Study on the Resistance Levels of Different Echinochloa crusgalli Populations to Penoxsulam and Bispyribac-sodium

Qi LEI, Bingjie LIU, Jingbo LI, Yunyun ZHOU, Kailing PENG, Chenzhong JIN, Xiu LIU, Kaifa GUO, Guiying LIU

Abstract [Objectives] This study was conducted to screen the resistance of Echinochloa crusgalli in some paddy fields in Changde City， Yiyang City and Yueyang City in Hunan Province to penoxsulam and bispyribac-sodium.

[Methods] The resistance to penoxsulam and bispyribac-sodium was screened in 11 E. crusgalli populations collected from rice production areas in Changde， Yiyang and Yueyang Cities， Hunan Province， and the resistance level of population HU-2 to penoxsulam was determined.

[Results] HU-1 to HU-6 and HU-8A， a total of seven populations showed high levels of resistance to penoxsulam and bispyribac-sodium. The resistance level of population HU-2 to penoxsulam was extremely high; population HU-7 showed low-level resistance to both drugs; HU-11 population showed low-level resistance to penoxsulam and was susceptible to bispyribac-sodium; and populations HU-8 and HU-9 were susceptible to penoxsulam and bispyribac-sodium.

[Conclusions] This study provides a technical basis for the rational use of penoxsulam and bispyribac-sodium and the selection of control chemicals for E. crusgalli in paddy fields.

Key words Echinochloa crusgalli; Population; Penoxsulam; Bispyribac-sodium; Resistance

Received： April 9， 2022  Accepted： June 10， 2022

Supported by Science and Technology Innovation Program of Hunan Province （2019NK4170）; Research Innovation Program for College Graduates of Hunan Province （CX2018B796）.

Qi LEI （1998-）， female， P. R. China， master， devoted to research about plant protection.

*Corresponding author.

Field weeds seriously affect crop yield and quality， and the use of herbicides is the most effective way to control field weeds. There are many types of herbicides， the largest of which is the acetolactate synthase inhibitor herbicides， which target acetolactate synthase （ALS）[1-2].  ALS is a key enzyme in the synthesis of branched-chain amino acids （valine， leucine and isoleucine）， which only exists in plants and microorganisms， but not in humans and animals， so it is an ideal herbicide target enzyme. Numerous studies have been conducted on the structure of ALS， the development of ALS inhibitors， and the mechanism of action of ALS inhibitors[3-8]. With repeated application of a single herbicide， resistant weeds gradually expand their range from initially isolated plants or fields and spread to control the population soil seed bank， resulting in the rapid evolution of herbicide resistance[9-10]. Echinochloa crusgalli was first discovered in Italy in 2005 to evolve resistance to ALS-inhibiting herbicides. Subsequently， E. crusgalli populations resistant to ALS-inhibiting herbicides were found in many countries around the world[11].

The herbicides penoxsulam and bispyribac-sodium are both ALS-inhibiting herbicides developed and promoted in the 21st century. They have been used for the control of E. crusgalli in paddy fields due to their low toxicity， high efficiency and safety to rice. Due to the long-term heavy use， the resistance of E. crusgalli to them in Hunan rice areas has increased year by year. In this study， the resistance of E. crusgalli in some paddy fields in Changde， Yiyang， and Yueyang Cities， Hunan Province to penoxsulam and bispyribac-sodium was screened， aiming to provide a technical basis for the rational use of penoxsulam and bispyribac-sodium and the selection of control chemicals for E. crusgalli in paddy fields.

Materials and Methods

Collection of test E. crusgalli

Collection of test E. crusgalli： In 2018， mature seeds of E. crusgalli were collected from paddy fields in Yiyang and Yueyang areas around Dongting Lake. The "W" random sampling method was adopted， and the seeds were collected at the maturity stage of E. crusgalli seeds before the seeds fell off naturally. A total of 11 groups of samples were collected （Table 1）. The collected samples were placed in a 37 ℃ seed drying oven for drying， and the dried seeds were cleaned and dehulled for use.

