Leafy greens are susceptible to contamination by human pathogens, and because ready-to-eat salads cannot undergo thermal decontamination, even initial contamination poses a food safety risk. To understand early bacterial colonization, we combined high-resolution fluorescence microscopy with Generalized Additive Models (GAMs) to investigate development of Escherichia coli micro-colonies on the leaves of romaine lettuce (Lactuca sativa L. var. longifolia) and wild lettuce (Lactuca serriola L.). Green Fluorescent Protein labelled bacteria were used as experimental tracers to visualize colonization dynamics under controlled conditions. Bacterial proliferation was quantified through fluorescence area, representing spatial expansion, and fluorescence intensity, reflecting aggregation; their combined metric provided a reliable proxy for micro-colony development. Over the 24 h incubation period, romaine lettuce consistently supported higher colonization than wild lettuce, reflecting differences in leaf surface micro-morphology. Maximum surface roughness emerged as a structural factor associated with bacterial proliferation. GAMs captured nonlinear growth patterns and species-specific responses, with romaine models explaining up to 91% of the observed variance. Comparison with Colony Forming Units (CFU) counts showed that fluorescence imaging resolved early spatial aggregation and colony expansion, whereas CFU measurements assessed culturability but lost spatial information due to tissue homogenization. Although fluorescence imaging based on genetically labelled bacteria is not directly applicable to post-harvest monitoring, it provides a platform for establishing predictive relationships between leaf surface traits and microbial growth. GAMs represent the transferable outcome of the study, enabling quantitative evaluation of temporal and morphological predictors of colonization dynamics and supporting approaches for improving microbiological safety in fresh-cut salad production systems.

Hyoseris radiata L., a Mediterranean wild edible species (WEP), was evaluated for the first time under hydroponic cultivation as microgreens and baby leaves. Plants were grown from wild-collected seeds using full- or half-strength Hoagland nutrient solutions (H and H/2). Growth and yield were assessed at each developmental stage, while chemical composition was analyzed across all stage × nutrient combinations. Wild plants were also analyzed as a reference for comparison. No significant differences in growth or yield were observed between H and H/2 treatments, suggesting the feasibility of reducing nutrient inputs for the hydroponic cultivation of this species without compromising productivity. Chemical composition was generally consistent with that reported for other leafy WEPs and vegetables. PCA clearly separated microgreens, baby leaves, and wild plants. Wild samples showed higher concentrations of phenols, flavonoids, soluble sugars, and iron, and substantially lower nitrate levels than cultivated plants. Baby leaves were characterized by higher chlorophyll content, vitamin B6, vitamin C, and iron than microgreens. The overlap between H and H/2 treatments indicated that developmental stage had a stronger influence on chemical composition than nutrient solution strength. Based on EDI%, cultivated H. radiata, especially baby leaves, proved to be a valuable source of vitamin A, vitamin B9, iron, and molybdenum. No concerns related to heavy metal accumulation were detected. Nitrate concentrations were higher in cultivated plants but were significantly reduced in baby leaves grown under H/2. Overall, H. radiata showed good productivity under reduced nutrient supply and promising nutritional quality, supporting its potential as a novel hydroponically grown leafy vegetable.

Duckweeds, such as Lemna minor L., are invasive aquatic species that can proliferate on the surface of the nutrient solution in hydroponic systems, requiring removal operations from the cultivation tanks and disposal as waste. Several studies have demonstrated the potential use of duckweeds as an organic fertilizer. Recycling plant waste as a nutrient source for crops may be a circular approach to enhancing the sustainability of intensive horticultural production systems. Two pot experiments were carried out to evaluate the possibility of using the biomass of Lemna as a fertilizer for lettuce. The following fertilization treatments were applied: Control (no fertilization), Lemna biomass (60, 120, and 180 kg ha−1 nitrogen), urea (60 kg ha−1 nitrogen), and commercial organic fertilizer (60 kg ha−1 nitrogen). Lettuce head diameter, fresh and dry weight, the number of leaves, and the contents of minerals, nitrates, chlorophyll and carotenoids were determined. In addition, nitrogen use efficiency was calculated. Fertilization with Lemna resulted in a significant increase in yield compared to control (+50% considering the average of the three Lemna doses) and both inorganic (+65%) and organic (+71%) fertilization treatments. No differences in yield and quality were observed between the three doses of Lemna, but the lowest one was the treatment with the best performance in terms of N productivity. These results suggest that Lemna biomass may be a proper source of nutrients for lettuce with advantages for yield and no effect on quality. Therefore, its use as an alternative to commercial fertilizers can allow farmers to profitably exploit a waste product and, at the same time, reduce the costs for fertilization, thus achieving environmental and economic benefits.

