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ARTICLE   Open Access    

A study on chilling hardiness of three persimmon genotypes by electrolyte leakage parameter

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  • Most fruit trees in temperate regions are exposed to chilling, which causes extensive economic losses. Low temperatures reduce biosynthesis activity of plants and the functioning of physiological processes, impose irreparable injuries, and finally, destroy the plants. So, to study chilling tolerance of three genotype of persimmon by electrolyte leakage parameter, a factorial experiment was carried out in a randomized complete block design with three replications. The first factor was assigned to persimmon cultivar at three levels of date-plum, 'Fuyu kaki', and Japanese persimmon. The second factor was assigned to chilling treatment at two levels of 4 °C and −16 °C. The third factor was assigned to three phenological stages including bud dormancy phase, bud swelling phase, and anthesis phase. The recorded traits included electrolyte leakage, electrical conductivity (EC), and proline content. The results showed that proline content, electrolyte leakage and EC of all samples were the highest at bud swelling phase, but they did not differ significantly between bud swelling and anthesis stages. The highest electrolyte leakage and EC was observed in plants exposed to −16 °C and the lowest in those exposed to 4 °C. The membrane of date-plum at −16 °C and bud swelling phase was most heavily injured by chilling. Among the trilateral effects of studied factors on the recorded traits, 'date-plum × −16 °C × bud swelling phase' showed the highest proline content, electrolyte leakage, and EC, and 'date-plum × −16 °C × bud dormancy' and 'Japanese persimmon × −16 °C × bud dormancy stage' exhibited the lowest proline content, electrolyte leakage, and EC.
  • The Trichoderma genus encompasses a wide-ranging collection of filamentous fungi, prevalent in various natural ecosystems[1]. Trichoderma species within this genus have earned acclaim for their exceptional capacity to inhabit plant roots, stimulate plant growth, and showcasing biocontrol attributes against a spectrum of fungal adversaries[2]. Employing tactics like mycoparasitism, antibiosis, competitive resource acquisition, and plant resistance induction, these species effectively manage fungal diseases[3]. Notably, they are increasingly utilized in agriculture as biofertilizers and biopesticides[1]. Trichoderma-based bio-fungicides, available in different formulations like wettable powders, granules, and flowable concentrates, offer a convenient application to seeds, seedlings, soil, and foliage[4,5]. Besides their disease-fighting properties, these bio-fungicides promote plant growth through various mechanisms such as phytohormone production, nutrient solubilization, and stress tolerance enhancement[3]. Recent progress in Trichoderma-based formulations has led to innovative materials, advanced nanotechnology strategies, and genetic engineering techniques aimed at boosting stability, shelf life, and efficacy[4]. Among these advancements, biochar has shown promise as an ideal carrier for Trichoderma formulations due to its high porosity, surface area, and soil stability maintenance abilities[6]. New research indicates that biochar can strengthen Trichoderma's biocontrol properties[7,8]. Experiments show that using Trichoderma bio-fungicides on soil blended with biochar is more effective in fungal disease control than on unamended earth[9]. Likewise, applying these bio-fungicides on biochar-coated seeds provides better resistance against fungal diseases in seedlings[7,10]. Such biotechnological advancements in Trichoderma-based formulations can promote sustainable agricultural practices by reducing reliance on chemical pesticides[11]. This, in turn, helps mitigate the ecological impact of agricultural activities and enhances food and feed safety[12]. By protecting plants from fungal diseases and improving soil fertility, Trichoderma-based bio-fungicides hold promise for enhancing crop yield[1]. Trichoderma formulations play a crucial role in minimizing harm to non-target organisms while maximizing the effectiveness of the active ingredient[13]. While Trichoderma is significant in ensuring agronomic safety, challenges in their formulation persist due to potential degradation of the biomass or bioactive metabolite caused by factors like exposure to air, light, and temperature[14]. Additionally, these products need to be easy to handle, apply, and produce[15,16]. To address this objective, the present study aims to offer a comprehensive examination of various technological advancements that enhance the efficiency of natural preparations. Distinguishing itself from typical literature reviews that predominantly delve into the biological attributes of metabolites, this review incorporates a bibliometric analysis of biopesticides and their formulations[17]. This analysis employs quantitative and statistical indicators to identify patterns related to the most critical pest issues, agriculture's susceptibility, sources of biological control, innovative methodologies, and the current status of Trichoderma formulations. The insights presented in this analysis significantly contribute to the bibliometric methodology, potentially promoting positive strides in the advancement of technology for Trichoderma formulation. Additionally, it offers valuable suggestions for researchers engaged in this field.

