3980 + 1.4686X	0.9935	12.3260 c	10.8457~14.0083
	1:1	Y = 3.6343 + 1.4300X	0.9917	9.0162 d	7.6979~10.5603
	1:2	Y = 2.7789 + 2.1848X	0.9948	10.3899 d	8.9957~12.0002
	1:4	Y = 2.3601 + 2.5423X	0.9529	10.9244 d	7.0933~16.8247
	0:1	Y = 2.4687 + 2.5420X	0.9970	9.9032 d	9.0402~10.8486
metamifop E. crus-galli var. mitis: E. crus-galli var. zelayensis	1:0	Y = −0.0581 + 2.9620X	0.9954	51.0134 a	45.6783~56.9716
	4:1	Y = 1.0548 + 2.2934X	1.0000	52.5134 a	51.9846~53.0477
	2:1	Y = 1.1166 + 2.7486X	0.9950	25.8731 b	22.7648~29.4057
	1:1	Y = 2.9343 + 1.4383X	0.9757	27.3014 b	20.6866~36.03149
	1:2	Y = 2.8387 + 1.6502X	0.9975	20.4036 c	18.3617~22.6726
	1:4	Y = 2.0271 + 2.2261X	0.9786	21.6492 c	16.0549~29.1927
	0:1	Y = 2.3867 + 1.9226X	0.9949	22.8702 c	20.1994~25.8941
metamifop E. crus-galli var. mitis: E. caudata	1:0	Y = 0.9637 + 2.0969X	0.9851	84.1160 a	67.1544~105.3617
	4:1	Y = 0.6250 + 2.2914X	0.9993	81.1594 a	77.5162~84.9739
	2:1	Y = 0.6676 + 2.3786X	0.9946	66.2814 b	58.9578~74.5148
	1:1	Y = 0.2146 + 2.7466X	1.0000	55.2436 b	54.9648~55.5238
	1:2	Y = 2.2966 + 1.7000X	0.9839	38.9267 c	30.2018~50.1720
	1:4	Y = 1.8724 + 2.2007X	0.9974	26.3743 c	24.2972~28.6290
	0:1	Y = 1.4763 + 2.4064X	0.9586	29.1276 c	20.4292~41.5297
propanil E. glabrescens: E. crus-galli var. zelayensis	1:0	Y = 0.2772 + 2.0243X	0.9661	215.3064 a	160.2969~289.1937
	4:1	Y = 0.7277 + 1.9250X	0.9985	165.7272 b	156.3469~175.6702
	2:1	Y = 1.397 + 1.6655X	0.9932	145.6589 b	127.6685~166.1846
	1:1	Y = 1.2011 + 1.8354X	0.9970	117.4235 c	106.7627~129.1489
	1:2	Y = 1.9868 + 1.5111X	0.9922	98.6234 c	83.1605~116.9613
	1:4	Y = 1.5821 + 1.8302X	0.9028	73.7042 d	40.3179~134.7370
	0:1	Y = 2.7067 + 1.2212X	0.9553	75.5064 d	51.2198~111.3087
quinclorac E. caudata: E. crus-galli var. zelayensis	1:0	Y = −0.0776 + 3.3438X	0.9941	33.0010 a	28.6176~38.0559
	4:1	Y = 1.5124 + 2.2586X	0.9902	35.0096 a	29.35181~41.7579
	2:1	Y = 0.3549 + 3.0226X	0.9985	34.4195 a	32.0882~36.9201
	1:1	Y = 1.4532 + 2.4561X	0.9789	27.8021 b	22.0934~34.9860
	1:2	Y = 2.1334 + 2.0953X	0.9979	23.3406 b	21.6217~25.1962
	1:4	Y = 1.2698 + 2.6918X	0.9848	24.3093 b	19.8260~29.8064
	0:1	Y = 1.0080 + 2.9992X	0.9449	21.4294 c	14.52078~31.6249
quinclorac E. caudata: E. crus-galli var. mitis	1:0	Y = 2.4716 + 1.6795X	0.9510	32.0211 a	20.7263~49.4712
	4:1	Y = 2.6965 + 1.5904X	0.9847	28.0807 a	22.8482~34.5115
	2:1	Y = 2.2990 + 1.9920X	0.9974	22.6945 b	20.9469~24.5878
	1:1	Y = 2.5484 + 1.8630X	0.9998	20.6991 b	20.1952~21.2156
	1:2	Y = 2.2268 + 2.4624X	1.0000	13.3721 c	13.3086~13.4359
	1:4	Y = 1.9424 + 3.1237X	0.9737	9.5247 d	7.2235~12.5589
	0:1	Y = 1.9706 + 3.2479X	0.9977	8.5651 d	7.8574~9.3365

