The aim of this educational review is to provide practical information on the hardware, methodology, and the hands on application of chlorophyll (Chl) a fluorescence technology. We present the paper in a question and answer format like frequently asked questions. Although nearly all information on the application of Chl a fluorescence can be found in the literature, it is not always easily accessible. This paper is primarily aimed at scientists who have some experience with the application of Chl a fluorescence but are still in the process of discovering what it all means and how it can be used. Topics discussed are (among other things) the kind of information that can be obtained using different fluorescence techniques, the interpretation of Chl a fluorescence signals, specific applications of these techniques, and practical advice on different subjects, such as on the length of dark adaptation before measurement of the Chl a fluorescence transient. The paper also provides the physiological background for some of the applied procedures. It also serves as a source of reference for experienced scientists.

Using chlorophyll (Chl) a fluorescence many aspects of the photosynthetic apparatus can be studied, both in vitro and, noninvasively, in vivo. Complementary techniques can help to interpret changes in the Chl a fluorescence kinetics. Kalaji et al. (Photosynth Res 122:121–158, 2014a) addressed several questions about instruments, methods and applications based on Chl a fluorescence. Here, additional Chl a fluorescence-related topics are discussed again in a question and answer format. Examples are the effect of connectivity on photochemical quenching, the correction of F V/F M values for PSI fluorescence, the energy partitioning concept, the interpretation of the complementary area, probing the donor side of PSII, the assignment of bands of 77 K fluorescence emission spectra to fluorescence emitters, the relationship between prompt and delayed fluorescence, potential problems when sampling tree canopies, the use of fluorescence parameters in QTL studies, the use of Chl a fluorescence in biosensor applications and the application of neural network approaches for the analysis of fluorescence measurements. The answers draw on knowledge from different Chl a fluorescence analysis domains, yielding in several cases new insights.

The net selection effect of herbicides on herbicide-resistance traits in weeds is conditioned by the fitness benefits and costs associated with resistance alleles. Fitness costs play an important evolutionary role preventing the fixation of adaptive alleles and contributing to the maintenance of genetic polymorphisms within populations. Glyphosate is widely used in world agriculture, which has led to the evolution of widespread glyphosate resistance in many weed species. The fitness of glyphosate-resistant and -susceptible perennial ryegrass plants selected from within a single population were studied in two field experiments conducted during 2011 and 2012 under different soil water availability. Glyphosate-resistant plants showed a reduction in height of 12 and 16%, leaf blade area of 16 and 33%, shoot biomass of 45 and 55%, seed number of 33 and 53%, and total seed mass of 16 and 5% compared to glyphosate-susceptible plants in 2011 and 2012, respectively. The reduction in seed number per plant resulted in a 40% fitness cost associated with the glyphosate-resistance trait in perennial ryegrass. Fitness costs of glyphosate-resistant plants were expressed under both conditions of water availability. These results could be useful for designing management strategies and exploiting the reduced glyphosate-resistant perennial ryegrass fitness in the absence of glyphosate selection. Nomenclature: Glyphosate; perennial ryegrass, Lolium perenne L., LOLPE.

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Knowledge about the mechanisms of herbicide resistance provide valuable insights into evolving weed populations in response to selection pressure and should be used as a basis for designing management strategies for herbicide-resistant weeds. The selection pressure associated with reactive management against glyphosate-resistant Lolium spp. populations would have favored the herbicide resistance to ACCase- and ALS-inhibitors. This work was aimed to determine the sensitivity of 80 Argentinean Lolium spp. populations to ALS- and ACCase-inhibitor herbicides for use in wheat or barley and to study the mechanisms of resistance involved. Sensitivity to pinoxaden and iodosulfuron-mesosulfuron were positively correlated ( r = 0.84), even though both affect different target sites. Inhibitors of cytochrome P450 monooxygenases (P450s) increased the sensitivity to pinoxaden and iodosulfuron-mesosulfuron in 94% of herbicide-resistant populations and target-site ACCase resistance mutations were detected only in two cases. Polymorphic variants were obtained with a pair primer designed on P450 sequences, cluster analysis discriminated around 80% of susceptible and P450-metabolic resistant plants sampled from a single population or different populations. Five markers corresponding to herbicide sensitivity were identified to be significantly associated with phenotypic variance in plants. Resistance to ALS- and ACCase-inhibitor herbicides were closely related, challenging the rotation of herbicides of both sites of action as a practice against resistance. In that sense, the use of pinoxaden and iodosulfuron-mesosulfuron would have provoked a selection on P450 genes that conduced a convergence of P450-metabolism based resistant Lolium spp. populations, which was detected by markers in a contribution to elucidate the molecular basis of this type of resistance.

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In Argentina, glyphosate resistance was reported in a Lolium perenne population after 12 years of successful herbicide use. The aim of the current paper was to put in evidence for the mechanism of glyphosate resistance of this weed. Susceptible leaves treated with different doses of glyphosate and incubated in vitro showed an accumulation of shikimic acid of around three to five times the basal level, while no changes were detected in leaves of glyphosate-resistant plants. The resistance mechanism prevents shikimate accumulation in leaves, even under such tissue-isolation conditions. The activity of the glyphosate target enzyme (EPSPS: 5-enolpyruvylshikimate-3-phosphate synthase) was quantified at different herbicide concentrations. EPSPS from resistant plants showed no difference in glyphosate-sensitivity compared to EPSPS from susceptible plants, and, accordingly, no amino acid substitution causing mutations associated with resistance were found. While the glyphosate target enzymes were equally sensitive, the basal EPSPS activity in glyphosate resistant plants was approximately 3-fold higher than the EPSPS activity in susceptible plants. This increased EPSPS activity in glyphosate resistant plants was associated with a 15-fold higher expression of EPSPS compared with susceptible plants. Therefore, the over-expression of EPSPS appears to be the main mechanism responsible for resistance to glyphosate. This mechanism has a constitutive character and has important effects on plant fitness, as recently reported.