Plant growth regulator and application thereof in relieving imazamox rice phytotoxicity

Abstract

The application discloses a plant growth regulator and application thereof in relieving imazamox herbicide injury to rice. The main components of the plant growth regulator include leucine, isoleucine and valine, and the plant growth regulator is combined with imazamox in a certain application mode and proportion, and is sprayed on rice seedling leaves, so that the effect of relieving imazamox herbicide injury is better than that of the commonly used protective agent, namely, bihuhu or brassinolide on the market, and the herbicide effect of the herbicide is not affected. The application also clearly defines the application conditions of the safety agent, and is a plant growth regulator which can effectively relieve imazamox herbicide injury to rice and does not reduce the herbicide effect on weeds.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of crop pesticide injury prevention, and particularly relates to a plant growth regulator and application thereof in relieving imazamox pesticide injury on rice. BACKGROUND

[0002] Rice is one of the most important food crops in China. In recent years, with the rapid development of the national economy and the adjustment of industrial structure, rural labor force is constantly transferred to cities, and rice production in China is developing towards scale, intensification and mechanization, so that light and simple cultivation measures such as machine transplanting and direct seeding have become the development trend (Zhang H C, Gong J L. Research status and development of rice planting mechanization and high-yield agronomy in China. Chinese Journal of Agricultural Science, 2014, 47(7): 1273-1289).

[0003] Direct seeding of rice is more time-saving and labor-saving than traditional transplanting, but direct seeding rice field is prone to breed weeds and weedy rice, which seriously affects the growth and development of rice and the final yield. With the increasing expansion of light and simple cultivation planting area such as direct seeding rice, the application of various herbicides is becoming more and more common, the use amount of herbicides is gradually increasing, and the problem of resistant weeds and soil high residue in rice field is becoming more and more serious (Wu Y Y, Xiao N, Yu L, Cai Y, Pan C H, Li Y H, Zhang X Q, Huang N S, Zhou C H, Ji H J, Dai Z Y, Li A H, Research progress on the development of herbicide-resistant rice germplasm in China. Journal of Plant Genetic Resources, 2021, 22(4): 890-899).

[0004] Weedy rice (Oryza sativa f. spontanea) and cultivated rice (O. sativa L.) belong to the same species. In fact, weedy rice is a close relative of domesticated rice and is a toxic weed that is ubiquitous in fields in rice-growing regions of the world. The use of common rice field herbicides does not have good removal effect on weedy rice which is closely related to cultivated rice, and the development and promotion of herbicide-resistant rice can solve this problem (Wang F Q, Xu Y, Li W Q, et al. Creating a novel herbicide-tolerance OsALS allele using CRISPR / Cas9-mediated gene editing. The Crop Journal, 2020, 9(2): 305-312).

[0005] Acetolactate synthase (ALS) is a key enzyme in the biosynthesis of branched-chain amino acids such as valine, leucine and isoleucine in plants. ALS inhibitors are a class of herbicides that target acetolactate synthase, by forming a complex with the enzyme in the plant, which blocks the substrate from entering the active site of the enzyme, preventing the synthesis of branched-chain amino acids, leading to inhibition of protein synthesis, eventually causing the plant to stop growing and developing, and even die (Ren Honglei. Acetolactate synthase and ALS gene research overview. China Agricultural Bulletin, 2016, 32(26): 37-42). Currently, five different chemical classes of ALS inhibitors have been commercialized: imidazolinones (IMI), pyrimidinylthiobenzoates (PTB), sulfonylamino-carbonyl-triazolinones (SCT) and triazolopyrimidines (TP), therefore, it is particularly effective for the control of weedy rice (Deng W, Yang Q, Zhang YZ, Hongtao Jiao HT, Mei Y, Li XF, Zheng MQ. Cross-resistance patterns to acetolactate synthase (ALS)-inhibiting herbicides of flixweed (Descurainia sophia L.) conferred by different combinations of ALS isozymes with a Pro-197-Thr mutation or a novel Trp-574-Leu mutation, Pesticide Biochemistry and Physiology, 2017, 136: 41-45).

[0006] Chinese scientists screened naturally (Wang F Q, Yang J, Fan F J, Li W Q, Chen Z H, Wang J, Tao Y J, Jiang Y J, Zhu Q H, Yang J. Development and application of functional markers for imidazolinone herbicide resistance gene ALS in rice. Crop Sci, 2018, 44(3): 324-331; Bi J G, Tan J S, Liu Y, Zhang A N, Wang F M, Liu G L, Yu X Q, Luo L J. Screening and identification of imidazolinone herbicide-resistant rice germplasm. J Plant Genet Resour, 2020, 21(4): 804-808), chemically mutagenized (Chen Z F, Wang C X, Liu W, Tang X Y, Deng X W. Rice herbicide-resistant protein and its application in plant breeding: CN201210037789.9.2013-04-17; Zhao B R, Yuan D Y, Shao Y, Mao B G, Yuan Z C, Hu Y Y, Peng Y, Luo W Z. A rice herbicide-resistant protein and gene and their application: CN106867977A.2017-06-20; Zhang B L, Chen T Z, Wang J Y, Ling X T. Application of ALS mutant gene and protein of japonica rice in herbicide resistance: CN106868027A.2017-06-20; Zhang B L, Ling X T, Wang J Y, Chen T Z, Deng H Q. Rice ALS mutant protein and gene conferring herbicide resistance in plants and their application: CN107090447A.2017-08-25; Zhang B L, Wang J Y, Ling X T, Chen T Z, Deng H Q, Wu K. Rice ALS mutant protein conferring herbicide resistance in plants and its application: CN108004224A.2018-05-08) and CRISPR / Cas9 gene editing (Sun Y W, Zhang X, Wu C Y, He Y B, Ma Y Z, Hou H, Guo X P, Du W M, Zhao Y D, Xia L Q. Engineering herbicide-resistant rice plants through CRISPR / Cas9-mediated homologous recombination of acetolactate synthase. Molecular Plant, 2016, 9(4): 628-631; Wang F Q, Xu Y, Li W Q, Chen Z H, Wang J, Fan F J, Tao Y J, Jiang Y J, Zhu Q H, Yang J. Creating a novel herbicide-tolerance OsALS allele using CRISPR / Cas9-mediated gene editing.Through methods such as The Crop Journal, 2021, 9(2): 305-312, a series of rice germplasms or materials resistant to ALS inhibitor herbicides were obtained (Tan SY, Evans RR, Dahmer ML, Singh BK, Shaner DL, Imidazolinone-tolerant crops: history, current status and future. Pest Manag Sci, 2005, 61: 246-257). Among them, the Tianjin Institute of Crop Science successfully bred rice varieties such as Jindao 372 and Jingeng 818, which are resistant to imidazolinone herbicides and are widely used in my country. A control strategy of using herbicide-resistant rice in combination with broad-spectrum herbicides to control weedy rice was established.