    Bibliometric analysis is a technique employed to scrutinize the characteristics and evolving patterns within academic literature using various mathematical and statistical methods[10]. Through this approach, we can quantitatively assess the overall state of the literature, collaborative relationships, research areas of interest, and the development trends in a specific research field[18]. A descriptive analysis of the corpus of published research pertaining to Trichoderma formulations was conducted. This analysis entailed the examination of co-occurring terms within the body of published articles, allowing for the elucidation of evolutionary trends in scientific themes[19]. The fundamental aim of this research is to conduct an exhaustive review of the existing body of literature on Trichoderma formulations and to project the areas of highest interest and potential for future investigation[20]. One of the primary objectives of bibliometric analysis is to assess the trends in research related to Trichoderma formulations, and to identify the most influential authors and institutions in the field of Trichoderma research. Determining the impact of research in terms of citations, patents, or applications in real-world scenarios.

    As a result, this study seeks to investigate the following research objectives: In the field of Trichoderma formulations, what are the key research themes and trends observed from 2016 to 2023 include:

    (1) How is research on Trichoderma formulations distributed geographically, and what regions exhibit the most active contributions to the field? (2) Can bibliometric analysis predict future trends and potential innovations in Trichoderma formulations research based on historical patterns? (3) The article follows a well organised structure[21]. Initially the research methodology adopted for the study is outlined. Subsequently, a well-organized article is crucial for effectively communicating research findings to the intended audience[22].

    Literature retrieval was performed online through the Science Citation Index Expanded (SCI-E) of the Web of Science Core Collection (WoSCC, Clarivate Analytics) from 2016 to 2023[21,22]. Scopus is a preferred data source for bibliometric analysis, and it provides comprehensive information and data from a multi-disciplinary field of literature[23]. To retrieve literature comprehensively and accurately on Trichoderma formulations, different search terms and retrieval strategies were assembled in this study. Finally, the optimal search items were set as follows: TS = ('Trichoderma formulation*') OR ('Bio formulation of Trichoderma*') OR ('Bio control') OR ('Antagonist') OR ('Rhizosphere fungus')[24]. The data range was set from 2016 to 2023, to collect all relevant publications. It is worth noting that as the Scopus database data network is constantly updated, the results may vary depending on the exact retrieval date.

    A detailed literature retrieval process was performed online through Science Citation Index Expanded (SCI-E) of the Web of Science Core Collection (WoSCC, Clarivate Analytics) from 2016 to 2023, and considered Scopus as a preferred data source for bibliometric analysis. Additionally, we outlined the search terms and retrieval strategies that are used in our study and set the range of data from 2016 to 2023 to collect all relevant publications. If we sum up our descriptions narrate on the following key points, like data sources, search terms, data range, constantly updated data base, and optimal search items[25]. The search strategy was designed to capture relevant literature on Trichoderma formulations. The search terms included Trichoderma formulation, bio-formulation of Trichoderma, biocontrol, antagonist and Rhizosphere fungus. The asterisks, in the search terms are used as wildcard characters to capture different word endings. The data range was set from 2016 to 2023 to collect all relevant publications within that timeframe. This was the period during which the literature retrieval was performed[25]. It is mentioned that as the Scopus database data network is constantly updated, and the results may vary depending on the exact retrieval date. This indicates that the study acknowledged the dynamic nature of the database and its potential impact on the results[26]. After assembling different search terms and retrieval strategies, the study determined the optimal search items, which were the selected search terms that would yield the most comprehensive and accurate results for the study's objectives. Overall, the present study took a systematic approach to literature retrieval, considering multiple data sources and employing a combination of search terms to ensure the retrieval of relevant publications on Trichoderma formulations. It is worth noting that as the Scopus database data network is constantly updated, to add upon, the results may vary depending on the exact retrieval date.

    To ensure the credibility of the research conclusions, this study gathered peer-reviewed English journal articles to summarize global research perspectives. It's important to mention that articles not aligned with this paper's purpose were manually omitted in the final phase of data collection[27]. For instance, some articles explored the relationship between plants and microorganisms on leaves. Eventually, a total of 287 articles that met all criteria were sourced from Scopus. These pieces represented almost all top-tier experimental studies on Trichoderma formulations from 2016 to 2023 worldwide. The variability among these articles could effectively indicate the trend of related research development. Therefore, these publications were prioritized for further analysis and assessment. Figure 1 illustrates the flowchart of the literature retrieved in this study[28].

    Figure 1.  Flow chart of literature review methodology.

    Table 1 presents the top 10 countries/regions, institutions, authors, and journals that published the most studies on Trichoderma formulations. As indicated in Table 1, China emerged as the country making the most significant contribution, with 1,231 publications, accounting for 20.33% of the total. India and Pakistan followed closely, ranking second and third, with 1,096 (18.10%) and 618 (10.20%) publications, respectively. Among the institutions, the Chinese Academy of Sciences held the top spot, boasting 160 (2.64%) publications. Following closely was the University of Agriculture, Faisalabad (144, 2.37%), and Nanjing Agricultural University (137, 2.26%). In the realm of scholarly contributions, prolific authors often set the tone for research trends. Identifying these influential scholars can shed light on the direction of the research field[29]. The leading author in the study of rhizosphere microorganisms was Wang Y, with 102 publications. Li Y, Zhang Y, and Hkan M were also highly prolific, each publishing nearly 90 studies. These works were predominantly featured in prominent journals in Ecology and Botany, such as Frontiers in Microbiology (3.73%), Frontiers in Plant Science (2.36%), and Plant and Soil (2.03%).

