Fluorescence Curves Research Articles

Reliable molecular detection of small hive beetles

Invasive species require adequate reliable detection methods to mitigate their further spread and impact. However, the reliability of molecular detection methods is often hampered by both false positives (Error type I) and false negatives (Error type II). At present, the reliability of the four published molecular detection methods for small hive beetles (SHB), Aethina tumida, has not been rigorously evaluated considering their extensive genetic diversity. Here, we performed intra- and interlaboratory comparisons of the four available methods using SHB samples representing 78 regions from 27 countries on five continents, beetles from the same genus (Aethina concolor, A. inconspicua, A. flavicollis and A. major), as well as western honey bees, Apis mellifera, and ectoparasitic mites Varroa destructor. The data show that the Idrissou et al. (2018) and Li et al. (2018) methods avoid both false positives and false negatives probably due to lower sensitivity to nucleotide mismatches on the primer and probe’s target sequences. Further, the Li et al. (2018) method can be considered more sensitive because the fluorescent amplification curve crosses the threshold at lower Cq values compared to the Idrissou et al. (2018) one. In light of our data, the Li et al. (2018) method is the most reliable molecular diagnostic tool for SHB. We therefore recommend using this method as it will contribute to management efforts of this invasive species.

Simultaneous Genotyping of Three SNVs, rs5471, rs5472, and rs2000999 Involved in Serum Haptoglobin Levels by Fluorescent Probe-Based Melting Curve Analysis.

Haptoglobin (Hp) is a hemoglobin-binding acute-phase serum protein. Several single nucleotide variations (SNVs) within the Hp gene (HP) or Hp-related protein gene (HPR), such as rs5471 (A>C) and rs5472 (A>G) in HP promoter region and rs2000999 (G>A) in intron 2 of HRP, are suggested to correlate with the serum Hp levels. To determine these three SNVs simultaneously, a genotyping assay based on duplex dual-labeled fluorescent probes was developed. The method was then validated by analyzing genomic DNA from 121 Ghanaian and two Japanese subjects who had been previously genotyped for rs5471, rs5472, and rs2000999. Both rs5471 and rs5472 could be determined as haplotypes with a single FAM-labeled fluorescent probe, and rs2000999 could be genotyped with a HEX-labeled fluorescent probe. The results obtained with the present method were consistent with the previous results except for those of three Ghanaian subjects. All three subjects appear to have multiple HPR copy number variants characteristic of African populations, which may have led to incorrect results during previous genotyping. This method allows us to genotype these three SNVs in a relatively large number of samples, especially in African populations where rs5471 is uniquely distributed.

Establishment of LAMP-CRISPR/Cas12a for rapid detection of Escherichia coli O157:H7 and one-pot detection

Escherichia coli O157:H7 is a pathogenic serotype of Escherichia coli. Consumption of food contaminated with E. coli O157:H7 could cause a range of diseases. Therefore, it is of great importance to establish rapid and accurate detection methods for E. coli O157:H7 in food. In this study, based on LAMP and combined with the CRISPR/cas12a system, a sensitive and specific rapid detection method for E. coli O157:H7 was established, and One-Pot detection method was also constructed. The sensitivity of this method could stably reach 9.2 × 10° CFU/mL in pure culture, and the whole reaction can be completed within 1 h. In milk, E. coli O157:H7 with an initial contamination of 7.4 × 10° CFU/mL only needed to be cultured for 3 h to be detected. The test results can be judged by the fluorescence curve or by visual observation under a UV lamp, eliminating instrument limitations and One-Pot detection can effectively prevent the problem of false positives. In a word, the LAMP-CRISPR/cas12a system is a highly sensitive and convenient method for detecting E. coli O157:H7.

