Viewpoint: Avoiding common pitfalls of chlorophyll fluorescence analysis under field conditions.

Phytophthora foot rot, caused by Phytophthora capsici, remains a significant challenge in the cultivation of black pepper (Piper nigrum L.) worldwide. In this study, we analyzed the impact of P. capsici infection on photosynthetic efficiency and gas exchange in six elite black pepper genotypes under field conditions. By monitoring chlorophyll fluorescence and foliar gas exchange at 24, 48, and 72 h post-inoculation (hpi) of P. capsici, we observed significant disruptions in PSII function and key physiological processes. The fluorescence parameters, including F0, F0/Fm, Fv/Fm, ΦPSII, qP and qL, showed sensitive responses to infection, effectively distinguishing tolerant genotypes from susceptible ones. The genotypes IISR-Shakti—the only released genotype of black pepper for cultivation, designated as ‘moderately resistant’ against Phytophthora foot rot and Kuruvantherivalli—the high yielding traditional cultivar of the crop, demonstrated greater resilience by maintaining higher photochemical efficiency and stable gas exchange, while genotypes such as ‘PMM’- the only reported ‘lemon scented’ genotype of black pepper and Edamalayar-III—a wild genotype of the crop experienced notable decreases in Fv/Fm, ΦPSII, Pn, Ci, and E values. Correlation analysis revealed strong negative correlations between disease severity index (DSI) and these physiological traits. The results highlight the potential of chlorophyll fluorescence and gas exchange measurements as practical, non-invasive tools for early disease detection and screening. Notably, IISR-Shakti shows significant promise for breeding programs aimed at enhancing resistance to foot rot.

Plants exposed to combined abiotic and biotic stresses often exhibit complex physiological responses that cannot be predicted from single stress factors. In this study, we evaluated the interactive effects of temperature stress and Erwinia amylovora infection on pear (Pyrus pyrifolia) leaves under five temperature conditions (10, 15, 25, 30, and 35 °C) with or without pathogen inoculation, using chlorophyll fluorescence analysis and RGB imaging over a 7-day period. Photosynthetic performance remained optimal at 25 °C under single temperature conditions, whereas pathogen inoculation alone caused PSII damage and reduced energy dissipation. Under combined stress, PSII responses exhibited temperature-dependent patterns: at 10, 15 °C, photoprotective mechanisms were partially maintained; at 25, 30 °C, severe structural and functional damage occurred; and at 35 °C, pathogen activity was suppressed while partial recovery of PSII was observed. By integrating chlorophyll fluorescence analysis with a linear mixed-effect model (LMM), distinct patterns of sensitivity were identified among fluorescence parameters, with ΦNO responding to single stress factors, and Fm, Fv, Fp, Fv/Fo, and qL showing significant three-way interactions. These findings highlight temperature-dependent strategies of pear leaves to cope with fire blight and emphasize the utility of chlorophyll fluorescence analysis for evaluating photosynthetic resilience. From an applied perspective, chlorophyll fluorescence could serve as a rapid, non-destructive tool for screening pear cultivars with enhanced tolerance to bacterial fire blight, contributing to more efficient orchard management strategies.

Salinity is one of the modern agriculture major obstacles, causing several physiological disturbances in plants, adversely affecting its growth and development. Therefore, some techniques are required in order to alleviate the negative effects of salinity stress on plants, as for example an adequate nitrogen fertilization. The aim of this study was to assess the ecophysiological responses of sugar apple plants (Annona squamosa L.) submitted to different salinity levels and nitrogen fertilization doses. The experimental design was a randomized block in an incomplete factorial scheme, with five electrical conductivities of the irrigation water (ECw: 0.5, 1.01, 2.25, 3.49 and 4.0 dS m-1) and five nitrogen doses (0, 101, 350, 598.2 and 700 mg dm-3), with four replicates, generated from the Box Central Composite experimental design matrix. Through a daily course, it was observed variations in gas exchange, chlorophyll content and fluorescence parameters. The gas exchange, chlorophyll content and fluorescence parameters were measured on photosynthetically active leaves. There was significant interaction between the salinity levels (ECw) and nitrogen doses for the chlorophyll indexes. The electrical conductivity of the irrigation water caused significant negative effects on chlorophyll fluorescence. Therefore, it can be stated that the sugar apple plants ecophysiology varies through the day and that salinity affects its chlorophyll content and fluorescence.