. As the density ratio of the two Echinochloa species changes, the ED₅₀ of the mixed population to herbicides also varies. When the density ratio of the two Echinochloa species is between 1:4 and 2:1, it may have a significant impact on the susceptibility of the mixed population to herbicides. For instance, under the treatment of quinclorac, when the density ratio of Echinochloa caudata and Echinochloa crus-galli var. zelayensis is 1:4, it begins to have a significant impact on the susceptibility of the mixed population. Under the treatment of propyrisulfuron, when the density ratio of Echinochloa crus-galli var. crus-galli and Echinochloa glabrescens is 1:2., it begins to have a significant impact on the susceptibility of the mixed population.

3.3. CDDP

Among the 21 primers, 7 primers with clear, stable and polymorphic bands were selected to amplify 22 populations of barnyard grass. A total of 82 loci were amplified, and an average of 11.71 loci were amplified by each primer. A total of 25 polymorphic sites were amplified, with an average of 3.57 per primer. The percentage of primer polymorphisms ranged from 15.38% to 60.00%, with an average of 34.5786% (

Table 6. CDDP primer information.

Primer	Primer Sequences (5′-3′)	Length (bp)	GC Content (%)	Annealing Temperature (°C)
WRKY-R2B	TGSTGSATGCTCCCG	15	67	53.7
ERF2	GCSGAGATCCGSGACCC	17	77	55.1
ERF3	TGGCTSGGCACSTTCGA	17	65	49.5
KNOX-3	AAGCGSCACTGGAAGCC	17	65	53.7
ABP1-1	ACSCCSA TCCACCGC	15	73	59.0
ABP1-2	ACSCCSA TCCACCGG	15	73	47.0
ABP1-3	CACGAGGACCTSCAGG	16	69	56.6

and

Table 7. CDDP amplification sequence information.

Primer	Number of Total Amplified Bands	Number of Polymorphic Bands	Polymorphism Band Percentage
WRKY-R2B	13	2	15.38
ERF2	15	7	46.67
ERF3	14	4	28.57
KNOX-3	15	3	20.00
ABP1-1	6	3	50.00
ABP1-2	14	3	21.43
ABP1-3	5	3	60.00
Total	82.0000	25	242.05
Average	11.7143	3.5714	34.5786

). In seven primers, primer KNOX-3 at 2000 bp produced a specific missing band that can distinguish E. caudata from other barnyard grass species (Figure 1).

The genetic similarity coefficient between the 22 barnyard weed populations ranged from 0.8840 to 0.9932. According to the genetic similarity coefficient among various populations, the Unweighted Pair Group Mean Arithmetic (UPGMA) was used for cluster analysis. At the similarity coefficient of 0.93, the 22 tested populations were divided into two groups, and the four populations of E. caudata were clustered into one group, while the other four barnyard grass species were clustered into another group (Figure 2).