PH-dependent binding of ATP aptamer to the target and competition strands: Fluorescent melting curve fitting study

The pH varies in different tissues and organelles and also changes during some diseases. In this regard, the application of molecular switches that use a competition-based aptamer switch design in biological systems requires studying the thermodynamics of such systems at different pH values. In this work, we studied the binding of the classical ATP aptamer to ATP and competition strands under different pH and ionic conditions using fluorescent melting curve analysis. We have developed an original approach to processing source data from a PCR thermal cycler. It is based on constructing a thermodynamic model of the melting profile and the subsequent fit of experimental curves within this model. We have shown that this approach enables us to narrow the temperature region under study to the width of the melting region without a significant loss in the quality of the result. This impressively expands the application area of this approach compared to frequently used techniques that require mandatory measurement of the signal outside the melting region. The results obtained by the method showed that the thermodynamic parameters of the ATP aptamer and its duplexes with competition strands change depending on pH. Therefore, molecular switches that use a competition strand to the ATP aptamer may have a pH-dependent sensitivity that has not been previously considered. This should be taken into account for future rational design of similar systems.

Establishment of a rapid detection method for Mycoplasma pneumoniae based on RPA-CRISPR-Cas12a technology

Mycoplasma pneumoniae can cause respiratory infections and pneumonia, posing a serious threat to the health of children and adolescents. Early diagnosis of Mycoplasma pneumoniae infection is crucial for clinical treatment. Currently, diagnostic methods for Mycoplasma pneumoniae infection include pathogen detection, molecular biology techniques, and bacterial culture, all of which have certain limitations. Here, we developed a rapid, simple, and accurate detection method for Mycoplasma pneumoniae that does not rely on large equipment or complex operations. This technology combines the CRISPR-Cas12a system with recombinase polymerase amplification (RPA), allowing the detection results to be observed through fluorescence curves and immunochromatographic lateral flow strips.It has been validated that RPA-CRISPR/Cas12a fluorescence analysis and RPA-CRISPR/Cas12-immunochromatographic exhibit no cross-reactivity with other common pathogens, and The established detection limit was ascertained to be as low as 102 copies/µL.Additionally, 49 clinical samples were tested and compared with fluorescence quantitative polymerase chain reaction, demonstrating a sensitivity and specificity of 100%. This platform exhibits promising clinical performance and holds significant potential for clinical application, particularly in settings with limited resources, such as clinical care points or resource-constrained areas.

Evaluation of the FPMC respiratory panel for detection of respiratory tract pathogens in nasopharyngeal swab and sputum specimens

ObjectivesThe performance of the new Respiratory Pathogen panel (fluorescent probe melting curve, FPMC) for the qualitative detection of 12 organisms (chlamydia pneumoniae, mycoplasma pneumoniae, adenovirus, influenza A virus, influenza B virus, parainfluenza virus, rhinovirus, etc.) was assessed.MethodsProspectively collected nasopharyngeal swab (NPS) and sputum specimens (n = 635) were detected by using the FPMC panel, with the Sanger sequencing method as the comparative method.ResultsThe overall percent concordance between the FPMC analysis method and the Sanger sequencing method was 100% and 99.66% for NPS and sputum specimens, respectively. The FPMC testified an overall positive percent concordance of 100% for both NPS and sputum specimens. The FPMC analysis method also testified an overall negative percent concordance of 100% and 99.38% for NPS and sputum specimens, respectively.ConclusionsThe FPMC analysis method is a stable and accurate assay for rapid, comprehensive detecting for respiratory pathogens.

Effects of nitrogen addition and Bothriochloa ischaemum and Lespedeza davurica mixture on plant chlorophyll fluorescence and community production in semi-arid grassland.