Trees in urban areas provide important ecosystem services and are an essential element of urban green space. The constant increase in artificial light from anthropogenic activities around the world creates photopollution that affects the phenology and physiology of plants. Here we conducted a field study to investigate the anthropogenic impacts on six urban trees (Saraca asoca, Terminalia catappa, Bauhinia variegata, Holoptelea integrifolia, Ficus benjamina and Thevetia peruviana) using chlorophyll fluorescence analysis. OJIP curve, maximum quantum yield of primary photochemistry (ΦPo), quantum yield of electron transport (ΦEo), probability that an absorbed photon will be dissipated (ΦDo), photosynthetic performance index (PIcsm) and reaction center photochemistry were assessed. According to the results, various parameters of chlorophyll fluorescence showed significant and important effects on different tree species. T. peruviana and F. benjamina were found to be tolerant to street lighting, while on the other hand, S. asoca, T. catappa, B. variegata and H. integrifolia were found to be sensitive to artificial light induced by street lamps. This study clearly indicates that chlorophyll fluorescence analysis is a potent method for screening the tolerance of tree species to photopollution induced by artificial lights.

Influence of ambient light and temperature on laser-induced chlorophyll fluorescence measurements

Chlorophyll fluorescence analysis as a measure of photosynthesis is increasingly used in eco-physiological studies. It is particularly useful in investigations on the photosynthetic performance of plants in stressful environments. Chlorophyll fluorescence, which can be measured rapidly and conveniently, provides insights into a plant's ability to tolerate environmental stresses such as those of a drought. Damage to the photosynthetic apparatus due to moisture or heat stress, which would reduce photosynthetic assimilation and eventually the yield, can be detected at a very early stage of the drought. This technique is widely used to identify stress tolerant plants and crop varieties. However, this technique has not been utilized to identify putative drought tolerant coconut varieties. Hence, the objective of this study was to set out the basic principles of the technique, discuss its applications in eco-physiological studies, and evaluate its potential to screen coconut varieties for drought tolerance. San Ramon, a tall form of coconut introduced to Sri Lanka few decades ago and a few other local tall accessions in the germplasm conservation blocks in the Coconut Research Institute of Sri Lanka appeared to possess a photosynthetic apparatus which was clearly more tolerant to moisture stress conditions than the other accessions. SRR, SRG, SA, CL PI & PW were the most drought tolerant of the 40 genotypes tested. The results generated by using Fv/Fm, were consistent with results of other important parameters such as the rate of photosynthesis and water use efficiency. Even the basic parameters of chlorophyll fluorescence, for instance the maximum quantum yield of photosystem II (Fv/Fm) measured on dark-adapted coconut leaves showed promise as a selection criterion in future screening programmes due to its quick, non-invasive and reliable nature. Similarities of fluorescence parameters observed between seedlings and adult palms enables fairly confident predictions on the performance of adult palms, based on studies with seedlings. In general, chlorophyll fluorescence appears as a promising tool for screening coconut palms for their drought tolerance. Key words: Coconut, drought tolerance, chlorophyll fluorescence, photosynthesis, Fv/Fm doi: 10.4038/cocos.v18i0.991 COCOS (2007), 18

Field experiments were conducted in Sicily (south Italy) to assess chlorophyll (Chl) fluorescence parameters in response of potato crop to nitrogen dose, to variation in genotype and in plant age, and to detect relationships between Chl content, fluorescence parameter Fv/Fm, and tuber yield. The experiment included five nitrogen doses (0, 10, 20, 30, and 40 g m-2) and four genotypes (Spunta, Sieglinde, Daytona, and Igea). Chl fluorescence parameters (initial fluorescence, F0, maximum fluorescence, Fm, variable fluorescence, Fv, Fv/Fm, Tmax (the time required to reach Fm), and Chl content were measured weekly between the appearance of the fifth and sixth leaves and the onset of plant senescence. A positive linear relationship was established between nitrogen supply and Chl content, F0, and Tmax. Nitrogen supply up to 10 g m-2 also had a positive effect on Fm and Fv, but above this rate it reduced Fv/Fm. Spunta had the highest Chl content, Fm, Fv, and Fv/Fm, but the lowest F0, whereas Sieglinde had the lowest Chl content, Fv, Fv/Fm, and Tmax and the highest F0. The cvs. Igea and Daytona exhibited intermediate Chl fluorescence parameters. Chl content and Tmax decreased with increasing plant age, whereas F0, Fm, and Fv increased until complete canopy development and thereafter declined until crop maturity. Tuber and plant dry matter yield were significantly correlated with Chl content, F0, and Tmax. Thus Chl fluorescence and content detect differences in the response of potato to N supply, can discriminate between genotypes, predict plant age, and yield performance under field conditions.

