Lignin and organic free radicals in maize (Zea mays L.) seeds in response to aflatoxin B1 contamination: an optical and EPR spectroscopic study.

BACKGROUND: Aflatoxin B1 (AFB1 ) is the most dangerous of the mycotoxins that contaminate cereal seeds naturally. A stress lignin formation is linked with the accumulation of reactive oxygen species causing a change in the redox status and formation of stable organic radicals, constituting the first layer of defense. The relationship between AFB1 and changes in lignin organic free radicals in seeds is not known, nor is the part of the seed that is more targeted. Using optical and electron paramagnetic resonance spectroscopy, we investigated AFB1 -induced changes in lignin and organic free radicals in seeds, and whether the inner and outer seed fractions differ in response to increasing AFB1 . RESULTS: Different changes in the content of lignin and free radicals with increasing AFB1 concentrations were observed in the two seed fractions. There was a significant positive linear correlation (R = 0.9923, P = 0.00005) between lignin content and AFB1 concentration in the outer fraction, and no correlation between the lignin content and the AFB1 concentration in the inner fraction. We found a positive correlation between the area of the green spectral emission component (C4) and the AFB1 concentration in the outer fraction. CONCLUSIONS: To the best of our knowledge, the results showed, for the first time, that maize seed fractions respond differently to aflatoxin with regard to their lignin and organic free radical content. Lignin content and (C4) area may be reliable indicators for the screening of lignin changes against AFB1 content in the seeds, and thus for seed protection capacity. © 2021 Society of Chemical Industry.

Screening of semi-volatile compounds in plants treated with coated cerium oxide nanoparticles by comprehensive two-dimensional gas chromatography.

Literature data about semi-volatile organic compounds in plants and the effect of cerium oxide nanoparticles on them are scarce. Surface modification of nanoparticles may change nanoparticle-environment interaction, and therefore affects compounds in plants. In this research, uncoated and glucose-, levan-, and pullulan-coated cerium oxide nanoparticles were used for wheat and pea treatment during the growth. The aim was the screening of semi-volatile organic compounds from plants' shoots using comprehensive two-dimensional gas chromatography-mass spectrometry, a powerful separation technique allowing to reach unique separation resolution, and investigation of qualitative changes after the treatment with coated cerium oxide nanoparticles. The results were analyzed by the identification of individual peaks and fingerprint analysis by image processing. Wheat samples contained a higher number of semi-volatile organic compounds (108) compared to pea (77) but were less affected by the treatments with coated nanoparticles. The highest number of compounds was detected in wheat after the treatment with levan- and pullulan-coated nanoparticles, and in pea after treatment with levan-coated nanoparticles. This article reports a successful application of a semi-volatile organic compounds profile presented only as categorical variables and unique fingerprint images for the inter-cultivar recognition. This method may be useful in screening nanoparticles' effects on different plants.

Differential Polarization Imaging of Plant Cells. Mapping the Anisotropy of Cell Walls and Chloroplasts.

Modern light microscopy imaging techniques have substantially advanced our knowledge about the ultrastructure of plant cells and their organelles. Laser-scanning microscopy and digital light microscopy imaging techniques, in general-in addition to their high sensitivity, fast data acquisition, and great versatility of 2D-4D image analyses-also opened the technical possibilities to combine microscopy imaging with spectroscopic measurements. In this review, we focus our attention on differential polarization (DP) imaging techniques and on their applications on plant cell walls and chloroplasts, and show how these techniques provided unique and quantitative information on the anisotropic molecular organization of plant cell constituents: (i) We briefly describe how laser-scanning microscopes (LSMs) and the enhanced-resolution Re-scan Confocal Microscope (RCM of Confocal.nl Ltd. Amsterdam, Netherlands) can be equipped with DP attachments-making them capable of measuring different polarization spectroscopy parameters, parallel with the 'conventional' intensity imaging. (ii) We show examples of different faces of the strong anisotropic molecular organization of chloroplast thylakoid membranes. (iii) We illustrate the use of DP imaging of cell walls from a variety of wood samples and demonstrate the use of quantitative analysis. (iv) Finally, we outline the perspectives of further technical developments of micro-spectropolarimetry imaging and its use in plant cell studies.

A perspective view on the nanomotion detection of living organisms and its features.

The insurgence of newly arising, rapidly developing health threats, such as drug-resistant bacteria and cancers, is one of the most urgent public-health issues of modern times. This menace calls for the development of sensitive and reliable diagnostic tools to monitor the response of single cells to chemical or pharmaceutical stimuli. Recently, it has been demonstrated that all living organisms oscillate at a nanometric scale and that these oscillations stop as soon as the organisms die. These nanometric scale oscillations can be detected by depositing living cells onto a micro-fabricated cantilever and by monitoring its displacements with an atomic force microscope-based electronics. Such devices, named nanomotion sensors, have been employed to determine the resistance profiles of life-threatening bacteria within minutes, to evaluate, among others, the effect of chemicals on yeast, neurons, and cancer cells. The data obtained so far demonstrate the advantages of nanomotion sensing devices in rapidly characterizing microorganism susceptibility to pharmaceutical agents. Here, we review the key aspects of this technique, presenting its major applications. and detailing its working protocols.

Using optical fibers to measure absorption in intact conifer leaves, relative numbers of chloroplasts, and pigment content.

For investigations of ongoing processes in plants, such as photosynthesis in conifer leaves, nondestructive and noninvasive measuring techniques are needed. In this paper, a novel approach has been developed for the measurement of chloroplasts' numbers and pigment contents in conifer leaves based on the measurements of leaf absorption spectra using optical fibers and an array spectrophotometer. To eliminate the effect of scattering on the measured absorption spectra, a strategy has been applied taking advantage of the combined use of thin optical fibers normal to the needle's longitudinal axis and the phenomenon that scattering is largest in the forward direction. The optical path in the leaf is nearly the distance between the fiber tips; thus, we were able to obtain the absorption spectrum of the pigments in situ. A effect of the measured absorption spectra, occurring due to the organization of pigments in the leaf and interaction between light and leaf interior, can be accounted for by using the so-called Duysens transformation. Using this transformation, pigment contents and the relative number of chloroplasts can be obtained from the measured absorption spectra. We applied the method to observe pigment concentrations in different stages of the greening process in the leaves of two conifer species, Taxus baccata and Picea abies. The presented method may be used to estimate changes in chloroplast number and pigment content during various phases of greening of a species and to observe differences among various species.

Variations in polyamine conjugates in maize (Zea mays L.) seeds contaminated with aflatoxin B1: a dose-response relationship.

BACKGROUND: Cereal seeds, such as maize seeds, are frequently contaminated with aflatoxin B1 (AFB1), one of the most dangerous naturally occurring carcinogens. In plants, phenolamides are involved in biotic stress response. The data on variations of phenolamides in AFB1-containing seeds are limited. RESULTS: Five polyamine conjugates, including two spermidine and three putrescine conjugates, were tentatively identified in methanolic extracts, using HPLC-DAD-MS. The ratio of putrescine to spermidine conjugates changed with increasing AFB1 concentration in a logistic dose-response manner, with a ratio of below 1 up to a concentration of 51.51 µg kg-1 , and approximately 2.54 and 3 at higher concentrations of 177.4 and 308.13 µg kg-1 , respectively. The observed variations of the total antioxidant activity and the total phenolic content may support this biphasic behaviour of the seeds against AFB1 stress. CONCLUSIONS: The obtained data are a contribution to the understanding of the roles of polyamine conjugates in seed defence to increasing AFB1 concentrations. According to our knowledge, this study reports for the first time the biphasic response of maize seeds to increasing AFB1 contamination level, comprising the induction of polyamine conjugate accumulation and variation in the ratio of conjugates. This dose-response relationship may provide useful information in the field of agricultural and food chemistry as an indicator of AFB1 contamination level and, hence, for selecting an appropriate seed quality. © 2020 Society of Chemical Industry.

Multivariate Curve Resolution - Alternate Least Square Analysis of Excitation-Emission Matrices for Maize Flour Contaminated with Aflatoxin B1.

In this preliminary study, we used the Multivariate Curve Resolution- Alternating Least Squares (MCR-ALS) algorithm to analyze the excitation-emission matrix for different samples of maize flour contaminated with aflatoxin B1 (AFB1) - uncontaminated, low-contaminated, high-contaminated and flour from the local market. We intended to see if there are differences in emission spectral parameters that depend on degree of contamination. The analysis used genuine emission of the fluorophores in the flour, in absence and presence of AFB1, which enables fast screening of the samples, without sample pre-processing. As a result of the analysis, two fluorescence components were derived from the emission spectra for all analyzed samples. The components' positions were the same for the uncontaminated reference sample and the commercial flour sample from the local market, whereas for the samples contaminated with the aflatoxin B1, the emitted peaks' positions were red-shifted. We found that the ratio of the areas of these two components is proportional to the intensity of contamination: 0.071 for uncontaminated sample, 0.090 for the sample from local market, 0.192 for low-contaminated sample and 1.431 for high-contaminated sample. These results indicate that fluorescence EEM coupled with MCR-ALS could be used for rapid and simple estimation of the degree AFB1 contamination in maize flour.

Tyramine-modified pectins via periodate oxidation for soybean hull peroxidase induced hydrogel formation and immobilization.

Pectin was modified by oxidation with sodium periodate at molar ratios of 2.5, 5, 10, 15 and 20 mol% and reductive amination with tyramine and sodium cyanoborohydride afterwards. Concentration of tyramine groups within modified pectin ranged from 54.5 to 538 µmol/g of dry pectin while concentration of ionizable groups ranged from 3.0 to 4.0 mmol/g of dry polymer compared to 1.5 mmol/g before modification due to the introduction of amino group. All tyramine-pectins showed exceptional gelling properties and could form hydrogel both by cross-linking of carboxyl groups with calcium or by cross-linking phenol groups with peroxidase in the presence of hydrogen peroxide. These hydrogels were tested as carriers for soybean hull peroxidase (SHP) immobilization within microbeads formed in an emulsion based enzymatic polymerization reaction. SHP immobilized within tyramine-pectin microbeads had an increased thermal and organic solvent stability compared to the soluble enzyme. Immobilized SHP was more active in acidic pH region and had slightly decreased K m value of 2.61 mM compared to the soluble enzyme. After 7 cycles of repeated use in batch reactor for pyrogallol oxidation microbeads, immobilized SHP retained half of the initial activity.

Fluorescence-Detected Linear Dichroism of Wood Cell Walls in Juvenile Serbian Spruce: Estimation of Compression Wood Severity.

Fluorescence-detected linear dichroism (FDLD) microscopy provides observation of structural order in a microscopic sample and its expression in numerical terms, enabling both quantitative and qualitative comparison among different samples. We applied FDLD microscopy to compare the distribution and alignment of cellulose fibrils in cell walls of compression wood (CW) and normal wood (NW) on stem cross-sections of juvenile Picea omorika trees. Our data indicate a decrease in cellulose fibril order in CW compared with NW. Radial and tangential walls differ considerably in both NW and CW. In radial walls, cellulose fibril order shows a gradual decrease from NW to severe CW, in line with the increase in CW severity. This indicates that FDLD analysis of cellulose fibril order in radial cell walls is a valuable method for estimation of CW severity.

Cell death parameters as revealed by whole-cell patch-clamp and interval weighted spectra averaging: changes in membrane properties and current frequency of cultured mouse microglial cells induced by glutaraldehyde.

The physiological and biochemical factors that lead to cell death have not been recognized completely. To our knowledge, there are no data on the bioelectric parameters that characterize early period of cell death, as well as on the appearance of related membrane current frequencies. We studied early parameters of glutaraldehyde (GA)-induced cell death, by examining the membrane properties of mouse microglia using the whole-cell patch-clamp technique. In addition, we investigated the GA-induced changes in the membrane current frequency, to see if characteristic frequencies would appear in dying cell. For data analysis, we applied a new approach, an improved multiple moving window length analysis and interval weighted spectra averaging (IWSA). We chose GA for its ability to induce almost instantaneous cell death. The 0.6% GA did not induce changes in the bioelectric membrane properties of microglia. However, the 3% GA caused significant decrease of membrane capacitance and resistance accompanied by the prominent increase in the membrane currents and nearly ohmic current response of microglial cells. These data indicate that 3% GA caused complete loss of the membrane function consequently inducing instantaneous cell death. The membrane function loss was characterized by appearance of the 1.26-4.62 Hz frequency peak in the IWSA spectra, while no significant increase of amplitudes could be observed for cells treated with 0.6% GA. To our knowledge, this is the first record of a frequency associated with complete loss of the membrane function and thus can be considered as an early indicator of cell death.

Soybean hull peroxidase immobilization on macroporous glycidyl methacrylates with different surface characteristics.

Soybean hull peroxidase (SHP, E.C. 1.11.1.7) was immobilized by a glutaraldehyde and periodate method onto series of macroporous copolymers of glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EGDMA), poly(GMA-co-EGDMA) with various surface characteristics and pore size diameters ranging from 44 to 200 nm. Glutaraldehyde immobilization method and poly(GMA-co-EGDMA) named SGE 20/12 with pore sizes of 120 nm gave immobilized enzyme with highest specific activity of 25 U/g. Deactivation studies showed that immobilization increased stability of SHP and that surface characteristics of the used copolymer had a major influence on a stability of immobilized enzyme at high temperatures and in an organic solvent. The highest thermostability was obtained using the copolymer SGE 20/12 with pore size of 120 nm, while the highest stability in dioxane had SHP immobilized onto copolymer SGE 10/4 with pore size of 44 nm. Immobilized SHP showed a wider pH optimum as compared to the native enzyme especially at alkaline pH values and 3.2 times increased K m value for pyrogallol. After 6 cycles of repeated use in batch reactor, immobilized SHP retained 25 % of its original activity. Macroporous copolymers with different surface characteristics can be used for fine tuning of activity and stability of immobilized SHP to obtain a biocatalyst suitable for phenol oxidation or polymer synthesis in organic solvents.

Therapeutic potential of low-molecular weight lignin model polymer fractions for treating skin lesions in animals: a pilot study.

Bacterial infections and resistance to antibiotics are increasingly severe problems. In recent years, Staphylococcus species have emerged as important pathogens in animals and humans. Current therapeutic methods against these species have serious disadvantages; therefore new agents with antibacterial potential, such as plant-based substances, are very important in therapy. We report a pilot study with new method of fractioning the dehydrogenate polymer DHP obtained from coniferyl alcohol and application of the low-MW fractions of 200-3000 Da for antibacterial activity in healing animal lesions. In vivo experiments were conducted on the dogs having a skin lesion. Dogs were treated with the suspension containing the low-MW DHP fractions as the active ingredient, in combination with alginate for 7 days. Cytological smears and microbiological analyses of the affected area were performed. Staphylococcus spp. was isolated from lesions in all dogs from our research. The results show that the low-MW DHP suspension in alginate promotes skin healing and reduction of the infection of the lesions in the affected animals. Pharmaceutical composition containing the low-MW DHP fractions exerts a soothing effect on the subject in wound treatment. Reduction in the number of bacteria by 30% and more were noticed in 6 dogs, while in 4 dogs this percentage is above 50%. No side effects were noticed. Synthesized lignin oligomers may have a significant place as antimicrobial and skin healing agents, especially since an increasing number of multidrug-resistant staphylococci are found on the skin lesions in animals.

Spectroscopic Investigation of Tomato Seed Germination Stimulated by Trichoderma spp.

Seed germination is a complex process that can be negatively affected by numerous stresses. Trichoderma spp. are known as effective biocontrol agents as well as plant growth and germination stimulators. However, understanding of the early interactions between seeds and Trichoderma spp. remains limited. In the present paper, Fourier-transform infrared spectroscopy (FTIR) and Raman spectroscopy were used to reveal the nature of tomato seed germination as stimulated by Trichoderma. A rapid response of tomato seeds to Trichoderma spp. was observed within 48 h on Murashige and Skoog medium (MS) substrate, preceding any physical contact. Raman analysis indicated that both Trichoderma species stimulated phenolic compound synthesis by triggering plant-specific responses in seed radicles. The impact of T. harzianum and T. brevicompactum on two tomato cultivars resulted in alterations to the middle lamella pectin, cellulose, and xyloglucan in the primary cell wall. The Raman spectra indicated increased xylan content in NA with T9 treatment as well as increased hemicelluloses in GZ with T4 treatment. Moreover, T4 treatment resulted in elevated conjugated aldehydes in lignin in GZ, whereas the trend was reversed in NA. Additionally, FTIR analysis revealed significant changes in total protein levels in Trichoderma spp.-treated tomato seed radicles, with simultaneous decreases in pectin and/or xyloglucan. Our results indicate that two complementary spectroscopic methods, FTIR and Raman spectroscopy, can give valuable information on rapid changes in the plant cell wall structure of tomato radicles during germination stimulated by Trichoderma spp.

Non-toxic orange carbon dots stimulate photosynthesis and CO2 assimilation in hydroponically cultivated green beans (Phaseolus vulgaris).

Continuous increasing leaf photosynthesis may enhance plant yield. As an evolutionary property, plants use less photosynthetic capacity than is theoretically possible. Plant nanobionics is a bioengineering field that improves plant functions using nanoparticles. We applied orange carbon dots (o-CDs) onto the foliage of green beans (Phaseolus vulgaris ) grown in hydroponics to improve their photosynthetic performance and CO2 assimilation. Photosynthesis parameters, photosynthetic pigments content, total phenolic content (TPC) and antioxidative activity (TAA) were measured. Results show that photosynthetic pigments remained unchanged, while photosynthesis was improved. Both o-CDs concentrations decreased TPC and TAA. The light response curve showed higher CO2 assimilation at both o-CDs concentrations, particularly at lower light intensity. Correlation analysis confirmed increased CO2 binding and assimilation at 1mg L-1 . This study demonstrated the potential of using o-CDs as a safe biostimulator through photosynthesis increase and CO2 assimilation without toxic effects on plants. This may stimulate yield increase that paves the way for their agricultural application.

Enhancement in statistical and image analysis for in situ µSXRF studies of elemental distribution and co-localization, using Dioscorea balcanica.

Synchrotron-based X-ray microfluorescence (µSXRF) is an analytical method suitable for in situ investigation of the distribution of micronutrient and macronutrient elements in several-micrometres-thick unstained biological samples, e.g. single cells and tissues. Elements are mapped and quantified at sub-p.p.m. concentrations. In this study the quantity, distribution and grouping/co-localization of various elements have been identified in straight and twisted internodes of the stems of the monocotyledonous climber D. balcanica Kosanin. Three different statistical methods were employed to analyse the macronutrient and micronutrient distributions and co-localization. Macronutrient elements (K, P, Ca, Cl) are distributed homogeneously in both straight and twisted internodes. Micronutrient elements are mostly grouped in the vasculature and in the sclerenchyma cell layer. In addition, co-localization of micronutrient elements is much more prominent in twisted than in straight internodes. These image analyses and statistical methods provided very similar outcomes and could be applied to various types of biological samples imaged by µSXRF.

Decomposition of complex fluorescence spectra containing components with close emission maxima positions and similar quantum yields. Application to fluorescence spectra of proteins.

Despite of widely application of multivariate analysis in chemometrics, problem of resolving closely positioned components in the fluorescence spectra remained unsolved, thus limiting the usage of fluorescence spectroscopy in analytical purpose. In this paper we have described a novel procedure, adapted especially for the analysis of complex fluorescence spectra with multiple, closely positioned components' maxima. The method was first tested on the simulated spectra and then applied on the spectra of proteins whose fluorophores have similar properties of both the excitation and the emission spectra. In this paper, simple but efficient modification of the method was applied. Instead of analyzing full size emission matrix (12 spectra), 9 spectra wide windows were analyzed, and 4 factors (greatest possible number of factors with physical meaning both for actin and simulated spectra) were extracted in each pass. Obtained factor scores were grouped by using the K-means algorithm. Groups of factor scores obtained from K-means algorithm were passed through the one more factor analysis (FA) in order to find one factor that represents each group. Our approach provides resolution of extremely closed spectral components, which is a vital data for protein conformation analysis based on fluorescence spectroscopy.

Intrinsic Fluorescence Markers for Food Characteristics, Shelf Life, and Safety Estimation: Advanced Analytical Approach.

Food is a complex matrix of proteins, fats, minerals, vitamins, and other components. Various analytical methods are currently used for food testing. However, most of the used methods require sample preprocessing and expensive chemicals. New analytical methods are needed for quick and economic measurement of food quality and safety. Fluorescence spectroscopy is a simple and quick method to measure food quality, without sample preprocessing. This technique has been developed for food samples due to the application of a front-face measuring setup. Fluorescent compounds-fluorophores in the food samples are highly sensitive to their environment. Information about molecular structure and changes in food samples is obtained by the measurement of excitation-emission matrices of the endogenous fluorophores and by applying multivariate chemometric tools. Synchronous fluorescence spectroscopy is an advantageous screening mode used in food analysis. The fluorescent markers in food are amino acids tryptophan and tyrosine; the structural proteins collagen and elastin; the enzymes and co-enzymes NADH and FAD; vitamins; lipids; porphyrins; and mycotoxins in certain food types. The review provides information on the principles of the fluorescence measurements of food samples and the advantages of this method over the others. An analysis of the fluorescence spectroscopy applications in screening the various food types is provided.

A fluorescence spectroscopic study of light transmission and adaxial-abaxial distribution of emitting compounds in leaves of Christmas star (Euphorbia pulcherrima).

In situ fluorescence measurements have been used to investigate relative amounts of blue-green pigments and their distributions in plant leaves from Euphorbia pulcherrima. Advantage was taken from the fact that this species has white leaves on the top, with low pigment concentrations, and green leaves on the stem with ordinary pigment concentrations. Excitation- and emission spectra below 410 nm from white leaves, where pigment absorption is low, are not distorted by self-absorption. Absorption- and reflection spectra from white and green leaves were measured using a spectrophotometer equipped with an integrating sphere. The absorption spectra were used to correct recorded fluorescence spectra for self-absorption. Self-absorption corrected photosystem fluorescence from green leaves, modeling light transmission in leaf tissue exponentially, matches to the excitation/emission spectra from white leaves, apart from small differences due to the pigment concentrations and selective scattering. The introduced exponentially decaying transmission relation also predicts that the ratio of excitation spectra from a white and green leaf is in proportion to the absorption spectrum of the green leaf, which was validated for Photosystem II particle fluorescence. This relation was also used to find a scaled absorption spectrum responsible for blue-green emission, which was assumed to originate from lignin. Excitation/emission spectra of the blue-green fluorescence were decomposed into five components and their relative amounts from adaxial and abaxial sides of the leaves have been quantified. Fluorescence lifetime measurements of the leaves, upon 403 nm excitation, revealed three decay times corresponding to the lignin fluorophores emitting in blue and green spectral region, and indicated that emissions at 500 and 550 nm may originate from the same fluorophore residing in the two physically different environments.

Trans-generational effect of cerium oxide-nanoparticles (nCeO2) on Chenopodium rubrum L. and Sinapis alba L. seeds.

Cerium oxide nanoparticles (nCeO2 ) are interesting nanomaterials due to their redox properties. Their wide application could result in unexpected consequences to environmental safety. Unlike acute toxicity, the trans-generational effects of carbohydrate-coated nCeO2 in the environment are still unknown. The main aim of this study was to investigate the effect of treating maternal plants of Chenopodium rubrum L. (red goosefoot) and Sinapis alba L. (white mustard) with uncoated (CeO2 ) and glucose-, levan-, or pullulan-coated nCeO2 (G-, L-, or P-CeO2 ) during seed germination on morphological and physiological characteristics of produced seeds in two subsequent generations. The plant response was studied by measuring germination percentage (Ger), total protein content (TPC), total phenolic content (TPhC), total antioxidative activity (TAA), and catalase (CAT) activity. Results showed that maternal effects of the different nCeO2 treatments persist to at least the second generation in seeds. Generally, C. rubrum was more sensitive to nCeO2 treatments than S. alba . The coated nCeO2 were more effective than uncoated ones in both plant species; L- and P-CeO2 were the most effective in S. alba , while CeO2 and G-CeO2 had a dominant impact in C. rubrum . Enhanced germination in all tested generations of S. alba seeds recommends nCeO2 for seed priming.

Using Front-Face Fluorescence Spectroscopy and Biochemical Analysis of Honey to Assess a Marker for the Level of Varroa destructor Infestation of Honey Bee (Apis mellifera) Colonies.

Varroa destructor is a parasitic mite responsible for the loss of honey bee (Apis mellifera) colonies. This study aimed to find a promising marker in honey for the bee colony infestation level using fluorescence spectroscopy and biochemical analyses. We examined whether the parameters of the honey samples' fluorescence spectra and biochemical parameters, both related to proteins and phenolics, may be connected with the level of honey bee colonies' infestation. The infestation level was highly positively correlated with the catalase activity in honey (r = 0.936). Additionally, the infestation level was positively correlated with the phenolic spectral component (r = 0.656), which was tentatively related to the phenolics in honey. No correlation was found between the diastase activity in honey and the colonies' infestation level. The results indicate that the catalase activity in honey and the PFC1 spectral component may be reliable markers for the V. destructor infestation level of the colonies. The obtained data may be related to the honey yield obtained from the apiaries.