Grass-legume mixture can effectively improve productivity and stimulate overyielding in artificial grasslands, but may be N-limited in semi-arid regions. This study investigated the effects of N addition on chlorophyll fluorescence and production in the grass-legume mixtures community. An N addition experiment was conducted in the Bothriochloa ischaemum and Lespedeza davurica mixture community, with seven mixture ratios (B0L10, B2L8, B4L6, B5L5, B6L4, B8L2, and B10L0) according to the sowing abundance of B.ischaemum and L.davurica and four N addition levels, N0, N25, N50, and N75 (0,25,50,75kgNhm-2 a-1), respectively. We analyzed the response of chlorophyll fluorescence parameters of the two species, the rapid light-response curves of chlorophyll fluorescence, as well as aboveground biomass (AGB) and overyielding. Our results showed that the two species showed different photosynthetic strategies, with L.davurica having significantly higher initial fluorescence (Fo), effective photochemical quantum yield of PSII (ΦPSII), and coefficient of photochemical fluorescence quenching (qP) than B. ischaemum, consisting with results of rapid light-response curves. N addition and mixture ratio both had significant effects on chlorophyll fluorescence and AGB (p<0.001). The ΦPSII and qP of L.davurica were significantly lowest in B5L5 and B6L4 under N addition, and the effect of N varied with mixture ratio. The photosynthetic efficiency of B. ischaemum was higher in mixture than in monoculture (B10L0), and ΦPSII was significantly higher in N50 than in N25 and N50 at mixture communities except at B5L5. The community AGB was significantly higher in mixture communities than in two monocultures and highest at B6L4. In the same mixture ratio, the AGB was highest under the N50. The overyielding effects were significantly highest under the N75 and B6L4 treatments, mainly attributed to L.davurica. The partial least squares path models demonstrated that adding N increased soil nutrient content, and complementary utilization by B.ischaemum and L.davurica increased the photosynthetic efficiency. However, as the different photosynthetic strategies of these two species, the effect on AGB was offset, and the mixture ratio's effects were larger than N. Our results proposed the B6L4 and N50 treatments were the optimal combination, with the highest AGB and overyielding, moderate grass-legume ratio, optimal community structure, and forage values.

Nitric oxide signal is required for glutathione-induced enhancement of photosynthesis in salt-stressed Solanum lycopersicum L.

Reduced glutathione (γ-glutamyl-cysteinyl-glycine, GSH), the primary non-protein sulfhydryl group in organisms, plays a pivotal role in the plant salt stress response. This study aimed to explore the impact of GSH on the photosynthetic apparatus, and carbon assimilation in tomato plants under salt stress, and then investigate the role of nitric oxide (NO) in this process. The investigation involved foliar application of 5 mM GSH, 0.1% (w/v) hemoglobin (Hb, a nitric oxide scavenger), and GSH+Hb on the endogenous NO levels, rapid chlorophyll fluorescence, enzyme activities, and gene expression related to the Calvin cycle in tomato seedlings (Solanum lycopersicum L. cv. 'Zhongshu No. 4') subjected short-term salt stress (100 mM NaCl) for 24, 48 and 72 hours. GSH treatment notably boosted nitrate reductase (NR) and NO synthase (NOS) activities, elevating endogenous NO signaling in salt-stressed tomato seedling leaves. It also mitigated chlorophyll fluorescence (OJIP) curve distortion and damage to the oxygen-evolving complex (OEC) induced by salt stress. Furthermore, GSH improved photosystem II (PSII) electron transfer efficiency, reduced QA - accumulation, and countered salt stress effects on photosystem I (PSI) redox properties, enhancing the light energy absorption index (PIabs). Additionally, GSH enhanced key enzyme activities in the Calvin cycle and upregulated their genes. Exogenous GSH optimized PSII energy utilization via endogenous NO, safeguarded the photosynthetic reaction center, improved photochemical and energy efficiency, and boosted carbon assimilation, ultimately enhancing net photosynthetic efficiency (Pn) in salt-stressed tomato seedling leaves. Conversely, Hb hindered Pn reduction and NO signaling under salt stress and weakened the positive effects of GSH on NO levels, photosynthetic apparatus, and carbon assimilation in tomato plants. Thus, the positive regulation of photosynthesis in tomato seedlings under salt stress by GSH requires the involvement of NO.