[0007] Because ALS enzymes exist only in plants and microorganisms, ALS inhibitor herbicides are highly safe for mammals; they are used in lower quantities compared to glyphosate and acetyl-CoA carboxylase inhibitors; and they also have strong selectivity, weed control, and long-lasting effects, which has led to their widespread attention and rapid application (Gao Jianqin, Pu Huiming, Qi Cunkou, Zhang Jiefu, Long Weihua, Hu Maolong, Chen Song, Chen Xinjun, Chen Feng, Gu Hui. Discovery and identification of imidazolinone-resistant rapeseed germplasm. Plant Genetic Resources). Journal of Pesticides, 2010, 11(3): 369-373), imidazolium ethanoate is one of the most prominent herbicides for soybeans, wheat and corn, but its fatal flaw is that it has a long residual time in the soil, which often causes phytotoxicity to sensitive crops in the following crop (Wang Xianfeng, Fan Zhiwei, Hu Rongjuan, Zou Wei, Tong Yucun. New progress and solutions to herbicide phytotoxicity. Pesticides, 2009, 48(5): 384-388), which brings great risks to the safe production of rice and wheat rotation areas in the lower reaches of the Yangtze River. Methoxyfenozide is another type of imidazolinone (IMI) herbicide (Gil-Monreal M, Giuntoli B, Zabalza A, et al. ERF-VII transcription factors induce ethanol fermentation in response to amino acid biosynthesis-inhibiting herbicides. Journal of Experimental Botany, 2019, 70(20): 5839-5851). It is more popular with farmers due to its low dosage and low residue. However, this type of effective herbicide is still a herbicidal agent. Most major rice varieties in China do not possess ALS mutations and are generally not resistant to methoxyprofon. Furthermore, the tolerance of rice varieties or lines to methoxyprofon varies among varieties, even among those resistant to ALS mutations. Therefore, applying methoxyprofon alone may cause varying degrees of phytotoxicity not only to rice varieties resistant to this herbicide but also to surrounding sensitive rice plants, affecting their growth, development, and yield. In our previous laboratory experiments, we found that high doses of the imidazolinone herbicide methoxyprofon can cause phytotoxicity to resistant rice seedlings. Therefore, it is necessary to use plant growth regulators to alleviate the phytotoxicity.

[0008] The standardized use of herbicide-resistant rice varieties and the development of their matching safeners are the most economical and effective measures to ensure the safe promotion of such herbicide-resistant rice varieties (Li Yanmin, Qi Xiantao, Liu Changlin, Liu Fang, Xie Chuanxiao. Research progress in breeding herbicide-resistant crops. Crop Journal, 2017(2): 1-6). Herbicide safeners are a class of special-purpose compounds that can protect gramineous crops from herbicide damage without reducing the activity of herbicides against target weeds (Huang Chunyan, Li Jing, Wang Yu, Chen Tiebao, Huang Yuanju, Cong Lin, Pu Dewan. Evaluation of the detoxification effect of 6 herbicide "safeners" on imidazoline. Pesticides, 2008, 47(9): 682-685). The successful development and application of safeners provide an effective way to solve the problems of herbicide damage and selectivity in weed control. Since the 1980s, the research and development of herbicide safeners has made significant progress. Scientists have experimented with various methods to protect crops from the damage caused by imidazolinone herbicides (Chai Chao, Ye Fei. Research progress of herbicide safeners [J]. Pesticide Science and Management, 2003, 24(4): 23-26). Currently, some herbicide safeners are under development, such as R-28725 (Ye Fei, Qu Hongyun. Mechanism study of safener R-28725 protecting corn from imidazolinone damage [J]. Acta Pharmaceutica Sinica, 2002, 4(1): 18-22), but they have not yet reached the level of application in production. It still requires some time for research and practice on application techniques and effects. According to statistics, in 2011, about 30% of herbicide products for corn and cereal fields contained safeners, and about 6% of herbicides for rice fields contained safeners. The market share of 80% of the safener is occupied by four agrochemical giants: Bayer, BASF, Syngenta, and Corteva (Chang Peng, Yang Hongwei, Cheng Guangbin. Synthesis of herbicide safener bis(oxazol) acid. Fine Chemical Intermediates, 2014, 44(2): 21-24). It can be seen that there are not many reports in my country on the development of effective protective agents and application methods to alleviate the herbicide damage of rice to methoxyfenozide.

[0009] Both Bihu and brassinolide belong to plant growth regulators. They can affect plant growth and development by changing the balance of endogenous hormone signaling molecules in plants and alleviate the damage caused by herbicides (Bian Qiang, Kou Junjie, Ju Guodong, Wang Manyi, Liu Guilong, Research progress of sulfonylurea herbicide safeners, Pesticides, 2011, 50(10): 703-710). Among them, Bihu contains a variety of natural plant endogenous hormones such as gibberellin and brassinolide, flavonoid catalytic balance components, amino acid compounds and stress inducers, which can induce crops to improve stress resistance, increase yield, improve quality and relieve herbicide damage (Yi Xurong, Qin Qingfang. Research on the application of Bihu in increasing rice yield. Southern Agriculture, 2017, 11(36): 13+15; Zeng Qing, Deng Xile, Zhou Shangfeng, et al. Detoxification effect and mechanism of gibberellin on metolachlor. Pesticides, 2019, 58(7): 519-522).

