The barnyard grass is a worldwide malignant weed[1], and one of the 15 kinds of serious weeds in farmland in our country[2], and the harm to the production of rice is particularly serious, and the harmed area of paddy rice field alone reaches 14 million hm2[3]. According to Wang Qiang et al [4] on the occurrence of weeds in rice paddies in Zhejiang Province and the harm of the survey showed that barnyard grass is the main malignant weeds in rice paddies. In the early rice field, the frequency of barnyard grass was 72%~100%, and the damage rate of the second grade and above reached 23.8%~50.0%. In the late rice field, the frequency of barnyard grass is 37.2%~74.7%, and the damage rate of the second grade and above is 8.3%~25.1%. The infestation in rice fields was basically similar to that in large fields. According to Hu Jinsheng et al [5], hybrid rice Shanyou 63 seedlings in each hole 3~4 clip 1 barnyard grass, rice yield reduction of 62.90% on average. The chemical weed control technology system in rice fields has the characteristics of good prevention and eradication effect, broad spectrum of grass killing, high safety and low cost. However, with the increase of herbicide use years, barnyard grass through the selection pressure of pharmaceuticals and
genetic role, produced the problem of drug resistance. The resistance ratio of barnyardgrass in the rice area of China was 1.27~5.42 in the area where butachlor was used for 8~12 years[6]. In the area of continuous use of grass dan for more than 10 years, the resistance ratio of barnyard grass to grass dan was more than 2.8 [7]. In Anxiang County, Hunan Province, barnyardgrass showed obvious resistance to dichloroquinolinic acid, and its resistance ratio was as high as 28.74 [8]. These studies showed that barnyardgrass in paddy fields in China showed some resistance to these three herbicides. At present, the main herbicides used in paddy fields in China are butachlor, acetamiprid, Gramoxone, dichloroquinolinic acid, Sauter, etc., as well as the mixtures of bensulfuron-methyl and barnyardicides[9], so it is imperative to adopt a rapid and sensitive bioassay to monitor the resistance and to select the suitable herbicides for the management of the resistance. In this paper, the sensitivity of three bioassays, namely, filter paper method, agar method and potting spray method, was compared with that of
dichloroquinolinic acid, and the resistance of barnyardgrass to pyrazosulfuron, pentafluorosulfuron, propargyl ketone, tetrazoylpyrifos
amine, diclofenac, zinc oxon, penconazole, and diclofenac were measured by the filter paper method and agar method according to the treatment time and the sensitivity of the agar method, The sensitivity of seven herbicides, namely, sulfosulfuron-methyl, propargyl, tetrazolium, dichloroquinolinic acid, zinhydrin, and pramipexole, was investigated, and the results were used as a basis for the bioassay of the herbicides, and for the control of barnyardgrass in paddy fields.
1 Materials and methods
1.1 Barnyard grass
The seeds of barnyard grass (Echinochloa crusgalli) were collected from the suburb of Zhuji City, Zhejiang Province, China, in 2002, dried in the sun, and then put into a paper bag and stored in a refrigerator
for use.
1.2 Test agents
10% pyrazosulfuron WP (pyrazosulfuron-ethyl, State-owned Kunshan Chemical Factory); 2.5% pentafluorosulfuron OF (penoxsulam, Dow Yick Yee Agro); 80% propargite. 80% Propargyl WG (oxadiargyl, trade name
for Inosida, Rh?ne-Poulenc Agrochemicals); 50% tetrazolium WP (fentrazamide, trade name Baytan, Bayer
Germany); 50% dichloroquinaldic acid WP (quinclorac, trade name
Company); 50% dichloroquinaldic acid WP (quinclorac, trade name
Company); 50% dichloroquinaldic acid WP (quinclorac, trade name
Company).