Test drugs and instruments

25 g/L Daojie penoxsulam dispersible oil suspension （Dow AgroSciences， USA）; 100 g/L Nongmeili bispyribac-sodium suspending agent （Japan Kumiai Chemical Industry Co.， Ltd.）.

Instruments： ZRG-450D intelligent artificial climate box （Shanghai Binglin Electronic Technology Co.， Ltd.）; intelligent greenhouse， self-made walking spray tower （provided by Hunan University of Humanities， Science and Technology）; seed dryer （Jiaduo Tangyin Jiaduo Industry and Trade Co.， Ltd.）; electric blast drying oven （Beijing Zhongxing Weiye Instrument Co.， Ltd.）.

Screening of resistance to ALS-inhibiting herbicides

According to the whole plant bioassay method， 200-300 grains of each population were selected， soaked at 4 ℃ for 24 h， then transferred to a petri dish with moist filter paper， and placed in an intelligent artificial climate box for germination under temperature 28/25 ℃ （day/night）， photoperiod L∶D=14∶10 （day∶night）， and light intensity： 3 000 LX. The seeds were transplanted into plastic pots of 210 mm×180 mm×100 mm after germination， and 25 plants were transplanted in each pot. After transplanting， they were transferred to a greenhouse （28 ℃， photoperiod L∶D=14∶10）.

When the E. crusgalli grew to the 3-4 leaf stage， the stem and leaves were sprayed with a self-made walking spray tower， and the spray pressure was 0.4 MP and the traveling speed was 0.8 m/s. The treatment doses of penoxsulam and bispyribac-sodium were 75 g and 175 g a.i/hm2， respectively. After the chemicals on the leaves were air-dried， they were transferred to the greenhouse for cultivation （28 ℃， photoperiod L∶D=14∶10）. After 21 d， the number of surviving plants in each treatment group was counted， and the survival rate was calculated. The resistant seeds were kept， and mature seeds were harvested and stored in a dry state at 37 ℃.

Determination of the resistance level of HU-2 to penoxsulam

The resistant HU-2 and susceptible HU-9 populations were pre-germinated respectively （the same method as "Screening of resistance to ALS-inhibiting herbicides"）， and transplanted into 110 mm×100 mm plastic pots， with 15 plants per pot， in three replicates. Appropriate dose concentration gradients were set separately for the resistant and susceptible populations （Table 2）.

When the rice seedlings grew to the 3-4 leaf stage， drug treatment was carried out. The number of surviving plants was counted 21 d after the treatment， and the above-ground parts were cut and bagged， placed in an electric heating blast drying oven with a constant temperature of 55 ℃ for 5 d， and then weighed to calculate the survival rate.

Statistical analysis of data

The dose-response of penoxsulam on E. crusgalli survival and dry weight inhibition rate was fitted using Sigma Plot 12.5 software four-parameter nonlinear regression model. The fitting equation was：

Y=C+D-C1+（X÷LD50）b

where Y is the survival rate or growth rate of E. crusgalli under different treatments compared with the blank control; C is the lower limit of dose response; D is the upper limit of dose response; X is the dose of penoxsulam; b is the absolute value of the slope. LD50 is the median lethal dose.

The t-test method in prism 5 software was used to analyze the significance between treatments （P<0.05）.

Results and Analysis

Resistance of E. crusgalli to penoxsulam and bispyribac-sodium in different areas

Among the 11 populations （Table 3）， the HU-1 to HU-6 and HU-8 populations showed overall high resistance to penoxsulam and bispyribac-sodium; population HU-7 was resistant to penoxsulam and bispyribac-sodium at low levels; HU-11 had low level resistance to penoxsulam， but susceptible to bispyribac-sodium; HU-9 and HU-10 were both susceptible to penoxsulam and bispyribac-sodium. Populations HU-1 to HU-8 were cross-resistant to both herbicides.