[0010] Brassinolide (BR), also known as brassinosteroid, is a novel plant growth regulator. This plant hormone was first isolated and extracted from rapeseed flowers in 1979. Subsequently, more than 40 structural analogs of it were discovered in other plants. They all have similar chemical structures and physiological activities, and are therefore collectively referred to as brassinosteroid compounds (Luo Wei. A brief analysis of plant growth regulator brassinolide. Guangzhou Chemical Industry, 2013, 41(15): 33-35). Its mechanism of action mainly includes that brassinolide can improve the photosynthetic capacity of crops, reduce transpiration, promote cell division and elongation in plants, accelerate the metabolism of herbicides in plants, and affect the absorption and translocation of herbicides by plants (Xia XJ, Zhang Y, Wu J X, et al. Brassinosteroids promote metabolism of pesticides incucumber. Journal of Agricultural & Food Chemistry, 2009, 57(18): 8406). There are already some examples showing that brassinolide has a certain detoxification effect on some herbicides in crops. For example, although brassinolide does not have a significant alleviating effect on glyphosate damage in cotton, it has a toxin-resistant effect (Zhang Xinghua, Li Jie. Detoxification effect of chlorpyrifos and brassinolide on glyphosate damage in cotton fields. Pesticides, 2008, 47(11): 834-835; Zhou Xiaomao, Bai Lianyang, Huang Kecheng, et al. Effect of natural brassinolide on mitigating herbicide damage to rice [J]. Weed Science, 2003, 21(1): 29-30; Song Cunyu. Repair effect of several agents on dichloroquinoline herbicide damage in rice [J]. Northern Rice, 2017, 47(1): 41-42). However, there are few studies on its use in the protection against herbicide damage from imidazolinone herbicides in rice, especially the lack of reports on its combined use for herbicide damage from methoxyfenozide. Summary of the Invention

[0011] Purpose of the invention: The technical problem to be solved by this invention is to study the safe agent developed by mixing methoxyfenozide with compound branched-chain amino acid agents and spraying it after phytotoxicity occurs. It was found that the safe agent developed by this invention not only has no effect on the weeding effect of methoxyfenozide in controlling weeds in direct-seeded rice fields, but can also significantly reduce the phytotoxicity of methoxyfenozide on rice and promote plant growth.

[0012] Technical solution: To solve the above technical problems, the present invention provides a plant growth regulator, which is made of the following components in parts by weight: 30-60 parts of L-leucine, 10-20 parts of L-isoleucine and 10-20 parts of L-valine.

[0013] The plant growth regulators include L-leucine, L-isoleucine, and L-valine in a mass ratio of 3:1:1.

[0014] The plant growth regulator, also known as a safener, is composed of three branched-chain amino acids: L-leucine, L-isoleucine, and L-valine, with a total mass percentage of 100%, of which L-leucine accounts for 60 parts, L-isoleucine for 20 parts, and L-valine for 20 parts.

[0015] The present invention also includes the application of the plant growth regulators mentioned above in alleviating the phytotoxicity of methoxyfenozide to rice.

[0016] The application rate of the plant growth regulator is 1125-3375 g / ha, of which L-leucine is 675-2025 g / ha, L-isoleucine is 225-675 g / ha and L-valine is 225-675 g / ha.

[0017] The concentration of the methoxyfenozide smoke is 14.4–336.0 g ai / ha.

[0018] The application also includes the application of brassinolide or Bismuth subsalicylate.

[0019] The application rate of brassinolide is 0.0072–0.126 g / ha.

[0020] The application rate of Bihu is 90-135 g / ha.

[0021] The mass ratio of brassinolide to amino acids is 0.0072-0.126:1125-3375.

[0022] The mass ratio of Bihu to amino acids is 90-135:1125-3375.

[0023] The application involves spraying the plant growth regulator on the stems and leaves 5-7 days after methoxyfenozide has caused phytotoxicity when applied to rice seedlings at the 4-5 leaf stage, and / or applying additional sprays.

[0024] The application rates of L-leucine, L-isoleucine, and L-valine are 675-2025 g / ha, 225-675 g / ha, and 0.0072-0.126 g / ha, respectively. The mass ratio of L-leucine, L-isoleucine, and L-valine is 3:1:1.

[0025] The rice varieties mentioned include, but are not limited to, Jinjing 818, 73119, and K37, or Nanjing 9108.

[0026] The three amino acids (leucine, isoleucine, and valine) of this invention, or in combination with brassinolide, when mixed with methoxyfenozide for spraying or as an additional spray after the onset of phytotoxicity, can alleviate the phytotoxicity of methoxyfenozide on rice without affecting its weed-controlling effect on weedy rice.

[0027] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0028] (1) Resistant rice varieties or lines can resist the phytotoxicity caused by treatment with 100g ai / ha-144g ai / ha methoxydimethalin alone, while sensitive varieties can only resist the phytotoxicity caused by less than 28.8g ai / ha methoxydimethalin.

[0029] (2) It can significantly alleviate the phytotoxicity of high doses of methoxydimethalin to resistant rice varieties and the phytotoxicity of low doses to sensitive varieties.

[0030] (3) After adding the safener, methoxyfenozide has no effect on the weed control effect of weedy rice. Attached Figure Description

[0031] Figure 1 The effects of different concentrations of methoxyfenozide on the seedling stage of the sensitive variety Nanjing 9108; Note: A. The left plastic box shows seedlings without methoxyfenozide spraying, and the right plastic box shows seedlings sprayed with 14.4 g ai / ha of methoxyfenozide; B. The left plastic box shows seedlings without methoxyfenozide spraying, and the right plastic box shows seedlings sprayed with 28.8 g ai / ha of methoxyfenozide; C. The left plastic box shows seedlings without methoxyfenozide spraying, and the right plastic box shows seedlings sprayed with 43.2 g ai / ha of methoxyfenozide; D. The left plastic box shows seedlings without methoxyfenozide spraying, and the right plastic box shows seedlings sprayed with 57.6 g ai / ha of methoxyfenozide.