    Table 1.  Leading journals contributing to the existing body of knowledge in the field of formulation of Trichoderma.
    Sl. no. Name of journal No. of publication Citations
    1 Journal of Applied Microbiology 2 40
    2 Applied Microbiology and Biotechnology 2 16
    3 Indian Phytopathology 3 2
    4 Biological Control 2 59
    5 Crop Protection 2 30
    6 Frontiers in Microbiology 2 23
    7 Journal of Biological Control 2 1
    8 Medicinal Plants 2 5
     | Show Table
    DownLoad: CSV

    The analysis of scientific production on Trichoderma formulations demonstrated the trend of publications per year on Trichoderma formulation studies. It was observed that published research showed a significant increase of 71.24% over the last decade (2016–2023) (Fig. 1). The increasing trend is possibly related to economic support from government programs, since funding for innovative, sustainable, and ecological research is being considered to meet the demand for food and mitigate environmental pollution[30]. Figure 2 illustrates a co-citation map of authors collaborating in the field of Trichoderma formulation. The purpose of conducting this co-citation analysis is to visually portray the knowledge base of the specific area of review. The analysis identifies three distinct clusters, each represented by different colored nodes: blue, red, and green. The green cluster stands out as it is associated with Harman, who has the highest collaboration, working with nine researchers on Trichoderma formulation research. The red cluster, on the other hand, signifies the second-highest collaboration, led by Mukherjee et al.[31] with six researchers. Lastly, the pink cluster represents the lowest level of collaboration among researchers, with only two researchers working together in this area.

    Figure 2.  Co-citation analysis of cited authors as the unit of analysis in the field of Trichoderma formulation.

    Table 2 showcases the country–wise citation analysis, and the series presented here seems to have large variability in distribution. In terms of total citations of studies dedicated to Trichoderma formulations. Citations count of articles by country as a unit of analysis represents the popularity of a field of research in a particular region. India with 20 publications having 115 citations topped the list and, therefore, is the most impactful country contributing to the existing body of knowledge in the said domain followed by Italy with four publications having 69 citations and Brazil with three publications having 51 citations. From the viewpoint of the total number of publications, India holds first position, having 20 publications, followed by Italy having four publications.

    Table 2.  Leading countries contributing to the existing body of knowledge in the field of formulation of Trichoderma.
    Sl. no. Country No. of publication Citations
    1 Argentina 1 20
    2 Belgium 2 30
    3 Brazil 3 51
    4 China 2 82
    5 Croatia 1 30
    6 Finland 1 37
    7 India 20 115
    8 Italy 4 69
    9 New Zealand 2 21
    10 Portugal 1 67
    11 South korea 1 54
     | Show Table
    DownLoad: CSV

    The top researchers working in the field of Trichoderma formulation are presented in Table 2. The authors' citation count represents the recognition of their research work in a particular field of research. It is quite clear from the list of 13 author's citations that all the authors have at least 10 citations to their name based on the total citation count. Among the 13 authors Park et al.[32] has the highest number of citations of 57 followed by Herrera-Téllez et al.[33] having 47 and Hewedy et al.[34] having 44 citations. The analysis of bibliographic coupling in the Trichoderma formulation domain is represented in Fig. 3. This technique utilizes references from existing publications to elucidate the relevant literature[35]. For this study, five thematic clusters have been identified, labeled green, red, blue, yellow, and purple. Among these interconnected groups, India stands out as the country with the most extensive collaboration network, linked with 15 other countries. Given that India also holds the highest number of published documents (115), it was anticipated to be the central node in this cooperation network. The findings demonstrate the strong relationships between researchers and their respective institutional affiliations, emphasizing the scientific cooperation aimed at developing sustainable and ecologically sound strategies for crop protection in a competitive manner[36].

    Figure 3.  Bibliographic coupling of articles in the field of Trichoderma formulation.

    In Fig. 4, a co-occurrence analysis of keywords with a minimum threshold of five occurrences is displayed. The network illustrates the most frequently utilized terms within the 'Trichoderma formulation' research domain, capturing the essence of the article's core content. The prevalence of these keywords can be indicative of the research direction and content within this specific field[37]. This analysis allows for the identification of developmental trends within a field and a comprehensive understanding of its current research status[38]. The co-occurrence graph of keywords reveals a total of four co-occurrence clusters (Fig. 4), encompassing themes like biocontrol, formulation, Trichoderma, and shelf life. Each cluster is further examined below, providing an in-depth portrayal of the prominent topics within the Trichoderma formulations landscape during the research period. Annual publication number leading years contributing to the existing body of knowledge in the field of formulation of Trichoderma is presented in Table 3.