Resistance level of population HU-2 to penoxsulam

Symptoms of E. crusgalli damage after spraying： About 3 d after spraying， the susceptible population HU-9 treated with high concentrations of penoxsulam 1X and 2X， and the resistant population HU-2 treated with high concentration 32X showed leaf yellowing and wilting phenomenon， other concentrations did not change significantly. After 21 d of treatment （Fig. 1）， different dose concentrations showed different degrees of damage symptoms. The E. crusgalli populations under high concentration treatments had stopped growth until death. There were no obvious changes in the E. crusgalli populations under low concentration treatments.

The LD50 values of HU-2 and HU-9 on penoxsulam are shown in Table 4 and Fig. 2， and the LD50 of the susceptible population HU-9 was 24.5 g a.i/hm2. Compared with HU-9， the HU-2 population developed a high level of resistance to penoxsulam， and itsresistance was 110 times higher than that of HU-9.

Conclusions and Discussion

ALS inhibitors have been used for 40 years since they were first used on the market in 1982[12]. The advantages of high efficiency and low toxicity make people rely more on these herbicides for weed control. Penoxsulam and bispyribac-sodium are typical representatives of ALS-inhibiting herbicides. Related reports show that by 2021， 169 weed biotypes have become resistant to ALS-inhibiting herbicides.

Riar et al.[13] reported that three rice field E. crusgalli populations collected from Arkansas and Mississippi， USA， produced 94 ， 30 and 9.4 times of resistance to penoxsulam， respectively. Song et al.[14] reported that E. oryzicola developed a 13.7 times of resistance to penoxsulam. Guo et al.[15] reported that E. crusgalli in Zhanjiang City， Guangdong Province， had 6.5 times of resistance to penoxsulam. Fang et al.[16] reported that rice field E. crusgalli in Jiangsu Province and Anhui Province produced 7.3 and 33 times of resistance to penoxsulam， respectively. Zhang et al.[17] reported that the resistance frequencies of E. crusgalli to herbicides such as penoxsulam and bispyribac-sodium in some rice areas in Anhui Province were 13.7% and 21.7%， respectively. El-Nady et al.[18] also reported that the GR50 of resistant Echinochloa colonum was 10.6 times higher than the susceptible population under bispyribac-sodium treatment. Li[19] measured the susceptibility of 24 E. crusgalli populations collected from paddy fields in different provinces to penoxsulam， and determined the resistance of 6 of them to penoxsulam. The results showed that the resistant populations developed 10.6 times of resistance to the susceptible populations.

In this study， 11 E. crusgalli populations collected from some rice fields in Hunan Province showed different resistance to penoxsulam and bispyribac-sodium. Among them， seven populations developed high levels of resistance to both penoxsulam and bispyribac-sodium; two populations in the penoxsulam-treated group developed low-level resistance， while one was observed to develop low-level resistance in the bispyribac-sodium-treated group; and only two populations were susceptible to both penoxsulam and bispyribac-sodium. In addition， the resistance level of population HU-2 to penoxsulam was extremely high （>110 times）， which deserves great attention. Therefore， in Hunan Province， the resistance of E. crusgalli to penoxsulam and bispyribac-sodium has developed to a very high level. Follow-up weed control measures in paddy fields need to adopt comprehensive management strategies， including scientific and reasonable rotation of pesticides， or synergistic control of resistant E. crusgalli with other control methods， such as pre-emergence sealing[20]， mechanical weeding， crop rotation[21] and other measures.

References

[1] GU LL. Penoxsulam， a triazolopyrimidine sulfonamide herbicide[J]. Modern Agrochemicals， 2015， 14（2）： 46-51. （in Chinese）.

[2] POWLES SB， YU Q. Evolution in action： Plants resistant to herbicides[J]. Annual Review of Plant Biology， 2010， 61（1）： 317-347.

[3] HEAP I. The international herbicide-resistant weed database[DB/OL]. [2022-3-23]. Available www.weedscience.org.

[4] HEAP I. The weed science society of America online[DB/OL]. [2022-3-23]. Available https：∥www.wssa.net.