[0032] Figure 2 This study investigated the effects of different concentrations of methoxyfenozide on the seedling stage of different rice varieties. Each plastic box contained four rice varieties, from left to right: Nanjing 9108 (NG9108, a sensitive variety), Jinjing 818 (JG818, a resistant variety), 73119 (a resistant variety), and K37 (a resistant variety). Note: A. The left plastic box shows seedlings without methoxyfenozide spraying, and the right plastic box shows seedlings sprayed with 48.0 g ai / ha of methoxyfenozide. B. The left plastic box shows seedlings without methoxyfenozide spraying, and the right plastic box shows seedlings sprayed with 144.0 g ai / ha of methoxyfenozide. C. The left plastic box shows seedlings without methoxyfenozide spraying, and the right plastic box shows seedlings sprayed with 240.0 g ai / ha of methoxyfenozide. Seedling images of methoxyfenozide at an ai / ha concentration; the plastic box on the left (D) shows seedlings without methoxyfenozide spraying, while the plastic box on the right shows seedlings sprayed with methoxyfenozide at a concentration of 336.0g ai / ha.

[0033] Figure 3 Effects of different safeners and methoxyfenozide mixtures on the growth of different materials at the 4-5 leaf stage; weed control effect of the safener developed in this invention in a plot; Note: (This experiment selected three experimental materials: weedy rice, sensitive variety Nanjing 49 (NG49), and resistant variety (K37). The planting of the materials was the same, only the treatments were different. The small pictures on the left and right are magnified pictures of each material; Among them: A: control without treatment; B: application of 100g ai / ha methoxyfenozide alone; C: combined application of 100g ai / ha methoxyfenozide and 135g / ha Bihu; D: combined application of 100g ai / ha methoxyfenozide and the protectant within the concentration range of this invention (the specific concentration of safener I applied in this experiment was: spraying 37.5g ai / ha branched-chain amino acids, of which 22.5g ai / ha L-leucine: 7.5g ai / ha L-isoleucine: 7.5g) (ai / ha L-valine = 3:1:1). Detailed Implementation

[0034] The experiments for this invention were conducted from May 2020 to October 2022, combining indoor and outdoor tests.

[0035] Test reagent: 4% methoxyfenozide, aqueous solution (Jiangsu Provincial Agricultural Hormone Engineering Technology Research Center Co., Ltd.);

[0036] Amino acids: L-valine, L-leucine, and L-isoleucine (Zhengzhou Zhongsi Food Co., Ltd.);

[0037] Bihu: Total active ingredient content 0.136%, gibberellin content 0.135%, indoleacetic acid content 0.00052%, brassinolide content 0.00031%, formulation: wettable powder (Agfle Agricultural, Forestry and Environmental Biotechnology Co., Ltd., Germany);

[0038] Brassinolide: Total active ingredient content 0.01%, aqueous solution (Jiangxi Weidi Biotechnology Co., Ltd.)

[0039] Test materials: Nanjing 9108 (abbreviated as NG9108), Jinjing 818 (abbreviated as JG818), 73119, K37 and Nanjing 49 (Su Shen Dao 201207, abbreviated as NG49). NG9108 is a high-quality japonica rice variety obtained by crossing Wuxiangjing 14 and Guandong 194 (abbreviated as NG9108, Wang Cailin, Zhang Yadong, Zhu Zhen, et al. Breeding and utilization of high-quality japonica rice variety Nanjing 9108. Jiangsu Agricultural Sciences, 2013, 41(9): 86-88). JG818 is a conventional japonica rice variety bred by Tianjin Rice Research Institute through hybridization of japonica rice 9618 and japonica rice 1007 (Wang Guangda, Gao Peng, Yang Wenyan, et al. Development and application of functional markers for the gene resisting imidazolinone herbicides in Jinjing 818. Chinese Journal of Rice Science, 2020, 34(4): 316-324). JG818, 73119 and K37 are herbicide-resistant rice varieties or lines (referred to as resistant rice materials) created by mutation of key amino acids in acetolactate synthetase (ALS), with NG9108 and NG49 without mutations as sensitive varieties.

[0040] Example 1: Comparative Test of the Effects of Plant Growth Regulators

[0041] 1. Concentration screening test of methoxyimidazolium on different rice varieties or lines

[0042] 1.1 Test Methods

[0043] 1.1.1 Test materials: There are three rice materials resistant to methicillin, including Jinjing 818, 73119 and K37, and the sensitive variety is Nanjing 9108 (abbreviated as NG9108).

[0044] 1.1.2 The dosage settings for the methoxyfenozide smoke concentration screening test are as follows:

[0045] Methoxyfenozide fumigation doses in sensitive rice materials: 0, 14.4 g ai / ha, 28.8 g ai / ha, 43.2 g ai. / ha and 57.6 g ai / ha;

[0046] Methoxyfenozide fumigation dosage for resistant rice materials: 0, 48.0 g ai / ha (this is the dosage used in production, 1N), 144.0 g ai / ha, 240.0 g ai / ha and 336.0 g ai / ha.

[0047] 1.1.3 Experimental Procedure

[0048] 1.1.3.1 Cultivation of rice materials:

[0049] Rice seeds were disinfected by soaking in 75% ethanol for 5 minutes, then rinsed repeatedly with deionized water to ensure no ethanol residue remained. After soaking in the dark at 28℃ for 48 hours, the sprouting rice seeds were placed in moist petri dishes for germination. When the sprouts reached approximately 0.5 cm in length, 200 uniformly germinated rice seeds were selected and sown in a transfer box (length × width × height: 53 cm × 37 cm × 14 cm) for a rice herbicide concentration screening experiment. Each transfer box was filled with paddy field clay loam, and direct seeding was used. Seedling boards were prepared before sowing to prevent waterlogging. After sowing, the seedlings were grown outdoors under natural temperature and light conditions. When the seedlings reached the 4-leaf stage, the stems and leaves were sprayed with the appropriate dose of methoxyfenozide. Each treatment had three biological replicates.