This study evaluates the use of biochar as a sustainable substitute to peat in the soilless cultivation of rocket salad (Eruca vesicaria (L.) Cav.). Biochar was added to a peat-based substrate at concentrations of 0% (control), 5%, 10%, 20%, 40%, and 70% v/v to assess its effects on seed germination, plant growth, mineral content, and nitrate accumulation. The results show that biochar concentrations up to 40% v/v maintained germination rates above 80%, similar to the control, while higher concentrations (70% v/v) drastically reduced germination to 29% and entirely compromised plant development and growth. A moderate biochar concentration (20%) had a positive effect on fresh weight and leaf area, while maintaining comparable levels of nutrient uptake, chlorophyll, and flavonols. In addition, biochar-enriched substrates (≥20% v/v) reduced nitrate accumulation in leaves by 26–30%, addressing a critical quality and safety concern. A high biochar content (≥40% v/v) altered the substrate’s physicochemical properties, including pH, porosity, and electrical conductivity, negatively affecting plant growth (a 38% reduction in plant growth and 42% in leaf area) and increasing heavy metal concentrations, such as that of zinc (~30%). These findings suggest that incorporating up to 20% v/v biochar in soilless substrates offers a sustainable alternative to peat, supporting rocket salad performance and improving leaf nitrate quality, without compromising yield or safety.

Reusing treated wastewater (TWW) in agriculture may reduce water use pressure. While TWW is often used for soil irrigation, its application in hydroponics remains limited. In these systems, TWW can serve as a source of nutrients for plants while also being further reclaimed. We evaluated two TWWs of different origin and composition for hydroponic rocket cultivation. Each TWW was tested in its native form (TWW1 and TWW2) and after dilution and supplementation with mineral salts (TWW1_DH and TWW2_DH), using a Hoagland nutrient solution as a control. Yield and qualitative aspects of the product, including health risk factors (nitrates and heavy metals), were assessed. Rocket grown in TWW1 reached the harvesting stage, but with a significant yield reduction compared to the control (−40%). In TWW2, plants reached only the cotyledon stage and were not harvested. Two harvests were obtained in TWW1_DH and TWW2_DH, with yields comparable to the control or even significantly higher (+25%) in the first harvest in TWW1_DH. No health concerns were detected, with values of Health Risk Index < 1 for all the heavy metals and nitrate levels (~3000 mg kg−1 FW) well below EU limits. The study highlights the potential of TWW for the hydroponic cultivation of rocket, but also highlights the need to tailor its use based on composition.

The impact of urban gardens on food production and nutrient supply is widely recognized in the literature but seldom quantified. In this paper, we present the results of a semi-structured interview conducted in the 'social gardens' of Prato (Italy), i.e. areas of land assigned by the Municipality to individual pensioners or unemployed people for the cultivation of vegetables intended for domestic consumption. Some demographic and socio-economic aspects, the cultivated crops and the related areas were investigated. Starting from the areas, the total production of vegetables and their minerals and vitamins contents were estimated. The typical gardener was male, retired, with an average age of 74, and a low level of education. Gardening enabled pensioners to utilize their free time, facilitated physical activity, promoted socialization, and stimulated self-esteem. A 50 m2 plot cultivated on 40% of the area produced an estimated amount of 90 kg of vegetables per year, equivalent to approximately 61.5% of a person's fruit and vegetable needs. Tomato, by far the predominant species, occupied more than 80% of the cultivated area. The highest contributions to nutrients intake concerned Vitamin C and Vitamin A, the lowest Ca and Na. A higher yield and a greater and more balanced nutrient supply could be easily obtained through better use of the land (reduction of uncultivated area and greater assortment of vegetables). In our view, raising gardeners' awareness of this aspect and involving them in training programs on agricultural practices, vegetables composition, and nutrition, could be helpful for increasing the nutrient productivity of the plots and, ultimately, for strengthening the productive function of social gardens.