A comprehensive understanding of the quantitative relationship among phenotypic and photosynthetic traits at key growth stages in wheat can guide trait selection and management strategies that can contribute to increased productivity. In this study, we quantified the physiological basis of morphological and phenological attributes of wheat using chlorophyll fluorescence analysis. Chlorophyll fluorescence parameters were measured using a handheld MultispeQ V2.0 device, connected to a PhotosynQ platform, for 72 genotypes grown in a randomized complete block design (RCBD). Days to heading (DH), percentage digital ground cover (DGC%), and flag leaf length and width were recorded at specific individual growth stages, and chlorophyll fluorescence parameters were measured at 95, 103, and 108days after sowing to the grain filling stage. Variance analysis revealed a significant difference among the three measurement time points in the fluorescence parameters. A notable increase in relative chlorophyll content was observed in the flag leaf, which plays a central role in photosynthesis; higher chlorophyll levels enhance light capture capacity and thereby support greater photosynthetic activity. The DGC% played a vital role in radiation absorption and photosynthesis. Significant positive correlations were observed among most fluorescence and physiological traits, although some traits showed negative correlations. Path analysis revealed that leaf temperature and ambient temperature indirectly affected morphophysiological traits via DH. Such relationships may assist breeders in selecting elite genotypes.

Chlorophyll fluorescence parameters can provide useful indications of photosynthetic performance in vivo. Coupling appropriate fluorescence measurements with other noninvasive techniques, such as absorption spectroscopy or gas exchange, can provide insights into the limitations to photosynthesis under given conditions. Chlorophyll content is one of the dominant factors influencing the conditions of a vegetation growing season, and can be tested using both fluorescence and remote sensing methods. Hyperspectral remote sensing and recording the narrow range of the spectrum can be used to accurately analyze the parameters and properties of plants. The aim of this study was to analyze the influence of lead ions (Pb, 5 mM Pb(NO3)2) on the growth of pea plants using spectral properties. Hyperspectral remote sensing and chlorophyll fluorescence measurements were used to assess the physiological state of plants seedlings treated by lead ions during the experiment. The plants were growing in hydroponic cultures supplemented with Pb ions under various conditions (control, complete Knop + phosphorus (+P); complete Knop + phosphorus (+P) + Pb; Knop (-P) + Pb, distilled water + Pb) affecting lead uptake via the root system. Spectrometric measurements allowed us to calculate the remote sensing indices of vegetation, which were compared with chlorophyll and carotenoids content and fluorescence parameters. The lead contents in the leaves, roots, and stems were also analyzed. Spectral characteristics and vegetation properties were analyzed using statistical tests. We conclude that: (1) pea seedlings grown in complete Knop (with P) and in the presence of Pb ions were spectrally similar to the control plants because lead was not transported to the shoots of plants; (2) lead most influenced plants that were grown in water, according to the highest lead content in the leaves; and (3) the effects of lead on plant growth were confirmed by remote sensing indices, whereas fluorescence parameters identified physiological changes induced by Pb ions in the plants.

