Garlic Ecotypes Utilise Different Morphological, Physiological and Biochemical Mechanisms to Cope with Drought 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.

Monitoring Drought Stress in Common Bean Using Chlorophyll Fluorescence and Multispectral Imaging.

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.

Study of High-Temperature-Induced Morphological and Physiological Changes in Potato Using Nondestructive Plant Phenotyping.

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.

Classification of high-throughput phenotyping data for differentiation among nutrient deficiency in common bean.

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.

Crop breeding for a changing climate in the Pannonian region: towards integration of modern phenotyping tools.

The Pannonian Plain, as the most productive region of Southeast Europe, has a long tradition of agronomic production as well as agronomic research and plant breeding. Many research institutions from the agri-food sector of this region have a significant impact on agriculture. Their well-developed and fruitful breeding programmes resulted in productive crop varieties highly adapted to the specific regional environmental conditions. Rapid climatic changes that occurred during the last decades led to even more investigations of complex interactions between plants and their environments and the creation of climate-smart and resilient crops. Plant phenotyping is an essential part of botanical, biological, agronomic, physiological, biochemical, genetic, and other omics approaches. Phenotyping tools and applied methods differ among these disciplines, but all of them are used to evaluate and measure complex traits related to growth, yield, quality, and adaptation to different environmental stresses (biotic and abiotic). During almost a century-long period of plant breeding in the Pannonian region, plant phenotyping methods have changed, from simple measurements in the field to modern plant phenotyping and high-throughput non-invasive and digital technologies. In this review, we present a short historical background and the most recent developments in the field of plant phenotyping, as well as the results accomplished so far in Croatia, Hungary, and Serbia. Current status and perspectives for further simultaneous regional development and modernization of plant phenotyping are also discussed.

Control of Early Blight Fungus (Alternaria alternata) in Tomato by Boric and Phenylboronic Acid.

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.

Detection, Transmission, and Characterization of Grapevine Virus H in Croatia.

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.

Multispectral Assessment of Sweet Pepper (Capsicum annuum L.) Fruit Quality Affected by Calcite Nanoparticles.

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., Soroksari 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 Soroksari, 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 Soroksari, 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 Soroksari, the opposite effects were observed.

Screening of Croatian Native Grapevine Varieties for Susceptibility to Plasmopara viticola Using Leaf Disc Bioassay, Chlorophyll Fluorescence, and Multispectral Imaging.

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.

Genome-Wide Association Studies of Mineral Content in Common Bean.

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.

Application of Phenotyping Methods in Detection of Drought and Salinity Stress in Basil (Ocimum basilicum L.).

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 (HUE2 D ), and higher reflectance in red (R Red ), reflectance in green (R Green ), and leaf inclination (LINC) indicated drought stress. At later stress stages, maximum fluorescence (F m ), HUE2 D , 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.

Physiological Responses of Basil (Ocimum Basilicum L.) Cultivars to Rhizophagus Irregularis Inoculation under Low Phosphorus Availability.

Arbuscular mycorrhizas (AM) can improve phosphorus (P) nutrition and could serve as an environmentally friendly approach for sustainable crop production under P-limiting conditions. The objectives of this study were to assess the effect of AM on different physiological traits and to quantify the responsiveness of different basil (Ocimum basilicum L.) cultivars to AM under low P availability. The basil cultivars 'Genovese', 'Sweet Basil', 'Dark Opal', and 'Erevanskii' were inoculated (AMI) using Rhizophagus irregularis. Photochemical efficiency and gas exchange were measured on AMI and non-inoculated (AMC) plants and, at harvest, the shoot biomass, shoot P concentration, root morphological traits, frequency of mycorrhizas in the roots (F%), and extent of root colonization (M%) were determined. Significant differences in F% and M% were found among the examined cultivars, with the highest found in 'Dark Opal' and the lowest in 'Erevanskii'. AMI reduced the shoot biomass and increased the shoot P concentration as well as other examined root traits in 'Genovese' and 'Erevanskii', whereas it did not affect those traits in 'Dark Opal' and 'Sweet Basil', indicating differences in responsiveness to AM. AMI positively affected the gas-exchange parameters in all examined cultivars, probably due to the increased sink capacity of a bigger root system and/or AM structures within the roots.

Beetroot mineral composition affected by mineral and organic fertilization.

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.

Genetic Diversity of Croatian Common Bean Landraces.

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.