IntroductionThe non-thermal plasma (NTP) technique has been suggested as a sustainable horticultural practice to promote biomass accumulation, nutrient uptake, N metabolism, and disease prevention in plants. In particular, the potentiality of this technique to promote the natural accumulation of nutrients into plants deserve to be explored as input saving is strongly recommended in the horticultural sector.MethodsThe nutrient solution supplied to a red coloured variety of rocket salad [Diplotaxis tenuifolia (L.) DC. ‘Dragon’s Tongue’] grown in a hydroponic close loop system was treated with NTP. Low, medium, and high concentrations of N (i.e., 1, 10, and 20 mM) of the nutrient solution were tested in control (no NTP) or NTP treated conditions in two consecutive growing cycles. Results and discussionResults highlighted a N-dependent effect of NTP treatment showing a biomass stimulation at 10 mM N while negative effects of this technique at 1 and 20mM N. The biomass boosting of NTP found at 10 mMN coupled with an increase in K and Zn showing positive effects also on the nutraceutical aspects. Interestingly, different mechanisms seemed to be involved in the detrimental effects found at low and high N levels, i.e., a lower sensibility to N deficiency at 1 mM and a synergic negative effect of N and NTP in promoting oxidative stress at 20 mM.

Forestry-waste biochar was tested as a commercial substrate (peat:lapillus 1:1 v/v) amendment in growing tomatoes (Solanum lycopersicum L.). Substrates were 0% (control), 5%, 10%, 20%, and 40% (% v/v) biochar-enriched and were characterized for their textural and physicochemical properties. After harvesting, tomato production (i.e., plant and fruits), quality (e.g., nutrition and nutraceutics), and safety (i.e., biochar-related pollutants) were assessed according to the different growing media. 10-to-40% biochar-enriched substrates only exceeded the pH threshold set by L.D. 75/2010. Ni and Mn exhibited a similar trend between substrates and fruits, while Cr, Pb, and Cd were absent. Plant biomass increased (up to 11-29%) according to biochar content, which conversely diminished fruit production (similar to 25-60% reduction). Only acenaphthene exhibited an increasing profile (11-12 mu g kg(-1)) according to the treatments, nevertheless complying with the European regulations. PLS-DA confirmed practice suitability by substrate-crop correlation, providing prediction models for quality and safety assessment.

Recycling plant waste as nutrients source for crops represents a circular approach to enhancing the sustainability of intensive horticultural production systems. Lemna minor L. is an invasive aquatic species which can proliferate on the nutrient solution in hydroponic systems requiring removal operations from the cultivation tanks. This study evaluated the possibility of using the biomass of this weed as an organic fertilizer for lettuce. The following fertilization treatments were applied: Control (0 kg N ha-1), Lemna (60, 120, and 180 kg N ha-1), Urea (60 kg N ha-1), commercial organic fertilizer (60 kg N ha-1). Fertilization with Lemna showed similar or better results than control and both inorganic and organic fertilization treatments. No differences in yield and quality were observed between the three doses of Lemna but L60 resulted to be the treatment with the best performance in terms of N productivity. These results suggest that Lemna biomass may be a proper source of nutrients for lettuce with advantage for yield and no effect on quality. Therefore, its use as an organic fertilizer can allow farmers to profitability exploit a waste product and, at the same time, reduce the costs of purchasing commercial fertilizers, thus achieving environmental and economic benefits.

Alstroemeria spp. and carnation (Dianthus caryophyllus L.) have considerable and increasing economic importance in the floriculture market, therefore breeders carry out intense breeding programs to select new superior varieties. However, poor germination of hybrid seeds remains a bottleneck. Based on this assumption, seed pre-treatments and in vitro germination protocols, using different germination substrates, were applied in Alstroemeria spp. and carnation to improve germinability. Seed viability was tested using the 2,3,5-triphenyl tetrazolium chloride (TTC) test, and resulted in 91.10% ± 2.33 and 86.66% ± 3.85 in Alstroemeria and carnation, respectively. In Alstroemeria, pre-treatment with potassium nitrate (KNO3) in combination with modified ½ Murashige and Skoog (MS) medium ensured high germination uniformity combined with high germination percentage, showing significantly higher values than the control. In carnation, a suitable seed sterilization procedure was set up (up to 95.8% sterility); treatments with gibberellic acid (GA3) and KNO3 did not influence germination percentage compared to the control. A high multiplication rate of seedling lines was obtained on hormone-free MS medium.