Ki Taoyuan Wang, Jiangsu Xinyi Zhongkai Agricultural Chemical Co., Ltd.);
1% Zinclorac SC (oxaziclomefone, trade name of de-weeding
An, Japan Aventis CropScience Co.); 40% Pargaclofen WP
(Prometryn, trade name of pargaclofen, Zhejiang Province, Changxing First Chemical
Chemical Co. 1.3 Test methods 1.3.1 Filter paper method Seeds of barnyard grass were germinated for 3~4 d, and the seeds of barnyard grass were white spare. Each herbicide to be tested was diluted with water into different concentrations . In a 9 cm diameter Petri dish lined with a round filter paper, 5.00 ml of each concentration was added to the petri dish, 20 seeds of barnyard grass were selected and covered with plastic wrap, and the petri dish was put into a LRH-250-G light incubator (Guangdong Provincial Medical Instrumentation Factory) and incubated for 24 h at (27±1)°C. (Guangdong Medical Instrument Factory), and incubated at (27±1)℃, 24 h light conditions . A blank control was set with water. Each treatment was repeated three times. After 2~5 d of treatment, the shoot length was measured and the inhibition rate was calculated[10]. 1.3.2 Agar method Referring to the method of Cao Au Cheng[11], the seeds of barnyard grass were germinated for 3~4 d, and the seeds of barnyard grass were prepared for use after whitening. The test chemicals were diluted with water into different concentrations. From the low concentration to the high concentration in turn, 30 ml of each solution was mixed with 60 ml of agar solution (with a mass fraction of 1.5%), and then poured into 3 petri dishes with a diameter of 9 cm in order of 20 ml each, with water as the control in 3 replicates, and then after the agar was cooled down and solidified, 12 neat newly bleached seeds were placed on the agar solution. After the agar was cooled and solidified, 12 neatly just-whitened barnyardgrass seeds were put into 1% agar culture medium with different concentration gradients, and the germs were placed upward in the same direction in the LRH-250-G light incubator at (27 ±1)℃ with 24 h of light exposure for cultivation. After a certain treatment time, the shoot length was measured and the inhibition rate was calculated. 1.3.3 Potting spray method In the 20 cm caliber plastic pots filled with about 80% of the full soil, each pot into the germination of barnyard grass seeds 20-30, covered with covered with a layer of fine sand. When the barnyard grass seedlings grew into 2 leaves and 1 heart, put in ASP-1098 automatic spraying device (Zhejiang University Electrical Equipment Factory, Pressure: 0.4MPa, Speed: 50 cm/s), spraying dichloro quinolinic acid of each concentration of the liquid to be tested. The water was used as a blank . Each treatment was repeated three times. The fresh weight of barnyardgrass plants was determined 15 d after the application of dichloro quinolinic acid, and the inhibition rate was calculated[10]. 1.3.4 Data processing Based on the logarithmic value of the test concentration and the inhibition rate of shoot length , the regression equation of the toxicity y=a+ bx, the inhibition of EC50, the 95% confidence limit of EC50 and the correlation coefficient r were determined by using the DPS software[12]. 2 Results and analysis 2.1 Determination of treatment time The sensitivity of barnyardgrass to seven herbicides, including pyrazosulfuron, pentafluorosulfuron, propargyl ketone, tetrazolium, dichloroquinaldic acid, zinclofenac, and parafoset was determined using the filter paper method under the light condition of (27±1)℃ and 24 h. The sensitivity of barnyardgrass to these seven herbicides was determined using the filter paper method. Sensitivity. The results showed that the sensitivity of barnyardgrass to the sulfonylurea herbicide pyrazosulfuron was lowest at the EC50 value of 13.07 mg/L for 2 d. The sensitivity of barnyardgrass to tetrazolylacetonitrile (Tetrazolylacetonitrile) and dichloroquinaldic acid (Zinclorac) was also higher than that of tetrazolylacetonitrile (Zinclorac). The sensitivity of barnyardgrass to the tri azolo-pyrimidinesulfonamide herbicide pentaflumizone, the diazolidinone herbicide propynil, the quinoline carboxylate herbicide dichloroquinoxaline and the triazine herbicide pargyline was the best at 3 d. The EC50 values of the triazolone herbicide were 0.4972,0.1042, , 0.3240,445.440, 0.1042 and 445.340, respectively. 0.3240,445.6 mg/L. The tetrazolone herbicide tetrazolium ammonium and the zinhydrin herbicide zinclorac were the best results at 4 d, with EC50 values of 0.02092,0.05064 mg/L, respectively (Table 1). 