SPECTRAL PROPERTIES OF CHLOROPHYLLS IN EDIBLE PLANTS INTENDED FOR DIAGNOSTICS

Abstract. This study examined the absorption and fluorescence spectra of chlorophylls and other pigments extracted in ethanol solutions from common edible plant species. Chlorophyll-a was the dominant fluorescent chromophore in all plant varieties. The fluorescence curves for all extracted solutions exhibit after optimizing by concentration dilution an increase in the fluorescence with a typical prominent leading Gaussian peak at a wavelength of 674 nm. This peak is followed by a decreasing shoulder extending into the near-infrared range beyond 724 nm. The dilution of the solutions with ethanol reduces the optical absorption from the other pigments and shows simultaneously an improved resolution of the chlorophyll-a spectral absorption peaks. This investigation provides new insights into the optical absorbance and other photo-excited processes that impact the fluorescence of chlorophylls, such as agglomeration, photobleaching and photo-aggregation. The findings may be used for biodiagnostics, fluorescent quantum dots and bio-sensors.

A Quantitative Method to Distinguish Cytosolic from Endosome-Trapped Cell-Penetrating Peptides.

Cell-penetrating peptides are known to penetrate cells through endocytosis and translocation. The two pathways are hardly distinguished in current cell assays. We developed a reliable, simple and robust method to distinguish and quantify independently the two routes. The assay requires (DABCYL) 4-(dimethylaminoazo)benzene-4-carboxylic acid- and (CF) carboxyfluorescein-labeled peptides. When the labeled peptide is intact, the fluorescence signal is weak thanks to the dark quenching property of DABCYL. A 10-fold higher fluorescence signal is measured when the labeled peptide is degraded. By referring to a standard fluorescent curve according to the concentration of the hydrolyzed peptide, we have access to the internalized peptide quantity. Therefore, cell lysis after internalization permits to determine the total quantity of intracellular peptide. The molecular state of the internalized peptide (intact or degraded), depends on its location in cells (cytosol vs endo-lysosomes), and can be blocked by boiling cells. This boiling step results indeed in denaturation and inhibition of the cellular enzymes. The advantage of this method is the possibility to quantify translocation at 37 °C and to compare it to the 4 °C condition, where all endocytosis processes are inhibited. We found that ranking of the translocation efficacy is DABCYL-R6-(ϵCF)K≫DABCYL-R4-(ϵCF)K≥CF-R9.

Universal organophosphate pesticides detection by peptide based fluorescent probes

In practical applications, the rapid and efficient detection of universal organophosphorus pesticides (OPs) can assist inspectors in quickly identifying the presence of OPs in samples. However, this presents a challenge for most well-established methods, typically designed to detect only a specific type of organophosphorus molecule at a time. In this proof-of-concept study, we draw inspiration from the structural similarities among OPs to develop innovative peptide-based fluorescence probes for the first time, which could efficiently detect a broad range of OPs within a mere 3 min. Analysis of fluorescence curve fitting reveals a clear linear correlation between the fluorescent intensity of the peptide probes and the concentration of OPs. Additionally, the selectivity analysis indicates that these peptide fluorescent probes exhibit an excellent response to various OPs while maintaining sufficient selectivity for detecting other pesticide types. Accurate sample analysis has also highlighted the potential of these peptide probes as practical tools for the rapid detection of OPs in actual vegetable samples. In summary, this proof-of-concept study presents an innovative approach to designing and developing ultrafast, universally peptide-based OP probes. These custom-designed peptide probes may facilitate rapid sample screening and offer initial quantification for OPs, potentially saving valuable time and effort in practical OP detection.

Mycorrhizal fungi reduce the photosystem damage caused by drought stress on Paris polyphylla var. yunnanensis.

