Resistance to graminicides in monocotyledons weeds Case studies of Lolium spp. and Phalaris paradoxa in Italy

Herbicide resistance is evolving fast in Italy, especially in cropping systems characterised by low diversity in space and time. In these systems there is often no rotation of herbicides mode of action (i.e. durum wheat and rice monoculture). Resistance is also evolving faster in cross-pollinating and genetically variable species (i.e. Lolium spp.). In Italy about 100,000 ha have been estimated to suffer resistant problems, mainly in durum wheat (Lolium spp., Avena sterilis and Papaver rhoeas) and paddy rice (mostly Schoenoplectus mucronatus and Cyperus difformis). ACCase is an essential enzyme in fatty acid biosynthesis in eukaryotes and prokaryotes. In plants, two forms of ACCase have been identified - the first is located in the chloroplast, the primary site of plant fatty acid biosynthesis, and the second in the cytosol. The homomeric ACCase in the cytosol of nearly all plant species and the heteromeric ACCase in the chloroplasts of dicots are insensitive to APP, CHD and DEN herbicides. In contrast, the plastidic homomeric ACCase in nearly all grass species is herbicide-sensitive, and this is the basis for selective control of grass-weeds by ACCase herbicides. The aims of the research were: (1) to monitor throughout the country the situation of resistance to Acetyl-CoA Carboxylase (ACCase) in Lolium and Phalaris species following complaints to the agrochemical industries; (2) to develop a reliable Petri dish quick test to detect ACCase-inhibitors resistance in Lolium and Phalaris species; (3) to characterise clethodim and pinoxaden resistance in Lolium spp. using different experimental approaches; (4) agronomic and molecular characterisation of a few populations of P. paradoxa resistant to ACCase inhibitors. Through classic pot screenings, populations with different patterns and levels of resistance have been selected to be studied with different approaches: outdoor dose-response, at molecular level using molecular markers and sequencing and at physiological level through enzyme bioassay. As pot experiments are costly and time consuming, a rapid test in Petri dishes based on survival data has been developed for detecting resistance to ACCase-inhibitors. Screening results revealed that among 41 tested Lolium populations, 23 were resistant to clodinafop, 19 to sethoxydim and 15 to pinoxaden. Among the 17 P. paradoxa populations, 3 were resistant to clodinafop, and one to sethoxydim and pinoxaden. Resistance to pinoxaden is associated to a wide pattern of cross-resistance among ACCase-inhibitors. The Petri dish seed quick test has been developed in order to find a discriminating herbicide concentration between susceptible and resistant populations; a herbicide concentration ("comparing dose") that gives results comparable to the recommended field dose used in greenhouse pot experiments; and a concentration discriminating between strong and weak resistance. The "comparing doses" has been validated using other populations not included in the set up experiments. The "comparing dose" for Lolium spp. have been: clodinafop 1 ?M, pinoxaden 0.2 ?M, clethodim 0.2 ?M and sethoxydim 0.1 ?M; for P. paradoxa, clodinafop 0.1 ?M, pinoxaden 0.05 ?M and clethodim 0.1 ?M. The Petri dish seed quick test revealed to be reliable with the two species in screening large numbers of samples compared with the costly and time consuming pot experiment. The quick test improves ACCase-resistance detection and can be adopted as a routine facility. Molecular investigation identified five ACCase mutations (Ile1781Leu, Trp2027Cys, Ile2041Asn, Asp2078Gly and Cys2088Arg) and revealed 12 genotypes in 14 clethodim resistant Lolium populations. Pinoxaden resistance has been reported associated to Lolium population mutants for five ACCase mutations (Ile1781Leu, Ile2041Val/Asn, Asp2078Gly, Cys2088Arg and Gly2096Ala) with specific homozygous and heterozygous status among mutations. In P. paradoxa two mutations have been associated to pinoxaden resistance: Ile1781Val and Asp2078Gly, always recognised at the homozygous status. The research has established that resistance to ACCase herbicides depends on the specific resistant allele(s), on the homo/heterozygous status of plants for the specific resistant allele(s), and on the combinations of different resistant alleles, as well as herbicide rates. To understand and devise resistance management strategies, knowledge of all these factors coupled with field records of herbicide and agronomic techniques used is essential. The overall situation of grass resistance in Italian durum wheat crops indicates that there is no single chemical that can solve all resistance problems. Therefore all stakeholders should be aware that IWM (or better ICM) is needed to properly manage resistance in the field. It must also be clear that IWM or ICM requires a higher technological level coupled with a deeper knowledge of all components of the cropping system.