[0050] 1.1.3.2 Screening test for methoxymethylene smoke concentration

[0051] To investigate the safety of the herbicide methoxyfenozide on different rice varieties, five herbicide concentrations were established to treat both sensitive and resistant varieties. The recommended dosage of 4% methoxyfenozide aqueous solution is 48 g ai / ha. For the sensitive variety NG9108, the recommended concentrations of methoxyfenozide were 0, 14.4 g ai / ha, 28.8 g ai / ha, 43.2 g ai / ha, and 57.6 g ai / ha, respectively. For the resistant varieties JG818, 73119, and K37, the recommended concentrations of methoxyfenozide were 0, 48.0 g ai / ha, 144.0 g ai / ha, 240.0 g ai / ha, and 336 g ai / ha, respectively. The appropriate dosages were applied evenly to the stems and leaves on a windless, sunny afternoon. The spraying was performed using a 0.3 MPa pressure atomizer (GARDENA imported household gardening fine mist watering can from Germany), with a dosage of 30 liters per acre. The treated plants continued to grow, and the dry weight of individual plants was measured 21 days after treatment. A water spray was used as a control, and the treatment was repeated three times.

[0052] 1.1.3.2 Determination of growth indicators

[0053] Survival rates were recorded for each treatment 14 days after methoxyfenozide treatment. At different days after methoxyfenozide treatment, harvested test materials were blanched at 105℃ for 15 min and dried at 70℃ to constant weight (Wang Gang, Yu Lihua, Wang Yuguang, et al. Effects of calcium nutrition on salt tolerance of sugar beet seedlings [J]. China Sugar Crops, 2021, 43(2): 40-46). The dry weight of each plant was measured using an electronic balance with a sensitivity of 0.0001 g, and the average value was used to calculate the dry weight inhibition rate. All experiments were biologically replicated three times.

[0054] Survival rate (%) = (Number of surviving plants in control - Number of surviving plants in treatment) / Number of surviving plants in control × 100%

[0055] Dry weight inhibition rate (%) = (Control dry weight - Treatment dry weight) / Control dry weight (without herbicide treatment) × 100%

[0056] 1.1.4 The test results are shown in Table 1:

[0057] Table 1. Effects of different concentrations of methoxymethylene smoke treatment on the survival rate and dry weight of NG9108.

[0058]

[0059]

[0060] In Table 1, lowercase letters indicate the significance of data differences between different treatments (Duncan P < 0.05).

[0061] from Figure 1It can be seen that after treatment with methoxyfenozide, seedlings of the sensitive rice variety NG9108 showed yellowing leaves and stunting. Furthermore, with increasing herbicide concentration, most plants withered and died after treatment exceeding 43.2 g ai / ha. Table 1 shows the survival rate, aboveground dry weight per plant, and dry weight inhibition rate of NG9108. This indicates that methoxyfenozide treatment significantly inhibits the growth of the sensitive rice variety, with higher concentrations resulting in more severe inhibition. The survival rate of NG9108 after treatment with 28.8 g ai / ha methoxyfenozide was 61.27%, significantly lower than that at 14.4 g ai / ha. When the methoxyfenozide concentration reached 43.2 g ai / ha and 57.6 g ai / ha, the survival rates of NG9108 were only 20.16% and 3.85%, respectively. With increasing herbicide concentration, after treatments of 14.4 g ai / ha, 28.8 g ai / ha, 43.2 g ai / ha, and 57.6 g ai / ha, the aboveground dry weight of individual plants was 74.7%, 39.2%, 9.9%, and 8.1% of the control, respectively, and the dry weight inhibition rates were 20.13%, 60.14%, 88.89%, and 91.05%, respectively. Considering phenotype, survival rate, and inhibition rate of aboveground dry weight, 28.8 g ai / ha of methoxyfenozide was selected as the screening concentration for the mitigation effect of adding a safener to sensitive varieties.

[0062] Depend on Figure 2 It can be seen that the higher the concentration of methoxyimidazolium fumigant, the shorter the seedlings of resistant rice varieties. However, unlike sensitive varieties, their leaves remained green. At doses exceeding 240 g ai / ha, K37 also showed significant wilting. Therefore, our study suggests that the optimal dose of methoxyimidazolium fumigant for resistant rice materials should not exceed 144.0 g ai.i. / ha. Tables 2-3 show that resistant rice varieties treated with 144.0 g ai / ha of methoxyimidazolium fumigant all exhibited a survival rate exceeding 50%, significantly higher than sensitive varieties, while sensitive varieties all died at 48.0 g ai / ha of methoxyimidazolium fumigant. However, it should be noted that the inhibition rate of single-plant dry weight of the aboveground parts of resistant varieties differed under the 144.0 g ai / ha methoxyfenozide treatment, which may be closely related to the different amino acid mutation sites in the mutant materials. However, it is clear that all resistant rice materials with ALS mutation sites are resistant to methoxyfenozide at a dose of 144.0 g ai / ha. Given that the recommended concentration of methoxyfenozide in production is 48.0 g ai / ha, and that it has already caused the complete death of plants in sensitive rice varieties without ALS mutation sites, K37, with the lowest resistance, was selected as the test material for subsequent herbicide mitigation trials in further safety agent development experiments.

[0063] Table 2. Effects of different concentrations of methoxymethylene smoke treatment on the survival rate of resistant rice varieties.

[0064]

[0065] In Table 2, lowercase letters indicate the significance of differences in data between different treatments (Duncan P < 0.05).

[0066] Table 3. Effects of different concentrations of methoxymethylene smoke treatment on the aboveground dry weight of resistant rice varieties.

