Alternative Herbicides to Manage Unintentionally Released Transgenic Canola

Introduction

Genetically modified (GM) crops are not allowed to cultivate in Korea but have officially been allowed to import for food and feed purposes (Park et al., 2007). In 2017, 2.28 million tonnes were imported for food, and 7.32 million tonnes as feed (KBCH, 2017). Transgenic crops are also being used as a trial for research activities in isolated areas (Kim et al., 2008). Public perception of GMOs is negative, and there are strong demands for regulation with regards to labeling, importing and distribution in advance (KBCH, 2017). In recent years, concerns regarding the unintentional escape of GM crops during transportation and manufacturing, and subsequent contamination between GM and non–GM plants have been raised (Park et al., 2010). Thus, the growing importance of GM crops in Korea has raised many concerns regarding the environmental risk (Lee et al., 2010). There are many risks associated with the GM and herbicide-resistant crops, including problems with segregation and introgression of herbicide-resistant traits and increased the reliance on herbicides for weed control (Han et al., 2016; Lee and Suh., 2011; Owen and Zelaya 2005). Another issue is the ecological impact that is simple weed management programs based on herbicide-resistant crops have on weed communities. Transgenic canola (Brassica napus L.) is the most concerning related to gene flow. Because it shows considerable outcrossing rate, gives rise to volunteer emergence in a field and can form temporary feral populations. In other word, the natural hybridization rate between canola and wild relatives is too high that unintentional exposure to natural ecosystems will not be ignored (Baek et al., 2008; Hoyle et al., 2007; Lim et al., 2015). Lee et al. (2007) conducted monitoring study to investigate the intentional or unintentional release of three herbicide resistant oilseed rape events (RT73, T45, and MS8/RF3) which have already been approved for consumption in Korea. Based on the PCR analyiss, no GM canola was detected in the surveyed areas. Since 2009, the Ministry of Environment has been announcing the environmental monitoring and survey every year to identify the unintentional release of transgenic canola to the environment. The environmental monitoring of glyphosate and glufosinate-ammonium tolerant GM canola was conducted through the nine provinces of Korea, from 2009 to 2013 (Shin at al., 2016). This study revealed that four and two crops were glufosinate- and glyphosate-resistant GM canola respectively contained T45, MS8, RT73, Rf3, and Topas 19-2 event genes. By the Ministry of Agriculture, Forestry and Livestock of Korea, 98 surveillance target sites of GM canola were approved throughout the country and done disposal actions in 2017. The identification of unintentionally infested sites and performance of some disposal actions might be important and could decrease the environmental risks associated with gene flow. In addition to them, the investigation of proper chemical management strategies against the canola would also be the best option. Hence, we carried out herbicide application experiments in the natura lecosystem of Korea with two consecutive in 2018 and 2019 years.

The purpose of this study was identification of alternative herbicides to eliminate unintentionally released herbicide resistant canola in the ecosystem of Korea.

Materials and methods

Herbicide application under greenhouse condition

The experiment was conducted under glasshouse condition located in the experimental farm station of Chungnam National University, Daejeon, South Korea. To begin, the mixture (1:1 (w/w) of horticultural nursery soil (Bunong, Korea) and rice nursery soil (Bunong, Korea) was placed into the plastic pots (20 cm inside diameter, 17.0 cm height, 0.2 cm thickness). Then, 50 canola seeds were sown individually in a 1 cm depth of the soil. Thereafter, pre-emergent herbicides (Table 1) applied at 1x and 2x recommended rate to the soil surface, as a soil apply, with three replications. The required amount of herbicides was calculated using the following formula.

Control and all treated plants were grown in the glasshouse and temperature was maintained at 25℃ and 17℃ (day/night) for 21 days. The number of seedlings and total fresh and dry weight were used as a measurement for control efficacy. In case of postemergence application, foliar apply herbicides (Table 1) were sprayed when seedlings were on 2-3 leaf stage (approximately 21 days after sowing; DAS). 14 days after treatment (DAT), the number of survived plants and total fresh weight were measured.

Herbicide application under field condition

A field experiment was conducted to define the control efficacy of pre- and post-emergence herbicides on canola under field condition. The experiment was carried out from April to late September in 2018 and 2019. The details of materials used and techniques adopted in the present investigation are described below.