Drought stress (DS) is one of the important abiotic stresses facing cash crops today. Drought can reduce plant growth and development, inhibit photosynthesis, and thus reduce plant yield. In this experiment, we investigated the protective mechanism of AMF on plant photosynthetic system by inoculating Paris polyphylla var. yunnanensis(P.py) with a clumping mycorrhizal fungus (AMF) under drought conditions. The drought environment was maintained by weighing AMF plants and non-AMF plants. The relative water content (RWC) of plant leaves was measured to determine its drought effect. DS decreased the RWC of plants, but AMF was able to increase the RWC of plants. chlorophyll a fluorescence curve measurements revealed that DS increased the OKJIP curve of plants, but AMF was able to reduce this trend, indicating that AMF increased the light absorption capacity of plants. DS also caused a decrease in plant Y(I) and Y(II). ETRI and ETRII, and increased Y(NO) and Y(NA) in plants, indicating that DS caused photosystem damage in plants. For the same host, different AMFs did not help to the same extent, but all AMFs were able to help plants reduce this damage and contribute to the increase of plant photosynthesis under normal water conditions.

Characteristics of the photosynthetic activity of corn using different drugs under stress

Goal. To study under controlled conditions the effect of hormonal preparations based on humus extracts and anti-stress stimulants with a high content of amino acids and biologically active substances on the development of corn under conditions of stress, as well as their effect on the photosynthetic activity of plants.&#x0D; Methods. The research was carried out in a climatic chamber (a room with adjustable control of temperature, daylight and air humidity) of the Ukrainian research plant quarantine station of the Institute of Plant Protection of the National Academy of Sciences. Photosynthetic activity was measured using a portable fluorometer «Floratest». During the growing season, two foliar feedings were carried out in the phase of development of corn VVSN 12, the first application of drugs during the growing season — VVSN 13, — the second application of drugs during the vegetation period — VVSN 14.&#x0D; Results. The analysis of the induction curves of chlorophyll fluorescence and the indicators of the maximum background fluorescence showed a difference between the plants that were under soil moisture of 70% and 50% RH. When corn plants were in a state close to stress for a long time, the difference between the curves of chlorophyll fluorescence intensity between the two controls increased. The use of the studied drugs provided the finding of the indicator of the maximum value of fluorescence induction above the dry control in all variants of the experiment. Best results provided: preparation B (high concentrated suspension for nutrition and plant stress decreasing based upon the humus extract. They contented N — 150 g/l, K2O — 300 g/l, Mg — 30 g/l, metal cations in В, Cu, Fe, Mn and Zn with additional adhesive effects and surfactant), at rate of application 0.5 and 0.25 l/ha; Axofol (high concentrated suspension from extract of brown algae Ascophyllum nodosum with microelements, B — 38.1 g/l, Mn — 10.2 g/l, Zn — 6.4 g/l) at rate of application 1.0 and 0.5 l/ha.&#x0D; Conclusions. As the time of corn plants being in a state close to stress increases, the difference between the curve of chlorophyll fluorescence intensity in the wet control and the dry control increases. The use of the studied drugs provided the finding of the indicator of the maximum value of fluorescence induction above the dry control in all variants of the experiment. The use of the researched preparations in conditions of soil moisture of 50% ensured the improvement of vegetation indicators of plants.

Development and Evaluation of a Rapid GII Norovirus Detection Method Based on CRISPR-Cas12a.

Norovirus is highly infectious and rapidly transmissible and represents a major pathogen of sporadic cases and outbreaks of acute gastroenteritis worldwide, causing a substantial disease burden. Recent years have witnessed a dramatic increase in norovirus outbreaks in China, significantly higher than in previous years, among which GII norovirus is the predominant prevalent strain. Therefore, rapid norovirus diagnosis is critical for clinical treatment and transmission control. Hence, we developed a molecular assay based on RPA combined with the CRISPER-CAS12a technique targeting the conserved region of the GII norovirus genome, the results of which could be displayed by fluorescence curves and immunochromatographic lateral-flow test strips. The reaction only required approximately 50 min, and the results were visible by the naked eye with a sensitivity reaching 102 copies/μl. Also, our method does not cross-react with other common pathogens that cause intestinal diarrhea. Furthermore, this assay was easy to perform and inexpensive, which could be widely applied for detecting norovirus in settings including medical institutions at all levels, particularly township health centers in low-resource areas.