[0067]

[0068] In Table 3, lowercase letters indicate the significance of differences in data between different treatments (Duncan P < 0.05).

[0069] 2. Detoxification effect of compound branched-chain amino acid safener on methoxyfenozide-induced herbicide damage in rice.

[0070] 2.1 Test Methods

[0071] 2.1.1 Test materials: resistant variety K37, weedy rice (collected in Huoqiu, Anhui in October 2020), and rice variety Nanjing 49 without ALS mutation.

[0072] 2.1.2 The dosage settings for mixed spraying treatments with different safeners and methoxyfenozide are shown in Table 4 below:

[0073] In actual production, methoxyfenozide is usually sprayed at 1 times the working concentration (50g ai / ha), and generally does not exceed 2 times the working concentration (100.0g ai / ha). In order to make the safe agent developed in this invention more in line with the actual production of rice, the examples of the safe agent developed in this invention are all treated with 2 times the working concentration of methoxyfenozide, that is, 100.0g ai / ha methoxyfenozide.

[0074] Table 4. Proportions of Compound Amino Acids and Methoxymidazine Smoke Spray Components

[0075]

[0076]

[0077] 2.2 Experimental Procedure

[0078] 2.2.1 Cultivation and treatment of rice materials:

[0079] 2.2.1.1 Material Cultivation

[0080] Cultivation in an artificial climate chamber: 49 seeds of resistant rice K37 were sown in a square plastic culture box (length × width × height: 5cm × 5cm × 6cm). The seeds had been soaked and germinated in advance. The culture was carried out in a greenhouse soil chamber, with each culture box containing paddy field clay loam. The artificial climate chamber was used for cultivation at 30℃ / 25℃ (day / night) and 14h / 10h (light / dark).

[0081] Cultivation in outdoor plastic buckets: After disinfection, rice seeds are soaked and germinated. Select germinated seeds with uniform growth and sow them in a water bucket (diameter: 21cm). The soil used is rice paddy clay loam. Sow 20 germinated seeds in each bucket and allow them to grow naturally under temperature and light.

[0082] Outdoor plot cultivation: Rice seedlings were placed in cement ponds within the netted greenhouse of the Jiangsu Academy of Agricultural Sciences' Institute of Food Crops, with each plot measuring 2m. 2 The experiment was repeated three times, with randomized block design in a cement pool containing clay paddy soil. The seeding rate of the variety was 4 kg per mu (approximately 0.067 hectares) to simulate the conditions of direct seeding in paddy fields. The plants were cultivated outdoors in Nanjing from May to July under natural light and temperature conditions. The experiment was repeated three times.

[0083] All three methods involve uniform treatment when the rice has grown to the stage of 4 leaves and 1 heart.

[0084] 2.2.1.2 Treatment of safety agents

[0085] When the seedlings have grown to four leaves and a central bud, select plants with uniform shape, growth, and leaves. On a sunny evening, spray the stems and leaves with the appropriate dosage of the mixed solution according to the ratio in Table 4. Use a 0.3 MPa pressure atomizer (GARDENA imported household gardening fine mist watering can from Germany), and spray at a rate of 30 liters per acre. After treatment, continue growth under the appropriate temperature and light conditions. Measure the dry weight of individual plants 14 days after treatment. Use water spray as a control (CK), and the biological replication is performed three times.

[0086] 2.2.2 Determination of growth indicators

[0087] Plant survival rate, single plant dry weight and dry weight inhibition rate were determined by referring to the method in "1. Concentration screening test of methoxyfenozide on different rice varieties or lines" in this example.

[0088] Detoxification effect (%) = [(Dry weight of rice after treatment with safener and herbicide - Dry weight of rice after treatment with herbicide alone) / Dry weight of rice in the blank control (without herbicide treatment)] × 100%

[0089] 2.3 Test Results

[0090] 2.3.1 Mitigation Test of Components and Ratios of Branched-Chain Amino Acid Plant Growth Regulators

[0091] 2.3.1.1 Greenhouse cultivation alleviates the effects

[0092] Table 5. Detoxification effects of different amino acid compositions and ratios on herbicide damage in K37 resistant rice cultured in artificial climate chambers.

[0093]

[0094] Lowercase letters indicate significant differences in each column of data across different treatments (Duncan P < 0.05).

[0095] Table 5 shows that at the 4-leaf stage, after spraying with a compound of 100g ai / ha methoxyfenozide and different ratios of amino acid components, stunted seedlings with leaf tip chlorosis were observed in K37 plants (5 days after application). After 21 days of treatment, some safeners significantly alleviated the phytotoxicity caused by 100g ai / ha methoxyfenozide on K37, with the alleviating effect ranking as follows: safener F > J > I > G > K. Other treatments showed no significant difference. For example, treatments F, G, I, and J all alleviated the phytotoxicity caused by 100g ai / ha methoxyfenozide on K37, with dry weight inhibition rates of 18.61%, 32.11%, 29.73%, and 26.78%, respectively, and detoxification effects of 18.99%, 5.50%, 7.89%, and 10.83%, respectively, i.e., F > J > I > G. The detoxification effects of other treatments were not significant. In this embodiment, the dry weight of individual plants after 14 days under the treatment with the safener formulation was higher than that under the treatment of applying 100g ai / ha methoxyfenozide alone, which showed a significant alleviating effect on K37. As has been clearly demonstrated in the methoxyfenozide concentration screening test in Example 1, the survival rate of resistant rice materials treated with 100g ai / ha methoxyfenozide alone was greater than 95%. Therefore, this embodiment focuses on the alleviating effect on plant growth, and uses the dry weight of individual plants as the screening index, so the survival rate is no longer recorded separately.

[0096] 2.3.1.2 Water tank cultivation alleviates the effect

[0097] Table 6. Detoxification effects of different amino acid compositions and ratios on herbicide damage in resistant rice material K37 cultured outdoors in water tanks.

