Co-existence of ALS-resistant Amaranthus species in north-eastern Italy: how to manage them

This PhD thesis comprises 6 chapters: Chapter 1 and 6 provide, respectively, a general introduction on ALS resistance in Amaranths and some general conclusions, whereas research questions are addressed in Chapters 2 to 5. All chapters have been organized as standalone publications and they can be read independently, therefore, some repetitions above introduction and methods sections might occur. At the moment, only Chapter II is under review (Pest Management Science). In the last ten years, a number of cases of Amaranthus spp. suspected ALS-resistant have been recorded in Italy, and in some cases more than one species appeared to be present in a single field. Three species were found: Amaranthus retroflexus, Amaranthus hybridus and Amaranthus tuberculatus, in one case living sympatrically. All populations were found to be ALS-resistant, and the main resistance mechanism was target-site mediated. A simplified identification key for weedy amaranths was devised. Herbicide resistant A. tuberculatus was found for the first time outside its native range (North America). The ALS substitution tryptophan 574 methionine was observed in dicots for the first time. Population genetics tools (microsatellites) and haplotype analyses were used to infer the origin of some Italian A. tuberculatus populations and the evolution of resistance among them. Two ancestral lineages and mainly two resistant (carrying mutation 574) haplotypes were found. Very likely at least a resistant allele was introduced from outside Italy, which lately spread to some other populations. Another resistant allele could have been selected in Italy. The presence of the same haplotype among geographically separated population clearly indicates that resistance has a common origin. Birds and the use of non-certified (possibly infested) seeds might be the main causes of A. tuberculatus seeds dispersal. The presence of more than one Amaranthus species in the same field can complicate weed management because different species can have different phenology and herbicide susceptibility. If A. tuberculatus is present in multiple Amaranthus species infestations, it should be the main target of herbicide treatments. Interventions should be rapid, because this species grows fast and therefore the herbicide application window is shortened. In 2018, a population of A. palmeri was found infesting a soybean field in North Eastern Italy. Whole plant herbicide assays demonstrated that this population was also resistant to ALS, and molecular analysis revealed a point mutation at position 574. This is the first case of herbicide resistant A. palmeri in Europe. Some A. retroflexus populations had a point mutation at position 376 of ALS gene, which conferred resistance to thifensulfuron-methyl, but not to imazamox. Further experiments clearly indicated that mutation 376 endows resistance to imazethapyr, but not to imazamox. Similar results were obtained with a Sorghum halepense population. An A. tuberculatus population was resistant to thifensulfuron-methyl, but no endowing-resistance mutations were found in the whole ALS gene. Further experiments suggested that resistance was not due to either a known point mutation or enhanced metabolism, therefore the resistance mechanism remained unknown. Four Amaranthus species were found to infest soybean fields in Italy: A. retroflexus, A. hybridus, A. tuberculatus, and A. palmeri. All these species have evolved resistance to ALS inhibitors. Herbicides with different SoA are still effective. Glyphosate (in the absence of a crop) and metribuzin can be used to control these resistant populations, whereas the use of bentazon should be further evaluated. Weed control should be focused on dioecious species (A. palmeri and A. tuberculatus), because they are associated with high risk of multiple-resistance evolution and crop losses. If A. palmeri or A. tuberculatus are present in fields, integrated weed management (crop rotation, mechanical weeding, etc.) must be adopted to limit their impact. The use of non-certified seeds should be avoided.