Comparative study of the efficiency of inducers of cotton resistance to verticillium wilt

The effect of pre-sowing seed treatment with immunostimulant Bisol-2, red light and low frequency electromagnetic field on the content of fungitoxic substances of phenolic nature – phytoalexins (isohemigossypol and gossypol-equivalent) in infected etiolated cotton seedlings of S-4727 cultivar infected with Verticillium wilt pathogen was studied. It was found that photostimulation of seeds by red light induces phytoalexin formation in cotton tissues infected by the pathogen 1.5–2 times more effectively in comparison with Bisol-2 preparation or inducer of electromagnetic nature. The correlation between the content of phytoalexins in the tissues of seedlings, parameters of induction curves of chlorophyll fluorescence and the number of plants with signs of wilt lesions grown from treated and untreated seeds with inducers was revealed. This indicates the possibility of using red light and weak low-frequency electromagnetic fields as factors contributing to the intensification of phytoalexin formation in response to Verticillium wilt infection of cotton.

An automated gradient titration fluorescence methodology for high-resolution identification of aqueous two-polymer phase extraction conditions for single-wall carbon nanotubes

A significant advance in rate and precision of identifying the co-surfactant concentrations leading to differential extraction of specific single-wall carbon nanotube (SWCNT) species in aqueous two-polymer phase extraction experiments is reported. These gains are achieved through continuous titration of co-surfactant and other solution components during automated fluorescence measurements on SWCNT dispersions. The resulting fluorescence versus concentration curves display intensity and wavelength shift transitions traceable to the nature of the adsorbed surfactant layer on specific SWCNT structures at the (n,m) species and enantiomer level at high resolution. The increased precision and speed of the titration method resolve previously invisible complexity in the SWCNT fluorescence during the transition from one surfactant dominating the SWCNT interface to the other, offering insight into the fine details of the competitive exchange process. For the first time, we additionally demonstrate that the competitive process of the surfactant switch is direction independent (reversible) and hysteresis-free; the latter data effectively specifies an upper bound for the time scale of the exchange process. Titration curves are compared to literature results and initial advanced parameter variation is conducted for previously unreasonable to investigate solution conditions.

Portable and simultaneous detection of four respiratory pathogens through a microfluidic LAMP and real-time fluorescence assay.

Respiratory pathogen infections are seasonally prevalent and are likely to cause co-infections or serial infections during peak periods of infection. Since they often cause similar symptoms, simultaneous and on-site detection of respiratory pathogens is essential for accurate diagnosis and efficient treatment of these infectious diseases. However, molecular diagnostic techniques for multiple pathogens in this field are lacking. Herein, we developed a microfluidic LAMP and real-time fluorescence assay for rapid detection of multiple respiratory pathogens using a ten-channel microfluidic chip with pathogen primers pre-embedded in the chip reaction well. The microfluidic chip provided a closed reaction environment, effectively preventing aerosol contamination and improving the accuracy of the detection results. Its corresponding detection instrument could automatically collect and display the fluorescence curve in real time, which was more conducive to the interpretation of results. The results showed that the developed method could specifically recognize the nucleic acid of influenza A(H1N1), Mycoplasma pneumoniae, respiratory syncytial virus type A, and SARS-CoV-2 with low detection limits of 104 copies per mL or 103 copies per mL. The test results on clinical samples demonstrated that the developed method has high sensitivity (92.00%) and high specificity (100.00%) and even has the capability to differentiate mixed-infection samples. With simple operation and high detection efficiency, the present portable and simultaneous detection assay could significantly improve the efficiency of on-site detection of respiratory infectious diseases and promote the accurate treatment, efficient prevention and control of the diseases.