2.2 Comparison of different bioassay methods The sensitivity of barnyardgrass to dichloroquinolinic acid (DQA) was determined by the filter paper method, the agar method and the potting spray method, and the results are shown in Table 2. The sensitivity of barnyardgrass to DQA in the filter paper method increased with time, and reached the highest level in 3 d. The results were summarized in Table 2. The sensitivity to DQA of barnyardgrass in the filter paper method increased in time, and reached the highest in 3 d, and the sensitivity of barnyardgrass in the potting spray method increased in time. The sensitivity of barnyard grass to dichloroquinoline in the filter paper method increased with time, and reached the highest at 3 d, and then the sensitivity decreased. Because both the filter paper method and agar method are one kind of Petri dish method, the agar method was chosen with a treatment time of 3 d. Comparison of the three bioassay methods showed that the sensitivity of barnyardgrass to diclofenac was the highest in the agar method, followed by filter paper method, and the lowest in the potting and spraying method, and the EC50 values were Table 1: Sensitivity to diclofenac in different treatment times Table 1: Sensitivity to diclofenac in different treatment times Table 1: Sensitivity to diclofenac in different treatment times Table 1: Sensitivity to diclofenac in different treatment times
Table 1 The susceptibility of barnyard grass to 7 herbicides under the conditions of different treating times (filter paper method)
The toxicity regression equation (y=a+bx)EC50/mg-L-195% confidence limit/mg-L-1 correlation coefficient (r)
Pyrazosulfuron2y =4.1618+0.7510x13.0700 3.039~ 160.00 0.9861
3y =4.0726+0.5945x36.3100 11.43~206.60 0.9772
4y =3.6725+0.6972x80.1600 15.18~2575.00 0.9619
5y =3.4566+ 0.7302x129.9000 29.50~1462.00 0.9715
Pentaflumizone 2y =5.0492+0.4919x0.7944 0.1746~3.3850 0.9562
3y =5.1536+0.5061x0.4972 0.2713~ 0.8661 0.9901
4y =5.2120+0.8236x0.5528 0.3285~0.9155 0.9895
5y =5.0787+0.9276x0.8225 0.5603~1.2030 0.9933
Propargyl 2y =5.4482 +0.5487x0.1724 0.09304~0.28420 0.9959
3y =5.4947+0.5100x0.1072 0.000028~0.543800 0.9625
4y =6.0368+1.3539x0.1715 0.00007~ 0.85850 0.9513
5y =5.2335+0.7600x0.4930 0.3043~0.7729 0.9943
Tetrazolium 2y =5.3576+0.3198x0.0762 0.005213~0.313800 0.9653
3y = 6.0225+0.8415x0.0609 0.000494~0.265800 0.9810
4y =5.7511+0.4472x0.0209 0.000389~0.106000 0.9712
5y =5.7992+0.6579x0.0610 0.008484~0.184100 0.9815
Dichloroquinolinic acid2y =5.1231+1.1628x0.7837 0.5832~1.0430 0.9938
3y =5.3580+0.7313x0.3240 0.2272~0.4413 0.9947
4y =5.2918+0.7718x0.4187 0.2835~0.5897 0.9930
5y =5.2924+1.0920x0.5398 0.4361~0.6603 0.9970
Azinocarbamate2y =4.9516+0.4019x1.3200 0.7563~2.4500 0.9957
3y =5.2154+0.3671x0.2589 0.06300~0.68330 0.9738
4y =5.9717+0.7500x0.0506 0.007248~0.163900 0.9703
5y =5.6014+0.7838x0.1709 0.01554~0.46730 0.9867
Puffer 2y =2.8918+0.6910x1125.0000 319.2~6315.0 0.9970
3y =3.9721+0.3880x445.6000 71.98~1.87x1040.9836
4y =-5.2733+2.7531x5390.0000 1360.000~4.053x1040.9976
5y =-8.9925+3.6817x6317.0000 2239.000~2.445x 1040.9907
-39-Wu Shengdang et al: Study on the susceptibility of barnyard grass to seven herbicides and their bioassay methods
Comparison of the susceptibility of barnyard grass to quinclorac by different bioassay methods
Table2 Comparison of the susceptibility of barnyard grass to quinclorac by different bioassay methods
Bioassay method Treatment time/d Toxicity regression equation (y=a+bx)EC50/mg-L-195% confidence limit/mg-L-1Correlation coefficient (r)
Filter paper method2y =5.1231+ 1.1628x0.7837 0.5832~1.0430 0.9938
3y =5.3580+0.7313x0.3240 0.2272~0.4413 0.9947
4y =5.2918+0.7718x0.4187 0.2835~0.5897 0.9930
5y =5.2924+1.0920x0.5398 0.4361~0.6603 0.9970
Agar method3y =5.4336+0.5826x0.1802 0.04343~0.46110 0.9753
Potting Spray method15y =-3.3407+ 3.1386x454.4 127.2~3961.0 0.9902
0.1802,0.3240,454.4 mg/L. And the time required to reach this sensitivity
was 3,3,15 d, respectively.
2.3 Comparison of barnyardgrass sensitivity to the seven herbicides
Application of agar method was carried out at ( 27±1)°C, 24 h light,
The sensitivity of barnyardgrass to seven
herbicides was determined by agar method at ( 27±1)°C, 24 h light,
according to the treatment time determined by filter paper method (Table 3). Bud length was chosen as an indicator parameter, and the order of susceptibility (based on EC50 values) was as follows:
zinclofenac> tetrazolium> propynil> diclofenac
ac> pentaflumizafop> pyrazosulfuron> and pramoxafen. The correlation coefficients of all the test
results were above 0.9682 (Table 3).