[0098]

[0099] Lowercase letters indicate significant differences in each column of data across different treatments (Duncan P < 0.05).

[0100] To further verify the mitigation effects of the four component safety agents developed in the examples, a water bucket culture experiment was conducted (Table 6). The results showed that the mitigation effect was F > G > I > J > K. The trend of the water bucket culture experiment was similar to that of the artificial climate chamber, with the mitigation effect being F > G > I > J > K. The mitigation effects of the F, G, I and J treatments were significant.

[0101] 2.3.1.3 The mitigation effect of small-scale cultivation

[0102] Table 7. Detoxification effects of different amino acid compositions and ratios on herbicide damage in resistant rice variety K37 cultured outdoors in cement ponds.

[0103]

[0104] Lowercase letters indicate significant differences in each column of data across different treatments (Duncan P < 0.05).

[0105] The results of the cement pool plot test are shown in Table 7. The mitigation effect was in the order of safety agent I > J > K > F > G, among which treatments I, J and K showed significant mitigation effects (P < 0.05).

[0106] In summary, both treatments I and J, i.e., at a working concentration of 100.0 g ai / ha methoxyfenozide (2N), combined with the application of 37.5 g ai / ha branched-chain amino acids, wherein the ratio of 22.5 g ai / ha L-leucine:7.5 g ai / ha L-isoleucine:7.5 g ai / ha L-valine is 3:1:1, or at a working concentration of 100.0 g ai / ha methoxyfenozide (2N), combined with the application of 37.5 g ai / ha branched-chain amino acids (commercially available branched-chain amino acid products), wherein the ratio of leucine / isoleucine / valine is 2:1:1, can significantly alleviate the growth inhibition of the resistant variety K37 by methoxyfenozide.

[0107] Example 2: Comparative test of the mitigation effects of the branched-chain amino acid plant growth regulator developed in this invention and commercially available safeners.

[0108] 1. Test Methods

[0109] 1.1 Test materials: The same as those in the section "1.2 Detoxification effect of compound amino acid growth regulators with different components and proportions on methoxyimidazolium-induced herbicide damage to rice" in Example 1.

[0110] 1.2 Effect of the mixture of compound branched-chain amino acids, Bihu, brassinolide and methoxyfenozide on the herbicidal effect of rice in planting plots

[0111] The dosage settings for the pharmaceuticals and adjuvants in Example 1 are as shown in Table 4. The actual treatment concentrations used in this example are shown in Table 8.

[0112] Table 8. Processing components and codes in this embodiment.

[0113]

[0114] 2. Experimental Procedure

[0115] The experiment was conducted from August to October 2021 in a cement strip pool within a netted greenhouse at the Institute of Food Crops, Jiangsu Academy of Agricultural Sciences. The effect of the mixture of Bihu, a safener, and methoxyfenozide on the herbicidal efficacy of the tested materials was determined using the outdoor plot whole-plant biomass assay.

[0116] 2.1 Cultivation of test materials and treatment of reagents: The method is the same as that in Example 1.

[0117] 2.2 Determination of growth indicators: The method is the same as that in Example 1.

[0118] 3. Test Results

[0119] Table 9. Effects of different safeners on the weed control of rice mulch by methoxymethyl smoke.

[0120]

[0121]

[0122] Lowercase letters indicate significant differences in each column of data across different treatments (Duncan P < 0.05).

[0123] As shown in Table 9, different concentrations of Bihu, brassinolide, and the compound branched-chain amino acid safener developed in this invention, when mixed with methoxyfenozide, reduced the weed control effect of 100g ai / ha methoxyfenozide on weedy rice to varying degrees. In terms of aboveground dry weight inhibition rate, compared with the application of 100g ai / ha methoxyfenozide alone, different concentrations of Bihu mixed with methoxyfenozide reduced the dry weight inhibition rate of weedy rice to varying degrees. Only when the mixture was applied to the stems and leaves of weedy rice at the 5-leaf stage did the inhibition rate show a significant difference compared to other safener-mixed treatments. In this example, the dry weight inhibition rate of the treatments, whether at the 4-leaf or 5-leaf stage, was between 88.35% and 95.11%, meeting the requirements for methoxyfenozide control of weedy rice in the field. Moreover, as the herbicide treatment time increased, all weedy rice died by 21 days, indicating that these safeners did not affect the weed control effect of the herbicide on weedy rice.

[0124] Under the same treatments as in Table 9, none of them significantly reduced the killing effect of methoxyfenozide on the sensitive variety NG49 at a dose of 100 g ai. / ha. Moreover, the dry weight inhibition was greater than that of weedy rice, and the symptoms of poisoning appeared earlier. Yellowing of leaves and stunted growth appeared 5 days after spraying, and all plants died by the 10th day of treatment. It can be seen that rice varieties without ALS mutations cannot resist the killing effect of methoxyfenozide at a dose of 100 g ai / ha. It can also be seen that the mixed application of safeners does not affect the killing effect on sensitive varieties.

[0125] Table 10. Herbicidal effects of different safeners on methoxyprobe-resistant rice K37.

[0126]

[0127] Lowercase letters indicate significant differences in each column of data across different treatments (Duncan P < 0.05).