Colony and bacterial LAMP: Simple alternative methods for bacterial determination

In this study, we developed colony and bacterial LAMP, which directly use bacterial colony and bacterial culture as the templates without DNA extraction for rapid and simple detection of bacteria. The end-point readouts were determined by naked eye under ultraviolet light, and real-time fluorescence curve was also used to confirm that the sensitivity of this method to Salmonella typhimurium and Bacillus cereus was 102 and 103 CFU/reaction, respectively. Results presented here provide alternative methods for colony and bacterial PCR that can greatly contribute to reliable and cost-effective diagnosis in resource-poor settings.

A Telechelic Fluorescent Indicator Based on Polymer Conformational Change for Free Copper(II) Ions

A novel copper(II) ion indicator based on polymer conformational change is designed and its chemo-response to the target analyte is tested in this paper. The word ‘telechelic’ in the title means that a polymer has two different fluorophores on either end. If one of them is a fluorescent donor and the other is a fluorescent acceptor, then the extent of Foerster resonance energy transfer (FRET) will depend on polymer conformation. The sensitivity of these sensors is tunable based on the chain length and the amount of the receptor on the polymer. This is revealed by the fluorescence response of 30mer, 50mer, and 100mer of poly(N-isopropyl)acrylamide with different amounts of metal chelation monomers. We also address the change in fluorescence over time due to the untangling of poly(N-isopropylacrylamide) in water. The fluorescent signal can maintain stability after metal binding. The photoluminescence results agree with the length calculation of polyelectrolytes. A fluorescent standard curve is created for the measurement of different concentrations of copper ions. The sensing limit can reach 10−10 M analytes, which is suitable for the measurement of chemicals in trace amounts in the environment.

Heat Stress Recovery of Chlorophyll Fluorescence in Tomato (Lycopersicon esculentum Mill.) Leaves through Nitrogen Levels

To study the impact of nitrogen application on the photosynthetic structure and photosystem activity of tomato (Lycopersicon esculentum Mill.) leaves during the recovery stage after heat stress, the OJIP curve and JIP parameters were determined through a control experiment in an artificial climate chamber. The tomato variety was “Jinfen No. 1”. Four day/night temperature levels (25 °C/15 °C as control CKT; 30 °C/20 °C, lightly high-temperature LHT; 35 °C/25 °C, moderate high-temperature MHT; 40 °C/30 °C, severe high-temperature SHT) were set for a duration of 7 days. Five nitrogen supply levels (N1–N5: 0, 1.3, 1.95, 2.6 and 3.75 g/plant, respectively; 2.6 g/plant is the recommended nitrogen application rate, CKTN4) were applied. The results showed that the O, K, J, I and P phases on the chlorophyll a fluorescence curve were significantly affected by different nitrogen treatments in heat stress recovery. Compared with CKT, with the increase in nitrogen supply, the fluorescence intensity of SHTN2-SHTN5 treatment increased significantly at P, I and J phases, while that of MHTN1-MHTN4 treatment decreased. The fluorescence intensity of SHTN5 and SHTN3 increased by 13.27% and 10.10% in the P phase, 13.52% and 12.1% in the I phase and 20.16% and 26.18% in the J phase, respectively. There were highly significant differences (p &lt; 0.01) in the impact of high temperatures and nitrogen levels on the fluorescence parameters. On the 1st day after short-term heat stress, N had no significant effect on Fv/FM, Fv, Fo and FM; however, their interaction was significant (p &lt; 0.05). On the 8th day, there were no significant interaction effects between HT and N for Fv/Fo, ABS/RC and DIo/RC. Fv/Fo proved to be sensitive to the application of both high temperatures and nitrogen. Under all five nitrogen applications, temperature played a significant role in increasing DIo/RC, especially for N2 and N3. The results indicated that decreasing the nitrogen application under SHT resulted in a higher number of active RCs and an increased value of specific energy flux (ABS/RC, TRo/RC and DIo/RC), indicating the enhanced ability of RC to reduce plastoquinone. The study provides a reference for the diagnosis of nitrogen nutrition under high-temperature stress using chlorophyll fluorescence methods.