[5] SINGH S， SINGH V， SALAS-PEREZ RA， et al. Target-site mutation accumulation among ALS inhibitor-resistant Palmer amaranth[J]. Pest Management science， 2019， 75（4）： 1131-1139.

[6] PAN L， GUO Q， WANG J， et al. CYP81A68 confers metabolic resistance to ALS and ACCase-inhibiting herbicides and its epigenetic regulation in Echinochloa crusgalli[J]. Journal of Hazard Mateiral， 2022（428）： 128225.

[7] MILANI A， SCARABEL L， SATTIN M. A family affair： Resistance mechanism and alternative control of three Amaranthus species resistant to acetolactate synthase inhibitors in Italy[J]. Pest Management Science， 2020， 76（4）： 1205-1213.

[8] YU Q， POWLES SB. Resistance to AHAS inhibitor herbicides： Current understanding[J]. Pest Management Science， 2014， 70（9）： 1340-1350.

[9] GUTTIERI MJ， EBERLEIN CV， MALLORY-SMITH CA， et al. DNA sequence variation in domain A of the acetolactate synthase genes of herbicide-resistant and -susceptible weed biotypes[J]. Weed Science， 1992， 40（4）： 670-677.

[10] BI Y， LIU W， GUO W， et al. Molecular basis of multiple resistance to ACCase- and ALS-inhibiting herbicides in Alopecurus japonicus from China[J]. Pesticide Biochemistry and Physiology， 2016（126）： 22-27.

[11] XIA W， PAN L， LI J， et al. Molecular basis of ALS- and/or ACCase-inhibitor resistance in shortawn foxtail （Alopecurus aequalis Sobol.）[J]. Pesticide Biochemistry and Physiology. 2015（122）： 76-80.

[12] HEAP J AND KNIGHT R. A population of ryegrass tolerant to the herbicide diclofopmethyl[J]. Journal of the Australian Institute of Agricultural Science， 1982（48）： 156-157.

[13] RIAR DS， NORSWORTHY JK， BOND JA， et al. Resistance of Echinochloa crusgalli populations to acetolactate synthase-inhibiting herbicides[J]. International Journal of Agronomy， 2012， 2012（3）： 1-8.

[14] SONG J， LIM S， YOOK M， et al. Cross-resistance of Echinochloa species to acetolactate synthase inhibitor herbicides[J]. Weed Biology and Management， 2017， 17（2）.

[15] GUO WL， FENG L， ZHANG C， et al. Resistance of barnyard grass Echinochloa crusgalli to penoxsulam in rice fields in Guangdong Province[J]. Acta Phytophylacica Sinica， 2020， 47（5）： 1131-1138. （in Chinese）. （in Chinese）.

[16] FANG J， LIU T， ZHANG Y， et al. Target site-based penoxsulam resistance in barnyardgrass （Echinochloa crusgalli） from China[J]. Weed Science， 2019： 1-7.

[17] ZHANG H， ZHANG Y， PAN YM， et al. Preliminary study on Echinochloa spp. resistance to herbicides in paddy fields of Anhui Province[J]. Weed Science， 2021， 39（3）： 44-50. （in Chinese）.

[18] EL-NADY M， HAMZA A， DERBALAH A. Echinochloa colonum resistance to bispyribac-soduim in egypt occurrence and identification[J]. Journal of Plant Protection Research， 2012， 52（1）.

[19] LI FL. Study on the resistance of rice field barnyardgrass to penoxsulam[D]. Wuhan： Huazhong Agricultural University， 2019. （in Chinese）.

[20] WANG ZJ. Control measures of barnyardgrass in paddy fields[J]. Nong Min Zhi Fu Zhi You， 2018（12）： 67. （in Chinese）.

[21] ANDRES A， CONCENO G， THEISEN G， et al. Management of red rice （Oryza sativa） and barnyardgrass （Echinochloa crusgalli） grown with sorghum with reduced rate of atrazine and mechanical methods[J]. Experimental Agriculture， 2012， 48（4）： 587-596.