Gas exchange, chlorophyll (Chl) fluorescence, and contents of photosynthetic pigments, soluble proteins (ribulose-1,5-bisphosphate carboxylase/oxygenase, RuBPCO), and antioxidant enzymes were characterized in the fully expanded 6th leaves in rice seedlings grown on either complete (CK) or on nitrogen-deficient nutrient (N-deficiency) solutions during a 20-chase period. Compared with the control plants, the lower photosynthetic capacity at saturation irradiance (Pmax) was accompanied by an increase in intercellular CO2 concentration (Ci), indicating that in N-deficient plants the decline in Pmax was not due to stomatal limitation but due to the reduced carboxylation efficiency. The fluorescence parameters ΦPS2, Fv'/Fm', electron transport rate (ETR), and qP showed the same tendency as Pmax in N-deficient plants. Correspondingly, a higher qN paralleled the rise of the ratio of carotenoid (Car) to Chl contents. However, Fv/Fm was still diminished, suggesting that photoinhibition did occur in the photosystem 2 (PS2) reaction centres. In addition, the activities of antioxidant enzymes on a fresh mass basis were gradually lowered, leading to the aggravation of membrane lipid peroxidation with the proceeding N-deficiency. The accumulation of malonyldialdehyde resulted in the lessening of Chl and soluble protein content. Analyses of regression showed PS2 excitation pressure (1 - qP) was linearly correlated with the content of Chl and inversely with soluble protein (particularly RuBPCO) content. There was a lag phase in the increase of PS2 excitation pressure compared to the decrease of RuBPCO content. Therefore, the increased excitation pressure under N-deficiency is probably the result of saturation of the electron transport chain due to the limitation of the use of reductants by the Calvin cycle. Rice plants responded to N-deficiency and high irradiance by decreasing light-harvesting capacity and by increasing thermal dissipation of absorbed energy.

Chlorophyll fluorescence measurements are providing insights into Photosystem II (PSII) quantum efficiency and hence are able to provide a good estimation of carbon assimilation under field conditions. A F2 generation of sibcross seedlings from a cross of `Goldspur' × `Redspur' were selected to identify genetic variations and the relationships among fluorescence parameters, carbon assimilation, and carbon partitioning in apple leaves. Mature leaves from extension shoots were analyzed for chlorophyll fluorescence with a CF-1000 chlorophyll fluorescence measurement system, photosynthetic rate with a LI-6200 portable photosynthesis system, and carbohydrates with a Shimadzu HPLC. Significant variations in leaf chlorophyll fluorescence parameters and photosynthetic rates were found. The ratio of Fv: Fm, an estimation of photochemical efficiency of PSII, decreased from ≈0.90 in June to ≈0.75 in September while the photosynthetic rates decreased from ≈8.5 in June to ≈4.5 μmol·m–2·s–l in September. The relationships between fluorescence parameters, photosynthesis, and carbohydrate partitioning were analyzed and the ratio of sorbitol to sucrose in relation to the efficiency of PSII and NADPH production will be discussed.

High light intensities raise photosynthetic and plant growth rates but can cause damage to the photosynthetic machinery. The likelihood and severity of deleterious effects are minimised by a set of photoprotective mechanisms, one key process being the controlled dissipation of energy from chlorophyll within PSII known as non-photochemical quenching (NPQ). Although ubiquitous, the role of NPQ in plant productivity is important because it momentarily reduces the quantum efficiency of photosynthesis. Rice plants overexpressing and deficient in the gene encoding a central regulator of NPQ, the protein PsbS, were used to assess the effect of protective effectiveness of NPQ (pNPQ) at the canopy scale. Using a combination of three-dimensional reconstruction, modelling, chlorophyll fluorescence, and gas exchange, the influence of altered NPQ capacity on the distribution of pNPQ was explored. A higher phototolerance in the lower layers of a canopy was found, regardless of genotype, suggesting a mechanism for increased protection for leaves that experience relatively low light intensities interspersed with brief periods of high light. Relative to wild-type plants, psbS overexpressors have a reduced risk of photoinactivation and early growth advantage, demonstrating that manipulating photoprotective mechanisms can impact both subcellular mechanisms and whole-canopy function.