[0128] As can be seen from Table 10, when different concentrations of BRIGHTEC, the safener I developed in this invention and imazamox were applied simultaneously at the 4-leaf and 5-leaf stages of resistant rice varieties, the phytotoxicity caused by 100 g a.i. / ha imazamox to resistant rice could be alleviated. When BRIGHTEC was added, 45 g / ha BRIGHTEC could effectively alleviate the phytotoxicity caused by 100 g a.i. / ha imazamox to the resistant rice material K37 at the 4-leaf stage. The dry weight inhibition rate after 14 days of treatment was 35.93%, which was not significantly different from the dry weight inhibition rate of 40.93% of K37 when 100 g a.i. / ha imazamox was applied alone, and the detoxification effect was 13.06%. After adding 90 g / ha BRIGHTEC, the dry weight inhibition rate of K37 was 15.65%, and the detoxification effect was 25.28%, significantly alleviating the phytotoxicity caused by 100 g a.i. / ha imazamox to K37. After adding 135 g / ha BRIGHTEC, the dry weight inhibition rate of K37 was 9.78%, significantly lower than the dry weight inhibition rate of K37 when 100 g a.i. / ha imazamox was applied alone, and the detoxification effect was 31.16%, which could also effectively alleviate the phytotoxicity of 100 g a.i. / ha imazamox to K37. It can be seen that BRIGHTEC can alleviate the phytotoxicity of imazamox to K37, and there is a dose effect. The treatment O with compound addition of brassinolide had little alleviating effect on the 4-leaf stage material, and there was no significant difference from the dry weight inhibition rate of K37 when 100 g a.i. / ha imazamox was applied alone. After adding the safener I with the composite branched-chain amino acid component developed in this invention, the dry weight inhibition rate of K37 was also significantly lower than that of K37 when imazamox was applied alone, and the detoxification effect was 39.08%, which was the highest value among the treatments in this example. Compared with the 4-leaf stage, spraying the test plants at the 5-leaf stage had a better detoxification effect. It can be seen that selecting plants with relatively larger leaf ages such as the 5-leaf age to spray herbicides is beneficial for the resistant rice to recover plant growth faster. The phenotypes of the test materials with the mixed application of safeners during growth are shown in Figure 3 A, Figure 3 B, Figure 3 C and Figure 3 D.

[0129] Example 3 Additional Test of Different Safener Components after the Appearance of Symptoms in Resistant Rice

[0130] 1 Test Method ]>

[0131] 1.1 Test Materials: K37

[0132] 1.2 Safener Components after the Appearance of Symptoms in Resistant Rice: The components and codes of the composite branched-chain amino acid, BRIGHTEC, and brassinolide mixed treatment are referred to Table 4.

[0133] 1.3 Cultivation and Treatment of Rice Materials: The method is referred to the method of Example 1

[0134] 1.4 Determination of growth indicators: The method is the same as that in Example 1.

[0135] 2. The test results are shown in Table 12 below:

[0136] Table 12. Detoxification effect of topdressing with safeners on herbicide damage in resistant rice material K37 that has already shown symptoms.

[0137]

[0138]

[0139] Lowercase letters indicate significant differences in each column of data across different treatments (Duncan P < 0.05).

[0140] As shown in Table 12, after spraying 100g ai / ha methoxyfenozide at the 5-leaf stage, and observing stunted seedlings with leaf tip chlorosis in the resistant variety K37 (5 days after application), subsequent application of Bihu and other safeners from this invention, along with 14 days of treatment, showed that all safener components alleviated the phytotoxicity of the resistant variety K37 under the 100g ai / ha methoxyfenozide treatment. The detoxification effect was S > T > Q > R > P > 1 > M > L. Clearly, the additional application of safeners resulted in a higher detoxification effect than the combined application of herbicide and safener. The increased total amino acid content suggests a dose-dependent detoxification effect. Considering the cost of the safety agent, we treated the safety agent with a low dose of compound amino acids (I) and different concentrations of brassinolide. This treatment significantly improved the detoxification effect of the safety agent I. However, it is worth noting that a higher brassinolide content is not necessarily better. The detoxification effect is best with a low dose of compound amino acids and a 2 / 3N working concentration of brassinolide.

[0141] In conclusion, this invention has developed a safe agent that can alleviate the phytotoxicity of 100g ai / ha methoxydimethoate smoke dose in ALS-resistant varieties, as well as a method for applying the safe agent.

Claims

1. The application of a plant growth regulator in alleviating the phytotoxicity of methoxyfenozide to rice, characterized in that, The active ingredient of the plant growth regulator is composed of L-leucine, L-isoleucine, and L-valine, with a mass ratio of 3:1:

1. The application rate of the plant growth regulator is 1125-3375 g / ha, of which L-leucine is 675-2025 g / ha, L-isoleucine is 225-675 g / ha, and L-valine is 225-675 g / ha. The concentration of methoxyfenozide is 14.4-336.0 g ai / ha. The application is performed 5-7 days after methoxyfenozide application at the 4-5 leaf stage of rice seedlings, resulting in phytotoxicity, by foliar spraying the plant growth regulator and / or additional spraying.

2. The application of a plant growth regulator in alleviating the phytotoxicity of methoxyfenozide to rice, characterized in that, The active ingredient of the plant growth regulator is composed of L-leucine, L-isoleucine, L-valine, and brassinolide, wherein the mass ratio of L-leucine, L-isoleucine, and L-valine is 3:1:1; the application rate of the plant growth regulator is 1125-3375 g / ha, wherein L-leucine is 675-2025 g / ha, L-isoleucine is 225-675 g / ha, and L-valine is 225-675 g / ha, and the application rate of brassinolide is 0.0072-0.126 g / ha; the concentration of methoxyfenozide is 14.4-336.0 g ai / ha; the application is performed 5-7 days after methoxyfenozide application at the 4-5 leaf stage of rice seedlings, followed by foliar spraying of the plant growth regulator and / or additional spraying.

3. The application of a plant growth regulator in alleviating the phytotoxicity of methoxyfenozide to rice, characterized in that, The active ingredient of the plant growth regulator is composed of L-leucine, L-isoleucine, L-valine, and Bihu, wherein the mass ratio of L-leucine, L-isoleucine, and L-valine is 3:1:1; the application rate of the plant growth regulator is 1125-3375 g / ha, wherein L-leucine is 675-2025 g / ha, L-isoleucine is 225-675 g / ha, and L-valine is 225-675 g / ha, and the application rate of Bihu is 90-135 g / ha; the concentration of methoxyfenozide is 14.4-336.0 g ai / ha; the application is performed 5-7 days after methoxyfenozide application at the 4-5 leaf stage of rice seedlings, followed by foliar spraying of the plant growth regulator and / or additional spraying.

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