    Figure 4.  Co-occurrence analysis based upon keywords from articles in the field of Trichoderma formulation.
    Table 3.  Annual publication number leading years contributing to the existing body of knowledge in the field of formulation of Trichoderma.
    Sl. no. Year No. of publication
    1 2016 8
    2 2017 13
    3 2018 21
    4 2019 16
    5 2020 20
    6 2021 17
    7 2022 15
    8 2023 8
     | Show Table
    DownLoad: CSV

    The shift in annual publication counts serves as a vital benchmark for gauging the progress of a research field, lending insights into potential development trends[28]. Figure 4 provides a clear portrayal of the publication distribution in Trichoderma formulation from 2016 to 2023, illustrating a noticeable increase in annual article output. This surge suggests a heightened interest in the field over the past few years. Scientific research fields typically undergo a four-stage evolution[39] ; (1) the inception phase, characterized by the introduction of novel research areas or directions by notable scientists; (2) the expansion phase, where scientists gravitate toward the emerging research direction, leading to a proliferation of discussion topics; (3) the stabilization phase, marked by the amalgamation of new knowledge to form a distinct research context; and (4) the contraction phase, in which the number of new publications diminishes. Notably, the research on Trichoderma formulation seems to be currently in the expansion phase[40].

    The publication pattern reveals a growing research interest in Trichoderma formulations, a relatively new field that is attracting increasing enthusiasm among scholars. Notably, the top contributors to this area, as identified through VOS viewer analysis, include key individuals, organizations, sources, and countries. Park emerges as the leading author based on citation count, followed by Herrera-Téllez and Hewedy. China, Portugal, Italy, and South Korea are recognized as major contributors to research in this field, as reflected in their citation counts. Conversely, India leads in terms of document count, demonstrating a significant contribution to the literature.

    Co-citation and bibliographic coupling analyses have identified three distinct thematic clusters. In the co-citation analysis, these clusters relate to application methods, types of Trichoderma formulations, and their biocontrol efficacy. Additionally, insights from the bibliometric analysis of biopesticide formulations have facilitated the integration of methods and strategies aimed at enhancing the effectiveness of Trichoderma formulations.

    This paper conducts a bibliometric analysis to critically examine articles related to biological control, focusing specifically on those published in various indexed journals, and offers a comprehensive overview of the evolution of Trichoderma formulations over time. The primary objective of this research is to investigate and characterize the key literature on this topic, covering historical, current, and emerging developments in this dynamic field. Bibliometric techniques are employed to visualize the Trichoderma formulation landscape.

    To achieve this goal, the study analyses a dataset of articles obtained from the core collection databases of Scopus and Web of Science. Within the scope of this study, significant publications on Trichoderma formulations are meticulously reviewed to highlight the potential trajectory of Trichoderma's role in biological disease control in plants. The study identifies and examines the various developmental phases of Trichoderma formulations, offering a comprehensive analysis that can shape the future of this critical research area. Given the scarcity of comprehensive bibliometric studies on Trichoderma formulation research, this study seeks to fill this gap, making a significant contribution to the extensive readership interested in Trichoderma.

    This study has several limitations and challenges that must be considered by future researchers. First, the study relies on a single database, which could restrict the amount of available data. Additionally, the search criteria were limited to research articles, and only those with specific phrases in the title were included, which may not represent the complete dataset. However, Trichoderma's biological control mechanisms against plant fungal and nematode diseases involve various strategies, including competition, antibiosis, antagonism, and mycoparasitism. In addition to these, Trichoderma enhances plant growth and induces systemic resistance in plants, making it effective in controlling a wide range of plant fungal and nematode diseases[41]. Although biological control is effective, it generally requires time to become established in the environment, making it a slower process. Therefore, optimizing the formulation of Trichoderma-based products is essential to ensure their stability and efficacy. It is important to ensure that these formulations are compatible with other agricultural treatments, such as chemical fertilizers and pesticides, to maximize their overall effectiveness. Proper formulation can improve the shelf life, ease of application, and survival of Trichoderma under varying environmental conditions. Ongoing research is necessary to refine these formulations for broader application in integrated pest management programs[42]. This is a significant limitation as the analysis was restricted to articles published in journals, excluding other valuable sources like reviews, conferences, books, and book chapters. To overcome this limitation, future researchers should consider utilizing additional databases such as Scopus and Science Direct, which can provide more comprehensive data. This limitation may have impacted the study's ability to provide a comprehensive overview of the field.

    The authors confirm contribution to the paper as follows: conceptualization: Kumar V, Mishra KK, Panda SR; writing − original draft preparation: Kumar V, Wagh AK, Mishra KK; writing − review and editing: Panda SR, Kumar V, Wagh AK; supervision: Kumar V, Panda SR, All authors have read and agreed to the published version of the manuscript.

    The data that support the findings of this study are available on request from the corresponding authors.

  • The authors declare that they have no conflict of interest.

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  • Cite this article

    Sedaghathoor S, Baladeh MK, Piri S. 2023. A study on chilling hardiness of three persimmon genotypes by electrolyte leakage parameter. Technology in Horticulture 3:10 doi: 10.48130/TIH-2023-0010
    Sedaghathoor S, Baladeh MK, Piri S. 2023. A study on chilling hardiness of three persimmon genotypes by electrolyte leakage parameter. Technology in Horticulture 3:10 doi: 10.48130/TIH-2023-0010

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ARTICLE   Open Access    

A study on chilling hardiness of three persimmon genotypes by electrolyte leakage parameter

Technology in Horticulture  3 Article number: 10  (2023)  |  Cite this article

Abstract: Most fruit trees in temperate regions are exposed to chilling, which causes extensive economic losses. Low temperatures reduce biosynthesis activity of plants and the functioning of physiological processes, impose irreparable injuries, and finally, destroy the plants. So, to study chilling tolerance of three genotype of persimmon by electrolyte leakage parameter, a factorial experiment was carried out in a randomized complete block design with three replications. The first factor was assigned to persimmon cultivar at three levels of date-plum, 'Fuyu kaki', and Japanese persimmon. The second factor was assigned to chilling treatment at two levels of 4 °C and −16 °C. The third factor was assigned to three phenological stages including bud dormancy phase, bud swelling phase, and anthesis phase. The recorded traits included electrolyte leakage, electrical conductivity (EC), and proline content. The results showed that proline content, electrolyte leakage and EC of all samples were the highest at bud swelling phase, but they did not differ significantly between bud swelling and anthesis stages. The highest electrolyte leakage and EC was observed in plants exposed to −16 °C and the lowest in those exposed to 4 °C. The membrane of date-plum at −16 °C and bud swelling phase was most heavily injured by chilling. Among the trilateral effects of studied factors on the recorded traits, 'date-plum × −16 °C × bud swelling phase' showed the highest proline content, electrolyte leakage, and EC, and 'date-plum × −16 °C × bud dormancy' and 'Japanese persimmon × −16 °C × bud dormancy stage' exhibited the lowest proline content, electrolyte leakage, and EC.

    • Losses by chilling may happen in all fruit-producing regions. On the other hand, low temperature is an environmental factor that restricts the planting, cultivation and production of horticultural crops. For example, the growing season of an annual crop is dictated by chilling-free time. Similarly, some limiting factors of producing permanent horticultural crops are low winter temperatures, early chilling in autumns, and late chilling in spring[1]. Adverse climatic conditions, especially winter frosts and spring chilling, are the most important parameters determining the distribution of species and, of course, the most important indicator of site selection for the construction of fruit orchards[2].

      Persimmon (Diospyros kaki L.), which belongs to the family Ebenaceae, is native to Sri Lanka and Southeast Asia. There are approximately 200 species in the genus Diospyros. Kaki, or the so-called oriental persimmon, is mainly grown for fruit production[3]. Among the various varieties of persimmon, only a few are important for commercial fruit production. The most important species of persimmon for commercial fruit production is Japanese persimmon (Diospyros kaki L.). In Iran there are genetic variations of 22 species of kaki and seven species of lotus and virginia (locally known as date-plum). Another persimmon cultivar in Iran is 'Fuyu kaki'[4]. Persimmon resembles pomegranate in winter chilling resistance so that it can resist the temperatures as low as −10 °C. The chilling requirement of the buds of this tree is 100−400 h of < 7 °C temperature. Some persimmon cultivars can tolerate the temperatures of as low as −18 °C[5].

      According Yu & Lee[6] chilling stress is a main ecological stress controlling the geographical distribution, growth, and development of temperate fruits. The range of freezing injury in the trees depends on the reduction rate of temperature, the lowest temperature reached, and the period of the stress conditions. They reported that freezing must be evaluated to predict the winter survival and productivity of the trees in particular regions, to monitor for tolerant genotypes, and to develop cultivation approaches that reduce chilling stress. Various methods are used to evaluate freezing injury in trees under field and laboratorial conditions, including estimate of tissue discoloration, thermal analysis, and determination of ion leakage. Low temperature reduces biosynthetic and physiological activity of plants and imposes permanent injuries and eventually death. Cold resistance can be defined as the ability of plants to tolerate freezing temperatures without incurring significant damages, and this is an important indicator for assessing the potential of planting species and cultivars[7]. Phenological stage can be vital for chilling injuries. Trees are more sensitive to low temperatures at flowering and petal shedding stages[8]. Low temperatures disrupt the balance between energy absorption and its use by target metabolites, so enzymatic activities are reduced to a greater extent than photophysical and photochemical processes involved in light absorption, energy transfer and transformation[9].