The evaluation of disease resistance is considered an important aspect of phenotyping for crop improvement. Identification of advanced lines of the common bean with disease resistance contributes to improved grain yields. This study aimed to determine the response of the photosynthetic apparatus to natural pathogen infection by using chlorophyll (Chla) fluorescence parameters and their relationship to the agronomic performance of 59 common bean lines and comparing the photosynthetic responses of naturally infected vs. healthy leaves. The study was conducted over two seasons under acid soil and high temperature conditions in the western Amazon region of Colombia. A disease susceptibility index (DSI) was developed and validated using chlorophyll a (Chla) fluorescence as a tool to identify Mesoamerican and Andean lines of common bean (Phaseolus vulgaris L.) that are resistant to pathogens. A negative effect on the functional status of the photosynthetic apparatus was found with the presence of pathogen infection, a situation that allowed the identification of four typologies based on the DSI values ((i) moderately resistant; (ii) moderately susceptible; (iii) susceptible; and (iv) highly susceptible). Moderately resistant lines, five of them from the Mesoamerican gene pool (ALB 350, SMC 200, BFS 10, SER 16, SMN 27) and one from the Andean gene pool (DAB 295), allocated a higher proportion of energy to photochemical processes, which increased the rate of electron transfer resulting in a lower sensitivity to disease stress. This photosynthetic response was associated with lower values of DSI, which translated into an increase in the accumulation of dry matter accumulation in different plant organs (leaves, stem, pods and roots). Thus, DSI values based on chlorophyll fluorescence response to pathogen infection could serve as a phenotyping tool for evaluating advanced common bean lines. Six common bean lines (ALB 350, BFS 10, DAB 295, SER 16, SMC 200 and SMN 27) were identified as less sensitive to disease stress under field conditions in the western Amazon region of Colombia, and these could serve as useful parents for improving the common bean for multiple stress resistance.

Seedlings of wheat (Triticum aestivum L.) cultivars Jing 411, Jinmai 30 and Yangmai 10 were exposed to 0, 10, 20, 30, 40 or 50 μM of CdCl2 in a solution culture experiment. The effects of cadmium (Cd) stress on wheat growth, leaf photon energy conversion, gas exchange, and Cd accumulation in wheat seedlings were investigated. Gas exchange was monitored at 3, 9, 24 days after treatment (DAT). Growth parameters, chlorophyll content, leaf chlorophyll fluorescence, and Cd concentration in shoot and root were measured at 24 DAT. Seedling growth, gas exchange, chlorophyll content, chlorophyll fluorescence parameters were generally depressed by Cd stress, especially under the high Cd concentrations. Cd concentration and accumulation in both shoots and roots increased with increasing external Cd concentrations. Relationships between corrected parameters of growth, photosynthesis and fluorescence and corrected Cd concentrations in shoots and roots could be explained by the regression model Y = K/(1 + exp(a + bX)). Jing 411 was found to be Cd tolerant considering parameters of chlorophyll content, photosynthesis and chlorophyll fluorescence in which less Cd translocation was from roots into shoots. The high Cd concentrations were in shoots and roots in Yangmai 10 which has been found to be a relative Cd tolerant cultivar in terms of most growth parameters.

The effects of a) glucose, b) mannose and 2-deoxyglucose (glucose analogues capable of triggering hexokinase-mediated photosynthesis repression) and c) sodium citrate on chlorophyll fluorescence parameters of the blue-green alga Spirulina platensis (Nordst.) Geitl. were investigated. The addition of glucose evoked, at first, the lowering of all photochemical fluorescence quenching parameters (FV/FM, FV'/FM', qP and ΦPS II). The decrease correlated with glucose concentration. The inhibition of photochemical activity relaxed after the third day of the experiment, perhaps, as the consequence of glucose utilization. If the concentration of glucose in the cultural medium was high enough (50 mM), on the seventh day all the indices of photochemical quenching began to diminish again, at which time qP reduction was more significant than that for FV'/FM'. The relief of glucose repressive effects was light-dependent. Glucose analogues caused the decrease in only one photochemical quenching parameter, qP. Unlike mannose, the effect of 2-deoxyglucose was more pronounced and did not weaken till the end of experiments. Nonphotochemical quenching fluorescence parameters (qN and NPQ) were also reduced under monosaccharide treatment. The influence of sodium citrate on chlorophyll fluorescence parameters was negligible. Therefore, the multiplicity of glucose effects in the course of inhibition of S. platensis photosystem II activity was demonstrated, while hexokinase-dependent repression mechanism was playing only a partial role and resulting in the blocking of electron flow from photosystem II reaction centers down the electron transport chain.