BACKGROUND:The first step in the contamination of leafy vegetables by human pathogens is their attachment to the leaf surface. The success of this is influenced strongly by the physical and chemical characteristics of the surface itself (number and size of stomata, presence of trichomes and veins, epicuticular waxes, hydrophobicity, etc.). This study evaluated the attachment of Salmonella enterica to 30 baby-leaf salads and tested whether the differences found among them were related to the following leaf traits: hydrophobicity, roughness, and epicuticular waxes. RESULTS:Differences in susceptibility to contamination by S. enterica were found between the 30 baby-leaf salads investigated. The lowest attachment was found in wild lettuce (Lactuca serriola L.) and lamb's lettuce 'Trophy F1' (Valerianella locusta [L.] Laterr.), with values of 1.63 ± 0.39 Log(CFU/cm2) and 1.79 ± 0.54 Log(CFU/cm2), respectively. Attachment was correlated with hydrophobicity (measured as contact angle) (r = -0.39) and epicuticular waxes (r = -0.81) but not with roughness (r = 0.24). The most important wax components for attachment were alcohols and, in particular, the three-dimensional (3D) wax crystals of C26 alcohol, but fatty acids probably also had a role. Both these compounds increased hydrophobicity. The presence of thymol, whose antimicrobial properties are well known, was found in lamb's lettuce. CONCLUSIONS:The findings of this study can help to predict and control the attachment and contamination of leafy salads by enterobacteria. They also provide useful information for breeding programs aiming to develop cultivars that are less susceptible to human pathogens, enhancing the food safety of vegetables. © 2024 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

BACKGROUND: Despite the optimal characteristics of peat, more environmental-friendly materials are needed in the nursery sector, although these must guarantee specific quantitative and qualitative commercial standards. In the present study, we evaluated the influence of biochar and compost as peat surrogates on yield and essential oil profile of two different varieties of basil (Ocimum basilicum var. Italiano and Ocimum basilicum var. minimum). In two 50-day pot experiments, we checked the performances of biochar from pruning of urban trees and composted kitchen scraps, both mixed in different proportions with commercial peat (first experiment), and under different nitrogen (N) fertilization regimes (second experiment), in terms of plant growth and volatile compounds profile of basil. RESULTS: Total or high substitution of peat with biochar (100% and 50% v.v.) or compost (100%) resulted in seedling death a few days from transplantation, probably because the pH and electrical conductivity of the growing media were too high. Substrates with lower substitution rates (10-20%) were underperforming in terms of plant growth and color compared to pure commercial peat during the first experiment, whereas better performances were obtained by the nitrogen-fertilized mixed substrates in the second experiment, at least for one variety. We identified a total of 12 and 16 aroma compounds of basil (mainly terpenes) in the two experiments. Partial replacement of peat did not affect basil volatile organic compounds content and composition, whereas N fertilization overall decreased the concentration of these compounds. CONCLUSION: Our results support a moderate use of charred or composted materials as peat surrogates.(c) 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Understanding the relation between the susceptibility of different leafy greens to human pathogen contamination and leaf traits can contribute to increase the food safety of the fresh vegetable industry. The aim of this research was to evaluate the susceptibility to E. coli ATCC 35218 attachment in 30 accessions of baby leaves, and to identify leaf traits potentially involved in the contamination. The accessions were surface inoculated with a bacterial suspension containing 1 × 107 cells/mL and the attachment was measured 1.5 h after inoculation. Significant differences in attachment were detected between the accessions for p ≤ 0.05. The three most and the three least susceptible accessions were selected and characterized for leaf micro-morphological traits (stomata density and size, surface roughness) and water content. Scanning electron microscopy was used to analyse the stomatal parameters. Roughness was measured by an innovative portable 3D digital microscope. No significant correlation between the attachment of E. coli ATCC 35218 and stomatal parameters was detected, while the attachment was positively correlated with roughness and water content. The E. coli ATCC 35218 population in surface-inoculated leaves was also measured after a UV treatment, which was found to be less effective in reducing bacterial contamination in the rougher leaves. This result suggested that roughness offers UV protection, further highlighting its impact on the microbiological safety of baby leafy greens.

BACKGROUNDPathogenic enterobacteria can travel through the plant vascular bundles by penetrating from cuts and persisting into ready-to-eat leafy greens. Because the cutting site is the main point of entrance and uptake, we tested how different cutting strategies can reduce bacterial internalization in leaves. Horizontal cuts at the base of the leaves were performed with two different types of tools: the first with a scalpel (by pulling the blade) and the second with a scissor-action that has blades that cuts by gliding against a thicker blade. Scissor-action generally makes closer border cuts. Blades of both types of tools have worked at 25 degrees C and 200 degrees C. The present study aimed to determine how these different types of cuts and temperatures affected bacterial uptake in leaves. Experiments were repeated on different plant genotypes and at different wilting stages. RESULTSOur findings showed that cutting baby-leaves with a scissor action at 200 degrees C significantly reduced the bacterial uptake compared to the not heated (which simulates a mechanized lettuce harvester). The most effective cutting treatments for reducing bacterial uptake were in the order: scissor 200 degrees C > scissor 25 degrees C > scalpel 200 degrees C > scalpel 25 degrees C. The scissor heated at 200 degrees C also prevented bacterial uptake on wilted baby-leaves. CONCLUSIONThe findings of the present study could provide a further contribution in terms of safety during harvest and suggest that a pre-heated blade supports safety during harvest of leafy greens. (c) 2022 Society of Chemical Industry.

By the year 2050, it is predicted that there will be 10 billion people on the planet, and along with this population growth, the need for food production will dramatically rise [...]

Wild edible leafy plants, thanks to their organoleptic characteristics and nutritional value that can make them be appreciated as salads by consumers, represent a good opportunity for growers and the fresh-cut industry, which are always looking for new crops to expand the number of products they offer. In this study, four wild species (dandelion, sorrel, wild chicory, and wild lettuce) were cultivated hydroponically up to the baby leaf stage in order to evaluate them as potential crops. At harvest, yield and antioxidant compounds, minerals, and nitrates content were assessed. The contribution to human mineral intake and the possible health risk associated with heavy metals were investigated. A characterization of the sensory profile was also carried out. Yield and chlorophylls and carotenoids content of the investigated species were comparable to those of common leafy vegetables. Variability in nitrate content was observed, with the lowest value in sorrel and the highest in dandelion. All species could contribute in Cr, Mg, and Se intake, and health risks due to heavy metals were excluded. Each species was well characterized by distinctive and peculiar sensory notes. In conclusion, the results of this preliminary study suggest that the four wild investigated species may be promising for baby leaf production.

The aim of this study was to assess whether selection breeding in chicory (Cichorium intybus L.) led changes in the susceptibility to Salmonella enterica and Escherichia coli contamination and whether the anatomical traits of the leaves are involved in the possible changes. Five chicory genotypes subjected to different intensities of selection were compared at the microgreen stage. Bacterial retention was evaluated after leaf incubation for 1.5 h on the surface of the bacterial suspension, followed by rinsing, grinding, plating on selective media, and colony forming unit (CFU) counting. The density of stomata and trichomes, total stomatal length and width, stomatal pit width, surface roughness and sharpness were evaluated. The intensively selected genotype (Witloof) was significantly more prone to contamination (2.9 +/- 0.3 lg CFU/cm(2)) as the average of the two bacterial types than the wild accession (Wild; 2.3 +/- 0.4 lg CFU/cm(2)) and the moderately selected genotypes (two leaf chicories, Catalogna type, and root chicory 'Magdeburg'; on average, 1.9 +/- 0.3 lg CFU/cm(2)). Witloof microgreens also showed larger stomata (on average +34% for stoma width and +44% for pit width), which could justify, at least in part, the higher susceptibility to enterobacterial contamination. In fact, when contamination was performed in the dark (closed stomata), the bacterial retention in Witloof was significantly reduced in comparison with the opened stomata (-44%) and in Wild (-26%). Differences in retention between Witloof and Wild were still observed after UV treatment. The hierarchical clustering performed by grouping the leaf anatomical features was consistent with the chicory genetic groups. Our results suggest that the domestication process can affect the safety of produce and that the micromorphological traits of the leaves may be involved.

This paper provides an overview of wild food plants traditionally used in the gastronomy of Tuscany, an Italian region with high biological diversity and whose cultural heritage is well known. Forty-nine bibliographic sources, including five unpublished studies, were reviewed. A list of species with ecological characteristics, plant parts used, use category (food, liquor, or seasoning), methods of preparation (raw or cooked), and recipes is presented. The use of 357 taxa (3711 use reports, URs), was recorded, belonging to 215 genera and 72 botanical families. Over the total taxa, 12 are new for Tuscany, 52 seem not to be present in other Italian regions, and 54 were not detected in the consulted European ethnobotanical literature. Of these taxa, 324 (3117 URs) were used as food, while 49 (178 URs) and 81 (416 URs) were used for liquor and seasoning, respectively. Of the 17 different food recipes, cooked vegetables constituted the largest group, followed by salads, omelets, snacks, and fillings. The chemical composition of the recorded food plants and the possible safety risks associated to their consumption, as well as their traditional medicinal use, are also shown. This review highlights the richness of ethnobotanical knowledge in Tuscany. Such biocultural heritage can be a “source of inspiration” for agriculture. As a reservoir of potential new crops, wild edible flora may contribute to the development of emerging horticultural sectors such as vertical farming and microgreens production. Moreover, the nutrient content and healthy properties of many wild food plants reported in this study has the ability to meet consumer demand for functional foods.

One of the major challenges for nursery growers is replacing peat with more sustainable materials that may also represent a worthy strategy to recycle organic refuse. Among organic matrices, coconut coir dust, green compost, and stabilized wood fibre are considered promising alternative to peat because of their suitable physical-chemical characteristics. These matrices were used to prepare substrate mixtures designed to achieve physical characteristics similar to those of the standard peat-based substrates usually adopted by growers (peat:pumice, 70:30 v v−1). An experiment was carried out in open field with Leucanthemum vulgare Lam., a plant species that could be used for ecological restoration, by using both the native and the ornamental variety of the same species (L. vulgare cv. ‘Filigran’), the latter as test plants. Six different growing media were tested: (i) peat:pumice 70:30 v v−1 (PP), used as commercial control; (ii) coconut coir dust:pumice, 70:30 v v−1 (CP); (iii) coconut coir dust:green compost 55:45 v v−1 (CGC); (iv) coconut coir dust:stabilized wood fibre 60:40 v v−1 (CW); v) green compost:stabilized wood fibre 30:70 v v−1 (GCW) and, (vi) coconut coir dust:green compost:stabilized wood fibre 40:30:30 v v−1 (CGCW). Plant biomass, biometric parameters, plant and matrix/substrate mineral content, and mineral composition of water drained out from pots were measured as main performance indicators of plants and growing media. All the tested peat-free substrates, with the exception of GCW, adequately supported plant growth and quality. Moreover, CP and CGCW improved some of the investigated biomass and biometric parameters compared with the control treatment. Substrates containing green compost were found to improve plant nutrition, because of the high availability of mineral elements in the raw material.

Vegetable produce can host human pathogens without exhibiting any sign of spoilage, raising concerns about food safety. Over the production chain, vegetables can be exposed to different routes of contamination to enteric pathogens, including during cultivation and at harvest. For this reason, foodborne disease control should start in the field. Agronomic practices, that have been used for long time to control the spread of phytopathogens in crops, are here reviewed in light of their contribution for limiting pre- and post-harvest contamination and proliferation of human pathogenic Enterobacteriaceae in vegetables. Based on up-to-date literature, this review aims to highlight the agronomic practices able to support produce safety: i) Variability in susceptibility to Enterobacteriaceae detected within vegetable species raises perspectives for selecting tolerant cultivars and grafting on tolerant rootstocks. ii) Soil solarization protects plants from Enterobacteriaceae contamination. iii) Correct fertilization and irrigation, including the use of safe fertilizers and water, as well as avoiding excess of nitrogen and water, are important, and hydroponics usually implicates a more controlled environment. iv) Pesticide and biocontrol can limit Enterobacteriaceae. v) Harvest not only requires hygienic conditions, but can be also modulated in relation to maturity stage and climatic conditions.