      According to Miranda et al.[10], the species of the genus Prunus, such as almonds, are resistant to chilling before flowering, but they become more susceptible at full-bloom and next stages than pre-flowering stage (deep dormancy of floral bud), but under identical phenological conditions, genotype is the determinant factor. In a study on the frost tolerance of eight olive cultivars, Barranco & Ruiz[11] concluded that electrolyte leakage was increased as the temperature was decreased to as low as −22 °C. Interestingly, this increase in electrolyte leakage was more pronounced in the susceptible olive cultivars. Electrolyte leakage method has been used to evaluate chilling injury in grape organs[12], raspberry branches[13], blueberry shoots[14], and peach tissues[15].

      Because of the importance of chilling resistance for permanent plants to pass through winters, there is a great interest in finding ways to determine the cold resistance of plants. Most of these methods have been designed to test controlled frosts and assess the resulting chilling injuries. But, researchers have long been looking for indirect ways to measure non-frosting cold[7]. Since persimmon has turned into an economic base for some orchard owners, including those in Guilan province (north of Iran), and given the economic significance of its wood, fruits, and its medicinal properties, it is imperative to explore factors limiting it's planting[4]. Understanding low-temperature hardiness as one of the environmental factors in order to find coping strategies to alleviate the damage caused by this phenomenon is one of the important research goals for horticulturalists and plant physiologists. As such, some persimmon genotypes were evaluated for cold resistance of dormant bud, swelled bud, and flower.

    • The experiment was conducted on three important persimmon commercial genotypes, including date-plum, 'Fuyu kaki', and Japanese persimmon in Talesh County (Guilan province, North of Iran) from mid-December to mid-March 2017. Persimmon trees in the same age of 10 years in the region were used in the experiment. The experiment was designed as factorial with three factors on the basis of a randomized complete block design with three replications. The first factor was assigned to persimmon genotype at three levels of date-plum, 'Fuyu', and Japanese persimmon. The second factor was assigned to chilling treatment at two levels of 4 °C and −16 °C. The third factor was assigned to three phenological stages including bud dormancy phase, bud swelling phase, and anthesis phase. The traits evaluated in this experiment included electrolyte leakage, electrical conductivity (EC), and proline.

      The change in EC of treated tissue minus the change in EC of the control at each temperature, assuming that the differential increase in ion leakage is due to the chilling temperature[14, 15]. The total capacity to leak can be determined by autoclaving the tissues and measuring the electrical conductivity of the aliquot[6]. To apply the experimental treatments, the selected tissues (branches and buds) were first sprayed with distilled water. Then, they were placed in an incubator and were cooled to 2 °C at a freezing rate of 10 °C/h. Then, they were cooled to the desired temperature at a freezing rate of 5 °C/ha and were kept at that temperature for 3 h. To this end, 0.5 g of the buds from each experimental plot was placed in closed container containing twice-distilled water and it was placed at the laboratory temperature for 24 h. Then, its EC was measured with an EC-meter to give EC1. To measure EC2, 0.5 g of the buds was frozen at −20 °C for 24 h. Then, they were placed at room temperature for 24 h. Then, EC2 was read and the electrolyte leakage (EL) was calculated by the following equation:

      EL = EC1EC2×100

      Proline was measured by Bates et al.'s[16] procedure. Fresh tissue (0.1 g) was ground in 10 mL of 3% sulfosalicylic acid to yield a uniform mixture. The resulting extract was centrifuged at 10,000 rpm for 5 min. Then, 2 mL of supernatant was mixed with 2 mg of ninhydrin reagent and 2 mL of pure acetic acid and was placed in a warm water bath at 100 °C for 1 h. The tubes containing the mixture were then cooled down in an ice bath to stop all reactions. Then, 4 mL of toluene was added to the mixture and the tubes were well shaken. After the tubes were held still for 15−20 s, two complete separate layers were formed in them. The colorful upper layer that contained toluene and proline was used to measure proline concentration. Then, its absorption was read at 520 nm and proline content of the samples was found out using a standard curve.

      Y=0.129X+0.009

      in which Y is the absorption read and X is proline concentration in mM/L. Data were analyzed by MSTATC statistical software and the means were compared by the LSD test.

    • Based on the results, proline content was significantly influenced by the simple effect of phenological stage (bud dormancy, bud swelling, and anthesis) and the trilateral interaction of 'genotype × chilling × phenological stage' at the p < 0.01 level and the interaction of 'genotype × phenological stage' at the p < 0.05 level. But, the impact of genotype, chilling, 'genotype × chilling', and 'chilling × phenological stage' was insignificant on this trait (Table 1).

      Table 1.  Analysis of variance for the effect of experimental factors on proline content.

      Sources of variationsdfMeans of squares
      Proline contentElectrolyte leakageEC
      Replication288.74**89.39ns32.14ns
      Genotype (A)219.48ns71.32ns117.79ns
      Chilling (B)121.37ns234.04*284.97*
      AB229.13ns138.85*461.82**
      Phenological phase (C)2369.23**6673.85**7819.65**
      AC440.96*26.31*115.24ns
      BC210.59ns143.87*285.73*
      ABC465.57**85.79+464.13**
      Error3414.4233.5959.83
      ns: insignificant difference; **: significant difference at 0.01; *: significant difference at 0.05; +: significant difference at 0.10.

      The results of means comparison (Table 2) revealed that the highest proline content was obtained at bud swelling phase, but it was lower at bud dormancy and anthesis phases among which there was not a significant difference. Means comparison of the data for the interactive effect of 'genotype × phenological stage' on proline content indicated that the highest proline content was related to the treatment of 'date-plum × bud swelling' and the lowest proline was obtained in 'date-plum × bud dormancy', 'date-plum × anthesis', 'Japanese persimmon × anthesis', and 'Japanese persimmon × bud dormancy' (Fig. 1).

      Table 2.  Means comparison for the effect of phenological stage on the studied traits.

      TreatmentProline
      (μmol/g FW)
      Electrolyte
      leakage (%)
      EC
      (μS/cm)
      Bud dormancy6.52 b2.54 b0.1 b
      Bud swelling14.66 a34.64 a36.23 a
      Anthesis7.15 b0.16 b0.16 b
      Similar letters in each column shows insignificant differences at the p < 0.05 level.

      Figure 1. 

      The effect of 'genotype × phenological phase' on proline content.

      With respect to the trilateral effect of 'genotype × chilling × phenological phase' on proline content, the highest proline content was obtained from 'date-plum × −16 °C × bud swelling' and the lowest from 'date-plum × −16 °C × bud dormancy', 'Fuyu kaki × 4 °C × anthesis', and 'Japanese persimmon × −16 °C × bud dormancy' (Fig. 2).

      Figure 2. 

      The effect of 'genotype × chilling × phenological stage' on proline content.

      It is argued that proline accumulation is an indicator of environmental stresses and plays a vital role in this respect[17]. By interacting with enzymes, proline contributes to protecting the structure and sustainability of their activity[18]. In a study on variations of injury level and proline content in the buds of some commercial genotypes of apricot at different phenological stages, the highest proline content was obtained at bud swelling phase and the lowest at anthesis phase[19], which is consistent with our findings.

      The analysis of variance for electrolyte leakage showed that the simple effect of genotype and the interactive effect of 'genotype × phenological phase' was not significant on this trait, but this trait was significantly influenced by the simple effect of phenological phase at the p < 0.01 level, by the simple effect of chilling and the interactive effects of 'genotype × chilling' and 'chilling × phenological phase' at the p < 0.05 level, and by the interactive effect of 'genotype × chilling × phenological phase' at the p < 0.10 level (Table 1). According to the results of means comparison, the highest electrolyte leakage was obtained from the fruits exposed to −16 °C and the lowest from those exposed to 4 °C (Fig. 3). Among different phenological phases, swelling had the highest electrolyte leakage and anthesis had the lowest but not differing from bud dormancy significantly (Table 2).

      Figure 3. 

      The effect of chilling on electrolyte leakage (EL).

      Means comparison for the interactive effect of 'genotype × chilling' on electrolyte leakage (Table 2) indicated that the highest and lowest rate of electrolyte leakage were obtained from 'date-plum × −16 °C' and 'Japanese persimmon × 4 °C' respectively, but showing insignificant differences with that of the other treatments. With respect to the interactive effect of 'chilling × phenological stage', it was revealed that '−16 °C × bud swelling' yielded the highest leakage and '4 °C or −16 °C × anthesis or bud dormancy' yielded the lowest (Table 3).

      Table 3.  Means comparison for the effect of 'chilling × phenological phase' on the studied traits.

      TreatmentIon leakage
      (%)
      EC
      (μS/cm)
      4 °C × bud dormancy1.60 c0.10 c
      4 °C × bud swelling29.34 b29.33 b
      4 °C × anthesis0.16 c0.16 c
      -16 °C × bud dormancy3.48 c0.11 c
      -16 °C × bud swelling39.94 a43.13 a
      -16 °C × anthesis0.16 c0.15 c
      Similar letters in each column shows insignificant differences at the p < 0.01 and p < 0.05 levels.

      The results for the interactive effect of 'genotype × chilling × phenological stage' (Fig. 4) indicated that the highest electrolyte leakage was related to 'date-plum × −16 °C × bud swelling' and all studied genotype had the lowest electrolyte leakage at 4 °C and −16 °C both at bud dormancy and anthesis phases.

      Figure 4. 

      The effect of 'genotype × chilling × phenological phase' on electrolyte leakage (EL).

      These results mean that electrolyte leakage is the highest at the early stage of dormancy breaking of trees in late winter (bud swelling phase). High electrolyte leakage reflects the inability of membrane in maintaining intra-cellular compounds and the outflow of ions from membrane, which injures cell membrane[20]. Therefore, it can be said that date-plum at −16 °C and bud swelling stage incurred the highest injury in cell membrane whereas all genotypes at bud dormancy and anthesis phase had the lowest electrolyte leakage and membrane injury, thereby showing hardiness to chilling-induced injury to their membrane. Since electrolyte leakage index is calculated on the basis of injury to cell membranes and this injury causes electrolyte leakage (especially K+) from cell[7], it can be inferred that the tissues of persimmons are damaged more at −16 °C than at 4 °C. In a study on frost tolerance of eight olive cultivars, Barranco & Ruiz[11] observed that electrolyte leakage was increased by applying colder temperature until the temperature of −22 °C, which is consistent with our findings.

      The results of variance analysis for the effect of experimental factors on electrical conductivity (EC) showed that the simple effect of the phenological stage (bud dormancy, bud swelling, and anthesis) and the interactive effect of 'genotype × chilling' and 'genotype × chilling × phenological stage' was significant on this trait at the p < 0.01 level and the effect of chilling and 'chilling × phenological stage' was significant at the p < 0.05 level. But, this trait was not affected by the simple effect of genotype and the interactive effect of 'genotype × phenological stage' (Table 1). The results of means comparison indicated that the highest EC was obtained from −16 °C and the lowest from 4 °C (Figs 5 and 6).

      Figure 5. 

      The effect of chilling on EC.

      Figure 6. 

      The effect of 'genotype × chilling × phenological stage' on EC.

      The plants had the highest EC at bud swelling phase and the lowest at bud dormancy phase, but this latter did not differ from that at anthesis stage significantly (Table 2). Means comparison for the interactive effect of 'genotype × chilling' on EC (Table 3) revealed that the highest EC was related to 'date-plum × −16 °C' and 'Japanese persimmon × −16 °C' and the lowest to 'kaki Fuku × −16 °C' showing insignificant difference with the treatments of 'Japanese persimmon × 4 °C' and 'date-plum × 4 °C'. Means comparison for the effect of 'chilling × phenological stage' on the EC of samples indicated that '−16 °C × bud swelling' had the highest EC while the lowest EC was related to bud dormancy and anthesis at both 4 °C and −16 °C (Table 4). The highest EC was obtained from 'date-plum × −16 °C × bud swelling' and 'Japanese persimmon × −16 °C × bud swelling' and the lowest from all studied genotypes at both 4 °C and −16 °C at bud dormancy and anthesis (Fig. 6).

      Table 4.  Means comparison for the effect of 'genotype × chilling' on the studied traits.

      TreatmentIon leakage
      (%)
      EC
      (μS/cm)
      Date-plum × 4 °C9.92 b8.68 b
      Date-plum × -16 °C19.56 a19.80 a
      Kaki Fuyu × 4 °C12.15 b12.84 ab
      Kaki Fuyu × -16 °C10.68 b5.77 b
      Japanese × 4 °C9.02 b8.07 b
      Japanese × -16 °C13.35 b17.82 a
      Similar letter(s) in each column shows insignificance differences at the p < 0.01 and p < 0.05 levels.

      In most trees, the EC of the solution leaking from chilling-exposed samples is a reliable parameter to evaluate chilling-induced injuries. To put it another way, when studying the injuries of chilling to plant tissue by electrolyte leakage, EC is a more reliable parameter than pH so that EC of samples (directly or indirectly) is used in the relevant formula to calculate both relative leakage at a certain temperature and injury index at a certain temperature. Chilling injuries to trees are assessed and measured by visual observations and electrolyte leakage[7]. Likewise, it was found that as chilling was intensified, EC was increased. The assessment of freezing resistance of fennel by measuring electrolyte leakage indicated that as freezing temperature was decreased, electrolyte leakage was significantly influenced in different parts and roots and leaves showed the highest and lowest electrolyte leakage percentage, respectively[21].

    • The results indicated that proline content, electrolyte leakage index, and EC of the samples was the highest in late winter when the dormancy of the trees started to break (bud swelling phase), but they were not statistically different between bud dormancy and anthesis phases. Since phenological phase can be crucial for chilling injuries, the recognition of phenological stages to find out the stage of chilling resistance the trees are at can be very helpful in avoiding chilling injuries. Also, it was revealed that these indices were increased as chilling was intensified, but the simple effect of genotype was not significant on any recorded traits. It may be said that chilling hardiness was similar across the studied genotypes. The results indicated that date-plum exhibited the highest membrane injury due to chilling (ion leakage) at −16 °C and bud swelling phase. Thus, it is more susceptible than other genotypes and the temperature of −16 °C at bud swelling stage can impose heavy damages to this genotype.

      • The authors declare that they have no conflict of interest.

      • Copyright: © 2023 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (6)  Table (4) References (21)
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    Sedaghathoor S, Baladeh MK, Piri S. 2023. A study on chilling hardiness of three persimmon genotypes by electrolyte leakage parameter. Technology in Horticulture 3:10 doi: 10.48130/TIH-2023-0010
    Sedaghathoor S, Baladeh MK, Piri S. 2023. A study on chilling hardiness of three persimmon genotypes by electrolyte leakage parameter. Technology in Horticulture 3:10 doi: 10.48130/TIH-2023-0010

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