Glyphosate has been applied in European countries for over a decade between rows in olive groves and grape vineyards to control Conyza species [hairy fleabane (C. bonariensis), horseweed (C. canadensis) and Sumatran fleabane (C. sumatrensis)], however poor control has been observed in recent years. Glyphosate susceptible (GS) or resistant (GR) populations were assayed in each species. In addition, Conyza spp. control with alternative herbicides (alone or in mixture with glyphosate) over two years was also assessed. The GS populations of the three species were controlled with glyphosate field doses (1080 g ae ha −1). The GR hairy fleabane, horseweed and Sumatran fleabane populations were 15.0, 15.7 and 19.8 times more resistant, respectively, than their respective GS population. The shikimic accumulation of GS populations was 4-6 times higher compared with the GR Conyza populations, confirming the glyphosate resistance of the latter ones. The increase in the glyphosate dose did not control the GR Conyza populations, despite providing a higher dry growth reduction. Glufosinate and flazasulfuron, alone or mixed with glyphosate, were the effective options to control GR and GS populations of hairy fleabane and Sumatran fleabane. However, the GR horseweed population might have evolved multiple resistance to glyphosate and flazasulfuron in Hungary. The other herbicides (PSI, auxinic and PPO) showed an additive effect together with the control provided by glyphosate in the GS and GR populations; however generally, these herbicides could be applied alone at the rosette stage. Effective herbicides with modes of action different from glyphosate, except flazasulfuron for controlling horseweed, should be used to delay the selection of herbicide resistance in perennial crops in Europe. around the world (Heap, 2018). Glyphosate [N-(phosphonomethyl)-glycine] is the world's most successful post-emergence and non-selective herbicide (Duke, 2018). In Spain it has been widely used to control weeds in citrus orchards, olive groves, grape vineyards, and others perennial and annual crops (González-Torralva et al., 2010, 2014), as well as in path borders, railway lines, recreation areas and derelict sites (Urbano et al., 2007). This herbicide is absorbed through leaves and other young-green tissues, and translocated via phloem into meristematic tissues (Preston and Wakelin, 2008). Glyphosate is a potent inhibitor of 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) by blocking the biosynthesis of phenylalanine, tryptophan, tyrosine and others aromatic compounds in susceptible plants (Maeda and Dudareva, 2012).

Conyza canadensis is a species invading large areas throughout the world, mainly due to its ability to evolve herbicide resistance. In Hungary, extensive areas have been infested by this species due to the difficulty in controlling it with glyphosate. To determine whether poor control was a result of misapplication or glyphosate resistance, eight suspected glyphosate-resistant C. canadensis populations from different Hungarian regions were studied. In whole-plant dose-response assays with glyphosate, the LD50 and GR50 values (survival and fresh weight reduction at 50% relative to the untreated control, respectively) indicated that resistance was confirmed in five of the eight populations (H-5 population being the most resistant). Additionally, the shikimic acid accumulation tests corroborated the results observed in the dose–response assays. 11 alternative herbicides from six different modes of action (MOA) were applied at field doses as control alternatives on populations H-5 and H-6 (both in the same regions). The H-5 population showed an unexpected resistance to flazasulfuron (ALS-inhibitor). The ALS enzyme activity studies indicated that the I50 for H-5 with flazasulfuron was 63.3 times higher compared to its correspondent susceptible population (H-6). Therefore, the H-5 population exhibited multiple-resistance to flazasulfuron and glyphosate, being the first case reported in Europe for these two MOA.

Conyza canadensis is a species invading large agricultural areas throughout the world, mainly to its ability to evolve herbicide resistance. Specifically, in Hungary, extensive areas have been infested by this species due to the difficulty in controlling it with glyphosate. To corroborate this fact as resistance and not as an incorrect herbicide application, eight suspicious glyphosate-resistant C. canadensis populations from different Hungarian regions were studied. In dose-response assays with glyphosate, the LD50 and GR50 values indicated that populations 1 to 5 were resistant to this herbicide (H-5 population the most resistant). Besides, the shikimic acid accumulation tests corroborated the results observed in the dose-response assays. 11 alternative herbicides from 6 different mode of action (MOA) were applied at field doses as control alternatives on populations H-5 and H-6 (both in the same regions). The H-5 population showed an unexpected resistance to flazasulfuron (ALS-inhibitor). The ALS enzyme activity studies indicated that the I50 for H-5 was 63.3 fold higher compared to its correspondent susceptible population (H-6). Therefore, the H-5 population exhibited multiple-resistance to flazasulfuron and glyphosate, being the first case reported in Europe for this two MOA. For that reason, the other herbicides with different MOA have to be tested here. Author Contributions: Conceptualization, R.P. and A.R.; Methodology, R.A., P.F. and A.R.; Software, R.A. and P.F.; Validation, R.P.; Formal Analysis, R.A and A.R.; Investigation, A.R., R.A., P.F., BT and C.P.; Resources, R.P.; Data Curation, P.F.; Writing-Original Draft Preparation, A.R.; Writing-Review & Editing, R.P. and A.R.; Visualization, R.A.; Supervision, R.P.; Project Administration, R.P.; Funding Acquisition, R.P." Funding: This research received no external funding Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: