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Crop production might suffer severe economic losses due to insufficient fertiliser availability. Specific signs of nutrient shortage influence plant morphology and physiology. This study pioneers the non-destructive tracking and characterization of nutrient deficiency symptoms in common beans using multispectral and chlorophyll fluorescence imaging, offering novel insights into the dynamic responses of plant morphology and physiology to specific nutrient shortages. Plants were cultivated in nutrient solutions with and without nitrogen (N), phosphorus (P), potassium (K), magnesium (Mg), iron (Fe) and control solution. Measurements were taken every three days for 12 days (MT1-MT4) of growth. K and N deficit plants had the earliest symptoms and most noticeable changes, whereas Fe deficiency plants had the slowest and least noticeable symptoms. Except for Fe, the most responsive chlorophyll fluorescence parameter was electron transport rate, which was reduced in plants from all nutrient deprivation treatments compared to control. All nutrient deficit treatments reduced leaf area at MT2, which was the most affected morphological parameter. The green leaf index, reflection in blue, and specific green were the most affected multispectral traits by nutritional deprivation. These findings demonstrate that plant nutrient deficit can be recognized and tracked non-destructively utilizing multispectral and chlorophyll fluorescence analysis. Overall, our work not only sheds light on the dynamics of nutrient deficiency in common bean plants but also offers practical implications for improving crop management strategies using non-destructive digital technology.

Abstract Soil acidity (pH

This research aimed to assess the effect of applying digestate fractions and conventional mineral nitrogen (N) fertilizers on plant and soil properties, as well as potential nitrate leaching, in acidic soil over two consecutive years (2018 and 2019). The digestate fractions were obtained after the anaerobic co-digestion of maize silage and liquid cattle manure (LCM). Seven different treatments were applied to the maize crop in four replicates: unfertilized control (C), mineral fertilizer (MF), LCM, solid fraction of digestate (SFD), liquid fraction of digestate (LFD) and a mixture of SFD and LFD with MF (50% of total N from digestate fractions + 50% of total N from MF). The highest maize yields were achieved on average in 2018. Statistically, the highest dry grain yield was observed in the MF treatment (12.1 t ha−1) and in the mixtures of MF+SFD (11.0 t ha−1) and MF+LFD (11.8 t ha−1), while the lowest yield was achieved in both years in the C treatment (7.9 t ha−1). The N fertilizer replacement value was statistically highest on average in the MF treatment (100%) and in the mixtures of MF+LFD (80.5%) for both years. The experiment results could inform legal guidelines and standardize digestate application on agricultural land in Croatia and the European Union (EU).

The lack of plant nutrients is a major problem for agriculture. Because of their essential role in the most important metabolic processes of plants, their deficiency depresses photosynthesis and disrupts the efficient operation of the photosynthetic apparatus. In this study, we investigated the gas exchange of common beans bean under the N, P, K, Mg, and Fe deficiency. The experiment was set up as a hydroponic, fully aerated, floating system in 6 hydroponic tubs. Each hydroponic tub contained 10 plants and was filled with a modified Hoagland nutrient solution. One group of plants was grown in a complete nutrient solution (control), while the other treatments lacked one of the following nutrients: N, P, K, Mg and Fe. During the experiment, gas exchange parameters: net photosynthetic rate (A), transpiration rate (E), stomatal conductance (gs) and intercellular CO2 concentration (Ci) were measured. At the end of the experiment, the nutrient content of the plant tissue was determined. Nutrient deficiency significantly affected all measured photosynthetic parameters and visual symptoms indicated that a lack of different nutrients affected the photosynthetic machinery at different points. Potassium deficiency lower stomatal conductance and increased mesophyll resistance to CO2 diffusion and/or RubisCO activity. Nitrogen and Mg deficiency affected chlorophyll synthesis and accelerated leaf senescence. Phosphorus deficiency caused less damage to gas exchange parameters probably due to protective mechanisms of reduced leaf area. Seed Fe content was surplus to sustain photosynthetic machinery during the early developmental phase.

Abstract Drought tolerance in Miscanthus sinensis is a desirable trait because of its potential use to develop new varieties, namely intra‐specific hybrids, adapted to drought‐prone marginal lands. In this study, drought tolerance was evaluated on 8 M. sinensis intra‐specific hybrids (GRC1–GRC8). Plants were grown in the growth chamber (14/10 h, 25/20°C, 70% relative air humidity and 300 μmol m−2 s−1 irradiance). Drought was induced by withholding irrigation for 21 days (stress phase) and after re‐watering (recovery phase) for 7 days. Nondestructive multispectral 3D images for plant morphology, color, and chlorophyll fluorescence imaging were used to quantify drought‐induced changes on a weekly basis for the entire duration of the experiment. Total leaf area (TLA) and digital biomass (DB) responded most rapidly to water deficits (7–14 days), followed by leaf senescence (14–21 days), and finally, a drop in the maximum efficiency of PSII (Fv/Fm; 21 days). Traits measured on the last day of the drought treatment were used to calculate the phenotypic plasticity. Significant differences in drought susceptibility and phenotypic plasticity were found among the studied hybrids. Drought treatment (21 days) reduced DB and TLA on average by 50%, normalized difference vegetation index (NDVI) by 20% and Fv/Fm from 0.79 (in control) to 0.69 in the less drought‐susceptible hybrids (GRC4 and GRC5), whereas in drought‐sensitive hybrids (GRC2 and GRC3), 21 days of drought reduced DB and TLA on average by 80%, NDVI by 45% and Fv/Fm dropped from 0.79 (in control) to 0.35. The more drought‐resilient hybrids showed lower phenotypic plasticity than their more sensitive counterparts, a stay‐green strategy enabled through lower biomass accumulation and by extension, reduced water usage. Multispectral imaging and image analysis enabled fast and nondestructive quantification of plant morphological and physiological responses under drought conditions and could be used as an effective screening tool for drought susceptibility.

In vitro plants that are free of pathogens are crucial for biotechnological breeding methods. The present study investigates the effects of sterilization with sodium hypochlorite (NaClO) and the addition of Plant Preservative Mixture (PPMTM) to the growth medium on pathogen elimination, germination, and seedling development of the winter wheat (Triticum aestivum L.) variety Mara. The sterilization treatments differed in the duration of seed sterilization in 4% NaClO and the PPM concentration added to the growth medium. Pathogenic fungi of the genera Aspergillus, Cladosporium, Penicillium, and Bipolaris were completely eliminated when the seeds were sterilized in NaClO and placed on growth media with the addition of PPM. Extending the duration of the sterilization treatment with NaClO to 50 min reduced Fusarium contamination, while the interaction between the 50 min sterilization treatment with NaClO and the addition of PPM to the growth medium reduced Alternaria contamination. Our results suggest that PPM could complement sterilization procedures with NaClO in the introduction of highly infected wheat seeds in vitro. Seed germination was not affected by sterilization with NaClO or by the addition of PPM. However, PPM at a concentration of 4 mL L−1 had a negative effect on seedling development.

Soil pH can affect the adsorption of mesotrione and exacerbate crop injury under non-acidic conditions. Soil samples collected from the same location were irrigated with water solutions of pH 7.5, 6.5, 5.5, and 4.5 and treated with 72, 36, 24, 18, 9, 4.5, 2.3, and 1.1 g a.i. (active ingredient) of mesotrione/ha. Bean growth was monitored over 28 days. Soil pH solution did not influence the effect of mesotrione on plant fresh weight, while herbicide-induced visual injury and reduction in carotenoid content were significantly mitigated under acidic conditions. The lowest rate (1.1 g a.i./ha) applied in slightly acidic soil (pH 6.5) caused visual injury of 45% 28 days after treatment, while visual injuries on plants grown in soils with pH 4.5 were only 20%. Further, bean plants grown at pH 4.5 showed only 3.3% lower carotenoid content compared to control plants since for those grown in a slightly alkaline environment (pH 7.5) reduction of this pigment was 35.5%. The mean effective dose (ED50 ± standard error) of mesotrione for inhibition of carotenoids were 5.25 ± 0.61 g a.i./ha at pH 7.5, 9.57 ± 0.74 g a.i./ha at pH 6.5, 13.07 ± 0.91 g a.i./ha at pH 5.5, and 14.98 ± 0.94 g a.i./ha at pH 4.5. Results indicate that the common bean is highly susceptible to the presence of mesotrione residue and that this sensitivity strongly depends on soil pH solution.

The development of automated, image-based, high-throughput plant phenotyping enabled the simultaneous measurement of many plant traits. Big and complex phenotypic datasets require advanced statistical methods which enable the extraction of the most valuable traits when combined with other measurements, interpretation, and understanding of their (eco)physiological background. Nutrient deficiency in plants causes specific symptoms that can be easily detected by multispectral imaging, 3D scanning, and chlorophyll fluorescence measurements. Screening of numerous image-based phenotypic traits of common bean plants grown in nutrient-deficient solutions was conducted to optimize phenotyping and select the most valuable phenotypic traits related to the specific nutrient deficit. Discriminant analysis was used to compare the efficiency of groups of traits obtained by high-throughput phenotyping techniques (chlorophyll fluorescence, multispectral traits, and morphological traits) in discrimination between nutrients [nitrogen (N), phosphorus (P), potassium (K), magnesium (Mg), and iron (Fe)] at early and prolonged deficiency. Furthermore, a recursive partitioning analysis was used to select variables within each group of traits that show the highest accuracy for assigning plants to the respective nutrient deficit treatment. Using the entire set of measured traits, the highest classification success by discriminant function was achieved using multispectral traits. In the subsequent measurements, chlorophyll fluorescence and multispectral traits achieved comparably high classification success. Recursive partitioning analysis was able to intrinsically identify variables within each group of traits and their threshold values that best separate the observations from different nutrient deficiency groups. Again, the highest success in assigning plants into their respective groups was achieved based on selected multispectral traits. Selected chlorophyll fluorescence traits also showed high accuracy for assigning plants into control, Fe, Mg, and P deficit but could not correctly assign K and N deficit plants. This study has shown the usefulness of combining high-throughput phenotyping techniques with advanced data analysis to determine and differentiate nutrient deficiency stress.

Drought negatively affects plants by altering morphological, physiological and metabolic processes and ultimately reducing yields. Garlic (Allium sativum L.), an important member of the Alliaceae family, is also sensitive to drought and maximizing the yield of garlic bulbs is largely dependent on water availability. The objective of this study was to determine the effects of drought stress on morphological and physiological characteristics, as well as on phenolic, sugar, inulin and free amino acid content and antioxidant activity in two Croatian garlic ecotypes, ‘Istarski crveni’ (IC) and Istarski bijeli (IB). Drought was induced by using polyethylene glycol 8000 (PEG) solution (−0.6 MPa) starting 21 days after clove planting and lasted for 20 days. Drought reduced plant height, number of leaves and plant weight, but increased root length in both ecotypes compared to the control treatment. Among the physiological parameters, significant differences were observed between the two ecotypes studied in the spectral characteristics of the leaves, namely reflection in red, green and blue, VAL, values of the vegetation indices related to the chlorophyll content (CHI, GI), and the anthocyanin content (ARI). Ecotype IC showed higher antioxidant activity in the control treatment due to higher total phenolic content (TPC), but under drought conditions higher DPPH radical scavenging activity was determined in ecotype IB and higher values of FRAP in IC. Sucrose and glucose generally decreased under drought, while inulin increased in IB but decreased in IC. Total free amino acid content increased under drought in both ecotypes. In conclusion, drought tolerance of IB might be associated with increased accumulation of inulin and higher levels of amino acids, especially those shown to contribute to drought resistance. In IC, drought tolerance is associated with an increase in some amino acid compounds and better root growth in depth, probably due to a more efficient translocation of sucrose to the underground part of the plant.

Drought is a significant constraint in bean production. In this study, we used high-throughput phenotyping methods (chlorophyll fluorescence imaging, multispectral imaging, 3D multispectral scanning) to monitor the development of drought-induced morphological and physiological symptoms at an early stage of development of the common bean. This study aimed to select the plant phenotypic traits which were most sensitive to drought. Plants were grown in an irrigated control (C) and under three drought treatments: D70, D50, and D30 (irrigated with 70, 50, and 30 mL distilled water, respectively). Measurements were performed on five consecutive days, starting on the first day after the onset of treatments (1 DAT–5 DAT), with an additional measurement taken on the eighth day (8 DAT) after the onset of treatments. Earliest detected changes were found at 3 DAT when compared to the control. D30 caused a decrease in leaf area index (of 40%), total leaf area (28%), reflectance in specific green (13%), saturation (9%), and green leaf index (9%), and an increase in the anthocyanin index (23%) and reflectance in blue (7%). The selected phenotypic traits could be used to monitor drought stress and to screen for tolerant genotypes in breeding programs.

Climate change has a significant impact on the agricultural sector, negatively affecting plants’ growth and development, with predicted strong consequences on food availability in the future. Although we are experiencing more frequent and intense heavy rainfall events, a major contributor to field flooding, there is still not much known about the impact of these events on different crops. In this study, we investigated the effects of waterlogging on a model plant white cabbage (Brassica oleracea var. capitata f. alba), with the aim to follow its response to both single and recurrent short-term (72-h length) waterlogging, as well as to track difference in the sensitivity between plants in different growth stages (38- and 48-day-old plants). In our 22-day experiment, settled under fully controlled conditions (16 h day/8 h night, 25 °C day/20 °C night, 60–70% relative air humidity, 250 µmol m−2 s−1 photosynthetic active radiation), with the aim to more comprehensively recognize consequences of waterlogging on plants, we measured changes in plants on multiple levels: (i) within its morphological traits (number and length of leaves, leaf area, and blade width), (ii) within chlorophyll fluorescence and multispectral traits (20 parameters), (iii) following the levels of plant stress parameters (salicylic acid, abscisic acid, proline, and total polyphenols), and (iv) following changes in the plants’ elemental and mineral composition. According to our results, white cabbage was shown not to be very sensitive to waterlogging, with only plants exposed to repeated waterlogging showing signs of the congestion stress. These signs, observed in the changes of molecular stress parameters salicylic and abscisic acids, were not so clearly evident at the aboveground level. We did not observe changes in the plants’ morphologies, nor their photosynthetic performance. In addition, removal of waterlogging stress resulted in complete recovery of our model plants, suggesting a prompt adaptation response of white cabbage. With the projected increased frequency of occurrence of flooding events, it will become increasingly more important to recognize crops being highly sensitive to flooding with the aim to try to adapt to the changing climate.

Potato (Solanum tuberosum L.) is vulnerable to high temperatures, which are expected to increase in frequency and duration due to climate change. Nondestructive phenotyping techniques represent a promising technology for helping the adaptation of agriculture to climate change. In this study, three potato cultivars (Agria, Bellarosa and Desiree) were grown under four temperature treatments: 20/15 °C (T1), 25/20 °C (T2), 30/25 °C (T3), and 35/30 °C (T4). Multispectral and chlorophyll fluorescence imaging, 3D multispectral scanning, and gas exchange analysis were used to study the effect of moderate heat stress on potato morphology and physiology and select phenotypic traits most responsive to increased temperatures. The most responsive morphological traits to increased temperatures are related to decreased leaf area, which were detected already at T2. Increased temperatures (already T2) also changed leaf spectral characteristics, indicated by increased red, green, and blue reflectance and decreased far-red reflectance and anthocyanin index (ARI). Regarding chlorophyll fluorescence, increasing temperatures (T2) caused an increase in minimal fluorescence of both dark-adapted (F0) and light-adapted (F0’) plants. Stomatal conductance, transpiration rate, photosynthetic rate, instantaneous water use efficiency (WUE), and intrinsic water use efficiency increased from T1 to T3 and decreased again in T4. Using recursive partitioning analysis, the most responsive potato phenotypic traits to increased temperature were leaf area projected (LAP), ARI, F0, and WUE. These traits could be considered marker traits for further studying potato responses to increased temperatures.

Viruses are responsible for more than 50% of annual potato tuber yield losses and cause great economic damage. The traditional Croatian potato cultivar ‘Brinjak’ is important for local growers because of its economically profitable production and as a gene pool for future breeding programs. However, the full genetic potential of the cultivar cannot be exploited due to virus infection. In this study, we attempted to eliminate potato virus M (PVM) and potato virus S (PVS) from potato cultivar ‘Brinjak’ and to evaluate the effects on physiological parameters and yield. Shoot apices were isolated from PVM + PVS-infected sprouts and cultivated for six weeks on MS medium with the addition of 50 or 100 mg L−1 ribavirin. The surviving shoot apices were micropropagated. The in vitro post-eradication period lasted 200 days. DAS-ELISA and RT-PCR were performed on R0 and R1 plants 90 days after acclimatization to determine the sanitary status of the plants. Chlorophyll fluorescence and multispectral imaging were performed on the R0 plants at the same time. The success of PVS elimination was 33% at both ribavirin concentrations. However, neither concentration was successful in eliminating PVM. Plants with mixed infection (PVM + PVS) had more severe disease symptoms compared to PVM-infected plants, affecting photochemistry and multispectral parameters and, consequently, yield. PVM + PVS plants had significantly lower number and weight of tubers per plant and lower average tuber weight than plants with single PVM infection in most of the generations studied. The results indicate a strong negative impact of PVS in mixed infections with PVM and show the importance of its elimination from potato plants.

Common bean (Phaseolus vulgaris L.) exhibits a wide range of seed coat colors and this morphological trait is widely used in cultivar identification and assessment of diversity within this species. With an advancement in technology and informatics, new methods of assessing seed color are emerging in addition to traditionally used visual observation. Due to a great variety of color measuring techniques, the evaluation of the agreement between methods is needed prior to using the methods interchangeably. Seed coat color in terms of CIE L*a*b* color coordinates of 100 common bean accessions belonging to five mono-colored landraces was assessed using two methods, colorimeter and Computer vision. The percentage difference between the two methods across all samples for L* color coordinate was 5.81%, for a* color coordinate 23.32% and for b* color coordinate 44.44%. According to Bland-Altman difference plot there is a considerable lack of agreement between the two methods. However, using stepwise discriminant analysis revealed that colorimeter method correctly classified 97% of accessions into their respective landrace, while the classification success of the Computer vision was 99%.

Micronutrient malnutrition is one of the main public health problems in many parts of the world. This problem raises the attention of all valuable sources of micronutrients for the human diet, such as common bean (Phaseolus vulgaris L.). In this research, a panel of 174 accessions representing Croatian common bean landraces was phenotyped for seed content of eight nutrients (N, P, K, Ca, Mg, Fe, Zn, and Mn), and genotyped using 6,311 high-quality DArTseq-derived SNP markers. A genome-wide association study (GWAS) was then performed to identify new genetic sources for improving seed mineral content. Twenty-two quantitative trait nucleotides (QTN) associated with seed nitrogen content were discovered on chromosomes Pv01, Pv02, Pv03, Pv05, Pv07, Pv08, and Pv10. Five QTNs were associated with seed phosphorus content, four on chromosome Pv07, and one on Pv08. A single significant QTN was found for seed calcium content on chromosome Pv09 and for seed magnesium content on Pv08. Finally, two QTNs associated with seed zinc content were identified on Pv06 while no QTNs were found to be associated with seed potassium, iron, or manganese content. Our results demonstrate the utility of GWAS for understanding the genetic architecture of seed nutritional traits in common bean and have utility for future enrichment of seed with macro– and micronutrients through genomics-assisted breeding.

Basil is one of the most widespread aromatic and medicinal plants, which is often grown in drought- and salinity-prone regions. Often co-occurrence of drought and salinity stresses in agroecosystems and similarities of symptoms which they cause on plants complicates the differentiation among them. Development of automated phenotyping techniques with integrative and simultaneous quantification of multiple morphological and physiological traits enables early detection and quantification of different stresses on a whole plant basis. In this study, we have used different phenotyping techniques including chlorophyll fluorescence imaging, multispectral imaging, and 3D multispectral scanning, aiming to quantify changes in basil phenotypic traits under early and prolonged drought and salinity stress and to determine traits which could differentiate among drought and salinity stressed basil plants. Ocimum basilicum “Genovese” was grown in a growth chamber under well-watered control [45–50% volumetric water content (VWC)], moderate salinity stress (100 mM NaCl), severe salinity stress (200 mM NaCl), moderate drought stress (25–30% VWC), and severe drought stress (15–20% VWC). Phenotypic traits were measured for 3 weeks in 7-day intervals. Automated phenotyping techniques were able to detect basil responses to early and prolonged salinity and drought stress. In addition, several phenotypic traits were able to differentiate among salinity and drought. At early stages, low anthocyanin index (ARI), chlorophyll index (CHI), and hue (HUE2D), and higher reflectance in red (RRed), reflectance in green (RGreen), and leaf inclination (LINC) indicated drought stress. At later stress stages, maximum fluorescence (Fm), HUE2D, normalized difference vegetation index (NDVI), and LINC contribute the most to the differentiation among drought and non-stressed as well as among drought and salinity stressed plants. ARI and electron transport rate (ETR) were best for differentiation of salinity stressed plants from non-stressed plants both at early and prolonged stress.

Winter savory (Satureja montana L.) is an important medicinal, aromatic, and honey plant. In Croatia, it is widely distributed along the Adriatic coast, where it is frequently exposed to droughts. First, the winter savory natural population with the highest germination across different drought treatments after hydropriming was selected. Nine hundred seeds from each of the three natural populations (P1, P2, and P3) were hydroprimed (dH2O) for 48 h. The seeds were then germinated in drought treatments with different concentrations of polyethylene glycol (PEG 6000) (−0, −0.2, −0.4, −0.8, −1.2, −1.6, −2, −2.5, −3.0 MPa). Since P1 showed the best results in germination parameters, it was used for the second phase of the experiment, where the effect of hormonal priming (100 and 400 ppm GA3, 48 h in the dark) and osmopriming (0.2% and 0.6% w/v KNO3, 72 h in the dark) on seed germination and seedling morphological parameters of the selected winter savory population under drought stress conditions (−0.8 and −2.5 MPa) was evaluated. Although winter savory grows in dry areas, this study showed that extremely dry conditions (−3.0 and −2.5 MPa) negatively affected seed germination, but this effect can be mitigated by priming treatments, especially with the hormonal priming (GA3 400 ppm).

In this study, near-infrared spectroscopy (NIRS) was used to predict the contents of essential macro- and microelements in common bean (Phaseolus vulgaris L.) accessions of most widespread Croatian landraces. Total of 175 samples were used for the model development by modified partial least square (MPLS), principal component regression (PCR) and partial least square (PLS) techniques. Based on the coefficients of determination (R2), standard error of calibration (SEC) and error of prediction (SEP) the models developed were (i) nearly applicable for nitrogen (N) (0.89, 0.12 and 0.45 respectively), (ii) poor for iron (Fe), cinc (Zn), potassium oxide (K2O) and potassium (K), (iii) usable for phosphorus pentoxide (P2O5), phosphorus (P), phytic acid (PA) and manganese (Mn). The MPLS regression statistics suggested the most accurate models developed comparing with PLS and PCR. It was concluded that a wider set of common bean samples needs to be used for macro- and microelements prediction by NIRS.

Finding a suitable alternative to the small pool of existing antifungal agents is a vital task in contemporary agriculture. Therefore, intensive research has been conducted globally to uncover environmentally friendly and efficient agents that can suppress pathogens resistant to the currently used antimycotics. Here, we tested the activity of boric acid (BA) and its derivative phenylboronic acid (PBA) in controlling the early blight symptoms in tomato plants infected with pathogenic fungus Alternaria alternata. By following the appearance and intensity of the lesions on leaves of the tested plants, as well as by measuring four selected physiological factors that reflect plant health, we have shown that both BA and PBA act prophylactically on fungal infection. They did it by reducing the amount and severity of early blight symptoms, as well as by preventing deterioration of the physiological traits, occurring upon fungal inoculation. Phenylboronic acid was more efficient in suppressing the impact of A. alternata infection. Therefore, we conclude that BA, and even more so PBA, may be used as agents for controlling early blight on tomato plants, as they are both quite effective and environmentally friendly.

Common bean (Phaseolus vulgaris L.) is an important legume crop. In Croatia, many different common bean landraces are grown mostly on small farms and in kitchen gardens. The aim of this research was to determine and compare nutrient content of Croatian common bean landraces and to test the existence of the relationship between common bean nutrient content with origin of accessions. A total of 226 common bean accessions from all over Croatia were collected, grown in unreplicated field trial at the experimental field in Zagreb, harvested and analyzed for nutrient content. Results showed high nutrient content, but clear difference between Mesoamerican and Andean gene pool regards the nutrient profile of Croatian common bean landraces was not determined. Regardless of gene pool, the average nutrient content in dry weight of Croatian common bean landraces was in range: 3.13–3.92% N, 0.49–0.57% P, 1.38–1.66% K, 0.34–0.42% Ca, 0.17–0.20% Mg, 63.75–75.77 mg Fe∙kg-1, 13.55–19.94 mg Mn∙kg-1 and 24.98–29.32 mg Zn∙kg-1. Present research provides a solid basis for future breeding programs and improvement of common bean traits.

A survey of recently discovered vitiviruses was performed on 113 Croatian autochthonous grapevine cultivars from the national collection “Jazbina” using one-step RT-PCR. The presence of grapevine virus H (GVH) was confirmed in nine (7.9%) cultivars and grapevine virus G in eight (7.1%), while the presence of grapevine viruses I and J were not detected. GVH was transmitted by the vine mealybug (Planococcus ficus) from a source plant to grapevine seedlings with a 10.5% transmission rate using a combination of 10 first and second instars per plant with 48 and 72 h of acquisition and inoculation access period, respectively. Transmission correlated with the presence of grapevine leafroll-associated virus 3 (GLRaV-3) in the GVH-source plant and recipient seedlings. No alternative GVH host was identified. A comparison of 356 nt fragments of the RdRP and CP coding regions showed nucleotide identity between the Croatian GVH isolates in the range of 95.5–99.2% and 97.5–99.4% and amino acid identity between 95.8 and 100% and between 98.3 and 100%, respectively. Comparison with foreign isolates revealed nucleotide sequence similarity in the RdRP and CP between 94 and 100% and between 97.7–100%, respectively. To the best of our knowledge, this is the first report of GVH in Croatia and the first identification of the vine mealybug as a vector of GVH.

Arbuscular mycorrhizal (AM) root colonization is known to have beneficial effects on plant growth especially under phosphorus (P) deficit conditions. The objectives of present study were: (i) to quantify changes in early wheat root development of AM-inoculated (AMI) and AM-free (AMF) roots under limited P availability; (ii) to assess possible mitigating effect of AM inoculation on photochemical efficiency under P deficit stress. AMI (inoculated with Rhizophagus irregularis) and AMF wheat plants were grown for 20 days in low (1 μmol/L) and high (50 μmol/L)P treatments. AM inoculation affected root morphology and shoot P concentration in low P treatment. AM inoculation alleviated reduction of the total root length in low P treatment, mainly due to an increase of fine roots length(< 0.5 mm). Contrastingly, shoot dry weight was reduced by AM inoculation in low P treatment. P deficiency decreased photochemical efficiency of wheat plants. However, due to increased sink capacity and facilitated nutrient concentrations AM inoculation alleviates phosphorus deficit stress and increased photochemical efficiency.

Sweet pepper (Capsicum annuum L.) is one of the most important vegetable crops in the world because of the nutritional value of its fruits and its economic importance. Calcium (Ca) improves the quality of sweet pepper fruits, and the application of calcite nanoparticles in agricultural practice has a positive effect on the morphological, physiological, and physicochemical properties of the whole plant. The objectives of this study were to investigate the effect of commercial calcite nanoparticles on yield, chemical, physical, morphological, and multispectral properties of sweet pepper fruits using a combination of conventional and novel image-based nondestructive methods of fruit quality analysis. In the field trial, two sweet pepper cultivars, i.e., Šorokšari and Kurtovska kapija, were treated with commercial calcite nanoparticles (at a concentration of 3% and 5%, calcite-based foliar fertilizer (positive control), and water (negative control) three times during vegetation). Sweet pepper fruits were harvested at the time of technological and physiological maturity. Significant differences were observed between pepper cultivars as well as between harvests times. In general, application of calcite nanoparticles reduced yield and increased fruit firmness. However, different effects of calcite nanoparticles were observed on almost all properties depending on the cultivar. In Šorokšari, calcite nanoparticles and calcite-based foliar fertilizers significantly increased N, P, K, Mg, Fe, Zn, Mn, and Cu at technological maturity, as well as P, Ca, Mg, Fe, Zn, Mn, Cu, and N at physiological maturity. However, in Kurtovska kapija, the treatments increased only Ca at technological maturity and only P at physiological maturity. The effect of treatments on fruit morphological properties was observed only at the second harvest. In Šorokšari, calcite nanoparticles (3% and 5%) increased the fruit length, minimal circle area, and minimal circle radius, and it decreased the fruit width and convex hull compared to the positive and negative controls, respectively. In Kurtovska kapija, calcite nanoparticles increased the fruit width and convex hull compared to the controls. At physiological maturity, lower anthocyanin and chlorophyll indices were found in Kurtovska kapija in both treatments with calcite nanoparticles, while in Šorokšari, the opposite effects were observed.

The aim of this study was to assess the influence of the application of mycorrhizal fungal inoculum on “Cabernet Sauvignon” (Vitis vinifera L.) leaf gas exchange, yield parameters, as well as grape berry composition, especially regarding phenolic compounds. The experiment was conducted over two years under natural vineyard conditions of the Zagreb wine-growing area, the continental region of Croatia. “Cabernet Sauvignon” grapevines were grafted on SO4 rootstock, both being commonly used in all wine production areas in Croatia. Results obtained demonstrated that symbiotic grapevines, in general, expressed improved leaf gas exchange parameters and higher yield parameters, especially regarding the number of clusters per vine. It should be emphasized that mycorrhizal fungi affected higher total flavan-3-ols, total anthocyanins, and total polyphenols in berry skin in both experimental years. Despite variation in some yield parameters, generally, it is possible to obtain higher yields together with the improved phenolic composition of grapes.

The present study was conducted to establish a protocol for the regeneration of virus-free garlic plants through somatic embryogenesis of two Croatian garlic ecotypes. Basal parts of cloves from mother plants were cultured on a full Murashige and Skoog (MS) or modified MS medium (¼ of KNO3 and NH4NO3 and 2xMgSO4) containing 0.1 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D) or 1 mg L−1 2,4-D + 0.5 mg L−1 kinetin (Kin) and representing four different treatments. Plants were regenerated in MS medium containing 0.1 mg L−1 2,4-D and rooted in a medium containing 0.05 mg L−1 1-naphthaleneacetic acid (NAA) + 0.005 mg L−1 6-(γ,γ-dimethylallylamino)purine (2iP). The presence of viruses (i.e., sanitary status) of the mother plants and regenerants was checked by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). The mother plants were infected with onion yellow dwarf virus (OYDV) and leek yellow stripe virus (LYSV). In addition, the presence of garlic common latent virus (GCLV) was confirmed in four mother plants. Embryogenic callus developed in all four treatments with success ranging from 55% to 81% depending on treatment and ecotype. Plant conversion was significantly higher in somatic embryos developed in media containing 0.1 mg L−1 2,4-D than those developed in media containing 1 mg L−1 2,4-D + 0.5 mg L−1 Kin. Virus elimination success ranged from 13.3% up to 62.5% depending on garlic ecotype and treatment. The overall rate of virus elimination by somatic embryogenesis for both treatments and ecotypes were 20.7%, 22.9%, and 30.5% for OYDV, GCLV, and LYSV, respectively. Based on these results, somatic embryogenesis has been shown to be equally or more successful in eliminating garlic viruses compared to other in vitro methods.

In the era of sustainable grapevine production, there is a growing demand to define differences between Vitis vinifera varieties in susceptibility to downy mildew. Croatia, as a country with a long tradition of grapevine cultivation, preserves a large number of native grapevine varieties. A leaf disc bioassay has been conducted on 25 of them to define their response to downy mildew, according to the International Organisation of Vine and Wine (OIV) descriptor 452-1, together with the stress response of the leaf discs using chlorophyll fluorescence and multispectral imaging with 11 parameters included. Time points of measurement were as follows: before treatment (T0), one day post-inoculation (dpi) (T1), two dpi (T2), three dpi (T3), four dpi (T4), six dpi (T5), and eight dpi (T6). Visible changes in form of developed Plasmopara viticola (P. viticola) sporulation were evaluated on the seventh day upon inoculation. Results show that methods applied here distinguish varieties of different responses to downy mildew. Based on the results obtained, a phenotyping model in the absence of the pathogen is proposed, which is required to confirm by conducting more extensive research.

In modern agriculture, besides providing high and stable yields, it is imperative to produce products with a high nutritive quality. The goal of this study was to determine the effect of different fertilization regimes on the macro- and micronutrients in beetroot. A 3-year field trial was set up according to a Latin square method with four types of fertilization (unfertilized control, 50 t stable manure ha-1, and 500 and 1,000 kg NPK 5-20-30 ha-1). The mineral content was determined as follows (mg 100 g-1 in fresh weight of beetroot): 14-29 P, 189-354 K, 18-34 Ca, 17-44 Mg, 0.67-1.83 Fe, 0.41-0.65 Mn and 0.28-0.44 Zn. The highest beetroot P content was determined for the treatment with stable manure, especially in a year with dry climatic conditions. The highest beetroot K content was determined for the treatment with 1,000 kg NPK 5-20-30 ha-1, but at the same time for the same treatment, a general decreasing trend of micronutrient content was determined, due to the possible antagonistic effect of added potassium. For better mineral status of beetroot, application of combined mineral and organic fertilizers supplemented with additional foliar application of micronutrients can be suggested.

In Croatia, the majority of the common bean production is based on local landraces, grown by small-scale farmers in low input production systems. Landraces are adapted to the specific growing conditions and agro-environments and show a great morphological diversity. These local landraces are in danger of genetic erosion caused by complex socio-economic changes in rural communities. The low profitability of farms and their small size, the advanced age of farmers and the replacement of traditional landraces with modern bean cultivars and/or other more profitable crops have been identified as the major factors affecting genetic erosion. Three hundred accessions belonging to most widely used landraces were evaluated by phaseolin genotyping and microsatellite marker analysis. A total of 183 different multi-locus genotypes in the panel of 300 accessions were revealed using 26 microsatellite markers. Out of 183 accessions, 27.32% were of Mesoamerican origin, 68.31% of Andean, while 4.37% of accessions represented putative hybrids between gene pools. Accessions of Andean origin were further classified into phaseolin type II (“H” or “C”) and III (“T”), the latter being more frequent. A model-based cluster analysis based on microsatellite markers revealed the presence of three clusters in congruence with the results of phaseolin type analysis.