Investigations on herbicide resistance in Apera spica-venti populations (2012)

Weeds belong to the main biotic yield-limiting factors in agricultural fields worldwide. Since the introduction of herbicides in the global market more than six decades ago, agro-ecosystems have been characterized by a strong reliance on herbicides for weed management. However, the selection pressure exerted on weed populations by long-term application of herbicides with the same mode of action has imposed selection for resistance within several weed species.\nApera-spica-venti (L.) Beauv. represents one of the most abundant annual grass weeds in autumn-sown crops of Central and Eastern European arable lands. Since the first report on herbicide-resistant A. spica-venti populations in 1994, several cases have been documented. Therefore, it is assumed that this species is evolving resistance to herbicides.\nThe main objectives of the present work were:\n1. The development of reliable testing procedures under greenhouse conditions for the verification of herbicide resistance in A. spica-venti populations;\n2. The quantification of resistance in A. spica-venti at different herbicide doses using dose-response assays and digital image analysis; \n3. The elucidation of resistance mechanisms in A. spica-venti populations at the molecular genetic level through laboratory experiments;\n4. The evaluation of the influence of farm management factors on the occurrence and spread of herbicide resistance in A. spica-venti populations through the use of statistical modeling;\n5. The development of a geo-referenced database for documenting the spatial and temporal distribution of herbicide-resistant A. spica-venti populations in Europe;\n6. The verification of the results obtained from the greenhouse assays under realistic field conditions and the detection of the introgression of herbicide resistance traits into the sensitive population after two generations.\nThe main resistance testing procedure consisted of growing plants in the greenhouse from seeds collected in the suspect fields and spraying them with herbicides. Applications were carried out with an automated precision sprayer either at a single dose or at a range of doses. Herbicide efficacy was then assessed at 15 and 30 days after treatment by direct comparison with untreated controls. Results from the greenhouse assays showed that most of the screened popula¬tions (~70%) have evolved resistance to herbicides, particularly acetolactate synthase (ALS)-inhibitors. \nDose-response assays revealed resistance factors at the ED90 (ED90 resistant / ED90 sensitive) of up to 140 after treatment with flupyrsulfuron-methyl, thus indicating that A. spica-venti is a resistance-prone grass weed. A novel method for quantifying resistance based on the assessment of percent canopy cover using digital image analysis has been developed in the greenhouse to provide a potential alternative to the labour-intensive and time-consuming dry weight assessments.\nLaboratory experiments conducted on over 70 ALS-resistant populations revealed that target site mutations were responsible for the observed resistance. Cleaved Amplified Polymorphic Sequences (CAPS) marker analysis and sequencing of the amplified 3? and 5? ends of the ALS gene by Rapid Amplification of cDNA Ends (RACE) revealed the presence of previously known as well as novel mutations endowing resis¬tance to ALS-inhibitors. \nThe development of a risk assessment model allowed the evaluation of the influence of farm management factors on the probability of resistance occurrence in A. spica-venti populations. Results showed that a high percentage of winter crops in the rotation (&gt;75%), together with reduced or no-till practices, early sowing and high population density significantly increase the risk of resistance emergence. An internet-based geo-referenced database was developed to document the spatial and temporal distribution of herbicide-resistant A. spica-venti populations in Europe. \nFinally, field experiments were carried out over the course of two years (2008/2010) by sow-ing ALS-resistant and susceptible A. spica-venti populations in winter wheat plots. Resistant plants survived herbicide application, completed their life cycle and set vital seeds, which showed a significantly higher germination rate compared to the sensitive population. However, maximum yield losses of only ~10% could be ob¬served at A. spica-venti panicle densities of &gt;400 m-2. Greenhouse bioassays conducted at the end of the first year with plants from seeds collected in the sensitive plots showed an increase in tolerance to ALS-inhibitors of ~20%, thus suggesting introgression of herbicide resis¬tance traits into the sensitive population already after one year. \nThe outcomes of this work add knowledge to the understanding of the mechanisms underlying resistance to herbicides in A. spica-venti populations and provide weed scientists and consultants with useful tools for the reliable diagnosis and prevention of herbicide resistance in weed populations.