The experiment was conducted at the confine field trial of Chungnam National University, Daejeon, South Korea (127°21′ E, 36° 22′ N, alt. 34 m). The experimental layout of the experiment was randomized complete block design (RCBD) with three replications. Canola seeds were sowed on April 10 in 2018 and May 10 in 2019 at the rate of 5 kg ha^-1 in a 1 m2 net plot area. Soil and foliar applyherbicides (Table 1) were applied at 1x and 2x recommended rates with three replications. Required amount of herbicides for the experiment was calculated by using the equation [1]. Thus, calculated amount of herbicide was sprayed to each plot with pressured sprayer. Pre-emergence herbicides were applied uniformly on the soil surface at the same day following the seed sowing. Postemergence herbicides were applied uniformly when canola plants formed 2-3 leaves (21 DAS). The number of seedlings, plant height and fresh and dry weights of plants were measured to evaluate the control efficacy of herbicides at 28 DAT and 14 DAT for pre-emergence and post emergence applications, respectively.

Herbicide dose-response study

The experiment was performed under greenhouse conditions from March to May in both 2018 and 2019. The dose-responses of canola to herbicides with different modes of action were determined in a glasshouse mentioned above. Firstly, 50 seeds were counted and sowed in plastic pots (20 cm inside diameter, 17.0 cm height, 0.2 cm thickness) containing the mixture of 1:1 (w/w) of horticultural nursery soil (Bunong, Korea) and rice nursery soil (Bunong, Korea). Followed by, pre-emergence herbicides were applied to the soil surface at eight doses: oxyfluorfen (protoporphyrinogen oxidase [PPO] inhibitors) at 0, 0.1175, 1.175, 11.75, 117.5, 1175, 11750, and 117500 g a.i. ha^-1; s-metolachlor (inhibition of cell division) at 0, 0.075, 0.75, 7.5, 75, 750, 7500, and 75000 g a.i. ha^-1; dichlobenil (inhibition of cell division) at 0, 0.67, 6.7, 67, 670, 6700, 67000, and 670000 g a.i. ha^-1; linuron (photosynthesis at photosystem II) at 0, 0.05, 0.5, 5, 50, 500, 5000, and 50000 g a.i. ha^-1. While, for post-emergence application bentazone (photosynthesis at photosystem II) at 0, 0.16, 1.6, 16, 160, 1600, 16000, and 160000 g a.i. ha^-1; fluthiacet-methyl (PPO inhibitors) at 0, 0.061, 0.61, 6.1, 61, 610, 6100, and 61000 g a.i. ha^-1; MCPA (action like indole acetic acid) at 0, 0.168, 1.68, 16.8, 168, 1680, 16800, and 168000 g a.i. ha^-1; glufosinate-ammonium (glutamine synthesis) at 0, 0.09, 0.9, 9, 90, 900, 9000, and 90000 g a.i. ha^-1; bentazone sodium (photosynthesis at photosystem II) at 0, 0.1, 1, 10, 100, 1000, 10000, 100000 g a.i. ha^-1 were applied individually to canola plants at 2-3 leaf stage using a sprayer (R&D Sprayer, USA). Temperature was maintained at 25/17℃ (day/night) until the experiment was ended up. Control efficacy of pre- and post-emergence applications was rated at 21 and 14 (DAT). Ratings were based on visual scale 0 to 100%, where 0 indicates for no visible plant injury and 100 means complete death or no germination. Data were subjected to analysis of variance and regression, and were fit to the logistic nonlinear regression:

where y = % weed control; x = herbicide dose (g/ha^-1), and a, b and c = equation estimated parameters, in which: a = amplitude between the minimum and maximum y points; b = rate that provides 50% of variable response; c = curve slope around.

Statistical analyses

ANOVA and Turkey’s HSD test were introduced perform statistical analyses using SAS 9.2 (SAS Institute Inc., Cary, NC, USA). Proc NLIN (SAS 9.2) was used for nonlinear regression analysis.