Table 3 The susceptibility of barnyard grass to 7 herbicides (agar method)
Table 3 The susceptibility of barnyard grass to 7 herbicides (agar method)
The name of the agentTreatment time/dToxicity regression equations (y=a+bx) EC50/mg-L-195% confidence limit/mg-L-1 Correlation coefficient (r)
Picosulfuron2y =4.3770+0.7327x7.0840 4.162~16.350 0.9952
Pentaflumizone3y =5.1433+0.3442x0.3835 0.1530~ 0.7697 0.9861
Propynil 3y =11.5803+4.1615x0.0262 0.01324~0.04424 0.9682
Tetrazolium 4y =7.0912+1.1118x0.0132 0.003699~0.030980 0.9881
Dichloroquinolinic acid3y =5.4336+0.5826x0.1802 0.04343~0.46110 0.9753
Azinopyrrolidone4y =7.2668+0.8750x0.0024 0.000011~0.017990 0.9729
Puffergrass 3y =3.5473+ 0.6123x235.9000 51.48~3410.00 0.9799
3 Conclusion and Discussion
The herbicidal activity of the compounds requires a certain amount of time to be demonstrated
, and the efficacy of the compounds could not be evaluated correctly if the treatment time is too long or too short, and the treatment time also determines the length of the whole testing cycle
.
The length of the test cycle is also determined by the treatment time. When the treatment time is too short, the test results of the weeds are not sensitive
and the regularity is poor. With the prolongation of the treatment time, the efficacy of herbicides
has a tendency to be exaggerated at higher concentrations and underestimated at lower
concentrations, thus decreasing the accuracy of the indication.
The sensitivity of barnyard grass to pyrazosulfuron in this experiment was the lowest with the EC50 value of 2 d. The sensitivity to pentaflumizone, propargyl ketone, dichloro
quinolinic acid and pramoxafen was the best with the result of 3 d, and the sensitivity to tebuconazole and zinclozuron was the most sensitive with the result of 4 d.
The results of this experiment are summarized in the table below. Because the herbicides in the same class have similar structure and action
characteristics, the authors believe that the results can be used as a reference for the treatment time of the same herbicides when barnyard grass is used as an indicator plant. Therefore, in order to achieve the goal of accuracy and speed, the authors recommended the following treatment times for barnyardgrass: 2 d for sulfonamide herbicides, 3 d for triazolopyrimidine sulfonamides, diazolidinones, quinoline carboxylates and triazines, and 3 d for tetrazolium
linolone herbicides, tetrazolium and tetrazinone herbicides, and 4 d for tetrazolium and tetrazinone herbicides.
Resistance monitoring is an important part of resistance management, so it is important to strengthen the monitoring of weed resistance to herbicides to understand the dynamics of resistance development and to identify the level of resistance, so as to take early measures to prevent or delay the emergence of resistant weeds, in which the method of resistance monitoring is crucial. The results of this experiment showed that the sensitivity of barnyard grass to dichloroquinolinic acid in the agar method was higher than that in the filter paper method and the
pot spray method, and the time required was shorter. Therefore, the agar method
is a rapid and sensitive method for herbicide bioassay, which can be applied
to rapidly monitor the resistance of barnyard grass in paddy fields and to screen
select herbicides for the control of resistant barnyard grass in paddy fields.
Based on the treatment time determined by the filter paper method, the sensitivity of barnyardgrass to seven herbicides was determined by the agar method, and the results showed that the sensitivity of zinc oxon was the best, the sensitivity of tetrazolium and propargyl oxon was the best, the sensitivity of diclofenac and pentaflumizone was the second best, and that of pyrazosulfuron was the second best. Vol. 19 (2007)
, and the worst one was penciclofenac. Thus, the high activity and low dosage of zincone and tetrazolium were further verified against barnyard grass [13,14]. The low sensitivity of paracetamol may be due to the fact that paracetamol is a selective systemic herbicide, which is mainly absorbed by roots, but can also be infiltrated into plants by stems and leaves. The absorbed pemphigus
conducts through the transpiration flow, inhibits the Hill reaction in photosynthesis
and causes the plant to lose its green color and dry up and die[15]. Since the shoot length was selected
as the indicator parameter in this experiment, it could not show its activity
well. Based on the above results, the authors concluded that in the
management of barnyardgrass resistance in paddy fields, herbicides with good sensitivity and different mechanisms of action should be used in rotation, so as to prevent and control resistant barnyardgrasses before they spread further, and thus eliminate them at the low resistance stage.