4. Discussion

Among the 15 herbicides tested, significant susceptibility differences (p < 0.05) were observed in five Echinochloa species against 12 of the herbicides, confirming the universality of interspecific susceptibility variations in Echinochloa to herbicides. Morphological disparities may account for the differential susceptibility. Studies have shown that compared with E. oryzicola, the cuticle of E. crus-galli is 1.8 times thicker, and its wax components are mainly alkanes and primary alcohols, forming a dense physical and chemical barrier, which greatly hinders the penetration of herbicides and enhances its resistance to herbicides [30]. In addition, the cuticle surface of E. crus-galli has obvious papillary protrusions, further strengthening the mechanical barrier function and making it difficult for herbicides to penetrate the epidermis and enter into the plant [31]. In terms of root system structure, significant differences also exist among different Echinochloa species. Field trials with butachlor show that E. colona remains poorly controlled due to its shallow root distribution and low root hair density. Conversely, E. crus-galli achieves effective herbicide uptake via its deep-reaching roots and dense lateral roots, leading to 65% higher butachlor accumulation in shoots compared to E. colona [32]. However, the differentiation of weed susceptibility to herbicides is an extremely complex process. In addition to morphological characteristics, physiological and biochemical properties may also play an important role. There are significant differences in the activity of detoxifying enzyme systems in different Echinochloa species, such as cytochrome P450 monooxygenases, glutathione S-transferases (GSTs), and carboxylesterases which resulted in differences in herbicide sensitivity [33]. Meanwhile, gene mutations at target sites may also be key factors contributing to the sensitivity differences among different Echinochloa species [34,35]. However, the specific causes leading to differences in herbicide susceptibility among different Echinochloa species still need further investigation.

Clarifying the interspecific susceptibility differences among Echinochloa species holds significant guiding significance for integrated weed management. For instance, when Echinochloa crus-galli var. mitis is detected in the field, penoxsulam is more recommended than cyhalofop-butyl. However, in practical field scenarios, different Echinochloa species often co-occur. Differential control trials have confirmed that when the density ratio of the two Echinochloa species is between 1:4 and 2:1, it will have a significant impact on the susceptibility of the mixed population to herbicides. This indicates that in mixed populations, when the density of relatively less susceptible Echinochloa species exceeds 20%, herbicide replacement or mixed application should be implemented. This threshold provides a critical decision-making basis for the precise management of mixed population of Echinochloa species. When propyrisulfuron is used to control the mixed populations of Echinochloa glabrescens and E. crus-galli var. crus-galli, a satisfactory control effect may can be achieved when the density of E. crus-galli var. crus-galli is below 20%. However, when its density exceeds 20%, it is advisable to consider replacing it with other herbicides, such as cyhalofop-butyl, or using a mixture of pyrazosulfuron and cyhalofop-butyl to enhance the weed control efficacy.

However, the successful implementation of such strategies hinges on accurate knowledge of the Echinochloa species composition in the field. Early and precise identification of Echinochloa species using molecular markers would empower growers to promptly optimize their herbicide application protocols. Although morphological identification of Echinochloa is challenging, no specific molecular markers have been found to resolve this issue. Similarly, in this study, CDDP (conserved DNA-derived polymorphism) markers only distinguish Echinochloa caudata from the other four Echinochloa species, and the generality and effectiveness of these markers in identifying other Echinochloa species still needs to be further evaluated. On the one hand, this is because the polyploid nature and complex genomes of most Echinochloa species significantly complicate the development and interpretation of molecular markers, often leading to ambiguous results due to gene redundancy and high sequence homology [14,36]. On the other hand, Echinochloa spp. from different geographical regions and ecological environments may exhibit substantial genetic divergence, limiting the universality of existing molecular markers across various Echinochloa ecotypes [23,37]. In the future, it is necessary to develop efficient molecular markers that are suitable for analyzing complex polyploid genomes to achieve the classification and identification of different Echinochloa species.

5. Conclusions

Our study results indicate that universal susceptibility to herbicides exists among five Echinochloa species. Among the 15 herbicides tested, 12 showed significant differences in susceptibility. This suggests that when controlling field Echinochloa weeds, it is necessary to first identify the specific Echinochloa species, then apply herbicides in a targeted manner based on their susceptibility profiles to reduce herbicide usage. If mixed populations of different Echinochloa species occur in the field, their occurrence densities should be surveyed to determine whether herbicide mixture or replacement is needed, ensuring overall control efficacy. This study used CDDP markers to distinguish Echinochloa colona from four other Echinochloa species. However, to identify the species composition of Echinochloa weeds in the field, additional effective molecular marker methods need to be explored.