Results and discussion

Herbicide application under greenhouse condition

Under greenhouse condition, the effect of pre- and post-emergence herbicides on the emerging and survival rate, plant height (cm), total fresh and dry weight (g/pot) of canola are presented in Table 2 and Table 3 respectively. In case of pre-emergence application, the control efficacy varied depending on the applied herbicides. The highest value was found in the pot that treated with s-metolachlor at 1x and 2x recommended rates having totally 22 and 17 seedlings per pot, respectively. While, untreated control line had only 26 seedlings per pot. Oxyfluorfen and dichlobenil completely inhibited the seed germination at both 1x and 2x recommended rates that growth was not observed. The experiments in greenhouse showed that when linuron was applied at 2x recommended rate, some of seeds could germinate. However, those seedlings had a defective growth and eventually leaded to a plant death.

Herbicide application under field condition

Canola population and other measurement values were at the highest in the untreated control plot. Under different weed control treatments, the number of live canola seedlings was decreased in both 2018 and 2019. MD. Shahidul et al. (2016), Al-Kothayri and Hasan (1990) and Rekha et al. (2003) also reported that the treated population was lower compared to the untreated control line. Similar to the greenhouse experiment, pre-application in the field condition also proved that s-metolachlor was less effective with 66 survived seedlings/plot, even double rate was used (Table 4). Full control efficacy was observed when oxyluorfen and dichlobenil was applied. However, herbicide application with linuron showed less effectiveness that 7 and 0 seedlings/plot could emerge and to develop at the 1x and 2x rates, respectively. While in the untreated control plot, there were 120 seedlings/plot, 28 DAS.

Regarding the control efficacy of foliar application, bentazone, MCPA and bentazone sodium were more efficient herbicides that could control canola considerably at both 1x and 2x recommended rates (Table 5). Followed by them, fluthiacet-methyl showed quite well efficacy. Whereas, totally, 80 and 22 live plants/plot were derived from the glufosinate-ammonium application at the 1x and 2x recommended rates, respectively. Our foliar application under field condition revealed that less efficacy might be observed from the same treatments which showed an excellent control under glasshouse condition.

Herbicide dose-response study

Dose-response curves adjusted to pre- (oxyfluorfen, s-metolachlor, dichlobenil, linuron) and post- (bentazone, MCPA, glufosinateammonium, fluthiacet-methyl, bentazone sodium) emergent herbicides are presented in Fig. 1. The least herbicide application rate of C80 values for oxyfluorfen, dichlobenil and linuron was 1/72x (16.3 g a.i. ha^-1), 1/110x (61 g a.i. ha^-1) and 1/58x (8.6 g a.i. ha^-1) respectively. Brassica napus was less susceptible to the treatment with s-metolachlor that the application rate of 8x (6,000 g a.i. ha- 1) should be applied to reach 80% control efficacy. Four out of five post-emergence herbicides showed an excellent effectiveness on Brassica napus (Fig. 1). The most brilliant inhibitions observed on Brassica napus (C80) were the post applications with bentazone, MCPA, fluthiacet-methyl, and bentazone sodium at the rate of 1/64x (24.9 g a.i. ha^-1), 1/95x (17.68 g a.i. ha^-1), 7.5x (81.3 g a.i. ha^-1), and 1/33x (30.3 g a.i. ha^-1), respectively. In contrast, glufosinate-ammonium could hardly inhibit the glutamine-synthesis under greenhouse condition. C80 was obtained at the recommended rate of 8.9x (8,010 g a.i. ha^-1).

Conclusion

After admittance of the importing of GM canola, concern on unintentional introgression with wild or family relatives as well as with other local weed or crop species is increasing among not only scientists but government and public sectors as well. Investigation of alternative and proper chemical treatments could be the best option to manage unintentional escaping of the transgenic canola to the environment. From our study, it could be concluded that except s-metolachlor and glufosinate ammonium, all the herbicides used in the study can be applied to control Brassica napus even at the lower dose than those recommended and/or labeled rate. If we want to be free from the herbicide resistant weeds caused by transgenic crops, further experiments need to be conducted to identify more effective herbicides in a certain area

Acknowledgements

This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries(IPET) through Agri-Bio industry Technology Development Program funded by Ministry of Agriculture, Food and Rural Affairs(MAFRA)(317073-02-2-HD030).